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After you work up an appetite biking the roads and woods around Lyme, CT, take time to enjoy the Tour de Lyme's fabulous picnic.
There will be good music, a tasty lunch and even stretching therapy to loosen up tight or sore muscles. The setting at Ashlawn Farm couldn't be better and we will have a tent in case the weather is damp or the sun is too hot. Lunch options will include a variety of well known food trucks (we are planning to add a few more to help manage the lines this year).
The Eightmile River Band will be joining us again this year to provide some great Bluegrass music.
The picnic is free to all registered riders – just show your wrist band. Family and friends who did not ride can purchase picnic tickets on event day. Adults $25 and Children under 13 $15.
ACTIVITIES: Select Physical Therapy and Vitality Spa & Holistic Health
Back by popular demand the staff from the Old Lyme branch of Select Physical Therapy will offer pre- and post ride manual stretching; foam rolling; injury & pain assessments & consultations by licensed physical therapists.
Muscles feeling tight? Stop by or tent for a relaxing massage by one of our licensed therapists.


text:
Avignon, May 4th 2015 - Naturex, world leader in speciality plant-based ingredients, has published its registration document for the year ended December 31st, 2014.
- 2014 Registration document
The 2014 Registration document was filed with the French Financial Markets Authority (Autorité des Marchés Financiers) on April 30th, 2015 under the number D.15-0452.
This document includes :
- the annual financial report for the year ended December 31st, 2014 according to article L 451-1-2 of Code monétaire et financier ;
- the Chairman of the Board of Directors' Report on Corporate Governance, internal control and risk management ;
- the report relating to the social, environmental and societal informations ;
- the information related to the Statutory Auditors'fees.
Copies of this document in French language are available free of charge, pursuant to applicable law, and can be downloaded from the company's web site www.naturex.fr, under the « Finance - Documents and Publications » section, as well as on www.amf-france.org.
English translation of this document will be soon available.
You can receive all financial information of Naturex free of charge by signing up at: www.naturex.com
- Financial schedule
Q1 2015 results: 26 May 2015
- Upcoming events
- AGM (Paris) 24 June 2015 - Strategy 2020 Presentation (Paris) 30 June 2015
Naturex has been listed since October 1996 on Euronext in Paris, Segment B
|Total number of shares comprising the share capital: 9,205,153 (March 2015)
NATUREX is a component of the CACT, Enternext CAC PEA-PME 150, CAC Small & Mid, CAC Small, Gaïa Index.
Naturex is eligible for the "long only" Deferred Settlement Service (SRD) and French equity plans (PEA and PEA-PME).
Naturex established a Sponsored Level 1 American Depositary Receipt (ADR) program in the United States. Under this facility, Naturex's ADRs are traded over-the-counter in the United States under the symbol NTUXY.
TICKER: NRX - Reuters: NATU.PA - Bloomberg: NRX:FP - DR SYMBOL: NTUXY
About Naturex
Naturex is the global leader in speciality plant-based natural ingredients. The Group is organised around three strategic markets (Food & Beverage, Nutrition & Health and Personal Care) and produces and markets speciality plant-based ingredients for the food, nutraceutical, pharmaceutical and cosmetic industries.
Naturex's head office is based in Avignon. The company employs 1,600 people with 8 sourcing offices located throughout the world and high-performance manufacturing operations across 16 sites in Europe, Morocco, the United States, Brazil, Australia and India. It also has a global commercial presence through a dedicated network of sales offices in more than 20 countries.
- Contacts
|Carole Alexandre
Investor Relations
Tel.: +33 (0)4 90 23 78 28
|Anne Catherine Bonjour
Actus Finance Press Relations
Tel.: +33 (0)1 53 67 36 93
Information réglementée
Communiqués de mise à disposition de documents :
- Modalités de mise à disposition de document de référence ou de ses actualisations
Full and original press release in PDF:
Si le fichier PDF ne s'affiche pas ou pour le télécharger, cliquez ici.
Pour consulter le fichier PDF en intégralité, cliquez ici.
Pour visualiser les documents au format PDF, vous pouvez télécharger gratuitement Acrobat Reader XI.
Valeur associée
|Euronext Paris
© Actusnews.

subset: commoncrawl


text:
'Winter Wonderbland': Families blast worst ever Winter Wonderland event
FURIOUS families have demanded their money back after a "rip-off" Winter Wonderland event.
Bakewell Winter Wonderland and Christmas Market near Derby opened up on Friday and excited guests were met with empty stages, pathetic rides, and a bog standard affair.
Among the many complaints were reindeers just being horses with antlers taped on to their heads, a bog in the centre of the fayre and a weird plastic smell wafting round the event.
To enter the Christmas themed park families were forced to fork out £7 per person on top of a £5 parking charge, with many coming from around Derbyshire and Yorkshire.
Simon Brown, who runs events himself, felt cheated by what was on offer.
"It was really embarrassing, the reindeer carousel was just a bit of cardboard antlers taped onto normal horses"Lee Atkins
The 37-year-old said: "I'm trying to work out if this was a parody or a joke, I was expecting a classy affair and it is bottom of the barrel stuff.
"It's the wrong kind of event and there has been outright false advertising, it was was sold as the Chatsworth show, whereas it's more like Blackpool Pleasure Beach."
Reveller Lee Atkins, 44, said: "It was really embarrassing, the reindeer carousel was just a bit of cardboard antlers taped onto normal horses."
Other disgruntled families claimed there were long queues both for the car park and to go in even though only 20-30 people were waiting at a time.
They also said children had to wait up to two-hours to see a grotto-less Santa.
Trip hazard cables and children funfair rides printed with cartoon women in bikinis also left parents angered with the shocking state of facilities.
Despite this, some families have praised the Bakewell Show for offering them a refund after the event.
But taking to social media, Peter Cunningham, 36, from Derbyshire, said: "There has been one girl in the ice rink the entire time I have been here, she was not skating either, she was just walking around.
"Every tent has a disgusting smell and the air smells like weird plastic.
"The water under the wood is so bad that when you step on it, it comes up and over your feet."
One disgruntled customer became so annoyed with the state of affairs that he decided to use his car to drift around the car park in a doughnut motion, before he was asked to leave by staff.
Another customer said: "There are so many problems here it is horrendous.
"There is a huge boggy field in the middle of the area and so many empty rides.
"A car ride, tea cup ride, and a ghost train have looked completely unused and I saw a freezing cold woman at a market stall wrapped in a blanket, she was definitely feeling sorry for herself."
William Haggyard, 22, from Matlock, said: "We didn't queue to get in but we definitely queued to get out.
"We had money to spend but there was nothing to buy, we were hoping to do our Christmas shopping here but we have left empty handed and disappointed.
Popular complaints are that there is cardboard on the floor to stop mud coming through and a £4 charge for 15 minutes on an ice rink which is in a severely bad condition.
The organisers posted on Facebook on Friday that there had been "a lot of set backs this morning due to the mini hurricane,' adding that 'we can only apologise for any inconvenience, caused however we cannot avoid the awful weather we have experienced."
The event is scheduled to close tonight then reopen for December 9 until December 11.
Daily Star Online has contacted the Bakewell Show organisers for their response.


text: Panera BreadA low-carb Mediterranean Chicken Salad not on the regular Panera menuChicken SaladTell them you want to order off of the Secret Menu. Then, ask for the "Power Mediterranean Chicken Salad".All Locations5 - Medium-High$7.69http://hackthemenu.com/panera-bread/secret-menu/power-mediterranean-chicken-salad/The Panera Bread secret menu was actually officially released by Panera a while back. The Panera Bread Secret Menu is a list of healthy options that they don't have oficially listed on their regular menu. The Power Mediterranean Chicken Salad comes loaded with all-natural, antibiotic-free chicken, romaine lettuce, baby spinach, ripe tomatoes, crisp apple-wood smoked bacon, chopped hard-boiled eggs, extra-virgin olive oil, and freshly squeezed lemon juice. You get all this flavor packed into one salad with just 360 calories, 12g carbs, and a whopping 35g of protein. Be sure to check out the Panera Bread Secret Menu as well as visiting our home page to check out the complete list of All the Secret Menus from all your favorite restaurants!


text:
Separate names with a comma.
Discussion in 'Homeowner Assistance Forum' started by gr8pyrs, Aug 8, 2003.
I'm in search of somebody in the Olney, MD area to quote aerating and seeding.
Post this in the commercial forum - someone will get with you
Thanks for the post.
I have found someone and did email the administrators to close this thread.


text:
- Date of birth: Feb 16, 1970
- Date of passing: May 23, 1970
|Let the memory of Vicki-Lee be with us forever
"To the Family of Vicki-Lee,
Please accept my deepest sympathy. It is so unnatural and painful to lose someone in death at such a young age. But be assured that Jehovah God yearns to reunite children with their parents by means of the resurrection. In the account at Mark 5:40-42, Jesus lovingly demonstrated what his Father will do for us in the future when he resurrected a 12-year old girl and returned her to her parents. I know that you will remember Vicki-Lee forever. Vicki-Lee is also in the memory of God. "The hour is coming in which all those in the memorial tombs will hear his voice and come out, those who did good things to a resurrection of life" (John 5:28,29)"
"Our cheeky little one lent to us for 3 wonderful months still so missed from your Mummy and Daddy"
to visit this memorial:
Subscribe to receive e-mail notifications when others contribute to this memorial.
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text:
Johns Hopkins University
Address: Baltimore, MD
People related to Johns Hopkins University:
Robert J. Abernethy - emeritus trustee
Leonard Abramson - emeritus trustee
Peter G. Angelos - emeritus trustee
C. Michael Armstrong - emeritus trustee
Jeffrey H. Aronson - trustee
Norman R. Augustine - emeritus trustee
Janie Elizabeth Bailey - trustee
Lenox D. Baker Jr. - emeritus trustee
H. Furlong Baldwin - emeritus trustee
Jeremiah A. Barondess - emeritus trustee
Ernest A. Bates - emeritus trustee
David H. Bernstein - emeritus trustee
Chris Beyrer - professor
Paula E. Boggs - trustee
George L. Bunting Jr. - emeritus trustee
David Calleo - professor
Constance Rose Caplan - emeritus trustee
Renee Chenault-Fattah - trustee
Eliot A. Cohen - professor
N. Anthony Coles Jr. - trustee
Ronald J. Daniels - president
Anthony W. Deering - emeritus trustee
Andreas C. Dracopoulos - trustee
Ina R. Drew - emeritus trustee
Roger C. Faxon - trustee
Pamela P. Flaherty - trustee
Louis J. Forster - trustee
Sanford D. Greenberg - emeritus trustee
Benjamin H. Griswold IV - emeritus trustee
David C. Hodgson - trustee
R. Christopher Hoehn-Saric - trustee
Charles J. Homcy - trustee
Stuart S. Janney III - trustee
Jeong H. Kim - emeritus trustee
David H. Koch - emeritus trustee
David Michael Lampton - professor
Roger C. Lipitz - emeritus trustee
Raymond A. Mason - emeritus trustee
Gail J. McGovern - trustee
Harvey M. Meyerhoff - emeritus trustee
Heather Hay Murren - trustee
Ralph S. O'Connor - emeritus trustee
Morris W. Offit - emeritus trustee
Sarah Brown O'Hagan - trustee
Linda L. Robertson - VP
Brian C. Rogers - trustee
Paul B. Rothman - VP
David M. Rubenstein - trustee
Mark E. Rubenstein - emeritus trustee
John F. Ruffle - emeritus trustee
Frank Savage - emeritus trustee
Charles W. Scharf - trustee
Mayo A. Shattuck III - trustee
Rajendra Singh - emeritus trustee
Alfred Sommer - professor
David M. Steiner - executive director, Institute for Education Policy
Shale D. Stiller - emeritus trustee
Morris Tanenbaum - emeritus trustee
Ruth Wedgwood - professor
Other current Johns Hopkins University relationships:
Johns Hopkins School of Medicine - medical school
Johns Hopkins University past relationships:
Fouad Ajami (deceased) - professor
Alfred R. Berkeley III - trustee
Bloomberg Family Foundation - funder
William R. Brody - president
Wm. Polk Carey (deceased) - emeritus trustee
Benjamin S. Carson - director of pediatric neurosurgery
A. James Clark (deceased) - emeritus trustee
Harvey P. Eisen - trustee
Milton S. Eisenhower (deceased) - president
Adam F. Falk - dean of arts & sciences
Marjorie M. Fisher - trustee
Francis Fukuyama - professor
Daniel Coit Gilman (deceased) - founding president
Gordon and Betty Moore Foundation - funder
Lincoln Gordon (deceased) - president
Johns Hopkins (deceased) - benefactor
Michael M.E. Johns - VP
Kristina M. Johnson - provost & SVP
Robert L. Johnson - trustee
Edward J. Kelly III - trustee
J. Barclay Knapp - trustee
Zanvyl Krieger (deceased) - benefactor
Joanne Leedom-Ackerman - trustee
Roger C. Lipitz - trustee
Edward J. Ludwig - trustee
John C. Malone - benefactor
Kweisi Mfume - trustee
Charles D. Miller - emeritus trustee
Steven Muller - president
Marion Pines - professor
Michael F. Price - trustee
William C. Richardson - president
Mark E. Rubenstein - trustee
Arthur Sarnoff (deceased) - trustee
Donald J. Shepard - trustee
Michael S. Steele - trustee
Relationship listings for Johns Hopkins University are selective, based on whether individuals have additional connections to other people and organizations in the Muckety world.
Muckety news stories featuring Johns Hopkins University
There are those in America's leadership elite who forge so many connections to so many other influential people that they become power centers themselves.
September 27, 2011


text:
Coaching with Paula Davis
Event date:
Jun 20 2017 - 6:45pm to 9:30pm
Paula Davis is currently the President of Sweet Adelines International. A Sweet Adelines member for 31 years, Paula is also an Expression Judge Specialist, Certified Expression Judge, Certified International Faculty, SAI Board of Directors member, and Chair of the International Review Board. She has sung with--and been the assistant director of--the RichTone Chorus, Houston Horizon Chorus and Velvet Hills Chorus. Additionally, Paula chartered and directed the Spindletop Sound Chorus in Beaumont, TX. She has the fairly rare distinction of having sung all four parts, but currently sings bass or baritone.


text:
NEW WARRIOR Lacrosse MLL Series Arm Guard 6.0 Lg
Sold for:
Start Free Trial
or Sign In to see what it's worth.
- Sold Date:
- Source: eBay
WARRIOR LACROSSE ARM GUARDCONDITION: New, MSRP $71.99 SIZE: Large BRAND NAME: Warrior MLL Series COLOR: Silver & White SHIPPING & INSURANCE: We typically ship within 5 days of cleared Paypal payment. Under certain circumstances, this time period may increase, especially if special packaging is required. We usually ship via USPS Priority or Express mail to the US only. We will use UPS for larger items. Insurance will be required on certain items of value over $100. While insurance on other items is optional, we highly recommend that you purchase it as an inexpensive way to assure that your items are covered if lost or damaged in the mail. We will provide insurance documents in the event of a loss but we cannot reimburse for goods damaged or lost by the carrier. RETURN POLICY: All of our items are sold AS IS. Please ask questions before auction close to be comfortable with the item you are bidding on. We are very focused on assuring that you receive a quality product and we strive for total customer satisfaction. Our products are guaranteed for authenticity and as described in listings. We will accept returns if the item is received back in the same condition it was sent and we preapprove the return. You must notify us within 3 days of your receipt of item and contact us ... read more


text:
Dakota Fanning...
I kind of find her pretty, witty and a great actor, really.
Here's a biography of Dakota Fanning via wikipedia:
Hannah Dakota Fanning, born February 23, 1994, better known simply as Dakota Fanning, is an American actress.
Dakota Fanning was born in Conyers, Georgia, the daughter of Joy (née Arrington), who played tennis professionally, and Steve Fanning, who played minor league baseball for the St. Louis Cardinals and now works as an electronics salesman in Los Angeles. Her maternal grandfather was former American footballplayer, Rick Arrington, and her aunt is former ESPN reporter Jill Arrington.Dakota is the older sister of Elle Fanning, also an actress.
Fanning is of half German descent and her last name is of Irish origin. Fanning and her family are Southern Baptist. She attends Campbell Hall School inNorth Hollywood, California, and is on the varsity spirit cheerleading squad.
Fanning's breakthrough performance was in I Am Sam in 2001. As a child actress, she appeared in high-profile films such as Man on Fire, War of the Worlds, and Charlotte's Web. Fanning began transitioning to more adult roles with Hounddog and The Secret Life of Bees. She has won numerous awards and is the youngest person ever to have been nominated for a Screen Actors Guild Award.
then and now...
the duckling has turned into a beautiful swan


text:
azuresin az·u·res·in (āzh'ə-rěz'ĭn)
A complex of an azure dye and a carbacrylic resin that is used to detect gastric achlorhydria without intubation.
Tagged: a
Read Also:
- Azurite
a blue mineral, a hydrous copper carbonate, Cu 3 (CO 3) 2 (OH) 2 : an ore of copper. a gem of moderate value cut from this mineral. Historical Examples She was twisting the azurite jewels on her bosom, and pressing the blunt points into her flesh. The White Peacock D. H. (David Herbert) Lawrence […]
- Azurmalachite
a blue-green ornamental stone consisting of a mixture of azurite and malachite.
- Azurophil
azurophil azurophil a·zu·ro·phil (ā-zhur'ə-fĭl', āzh'ə-rō-fĭl') or a·zu·ro·phile (-fīl', -fĭl') adj. Staining readily with an azure dye. Used especially in reference to certain cytoplasmic granules in white blood cells.
- Azusa
a city in SW California, near Los Angeles.


text: NASSAU COUNTY List Grid Map We have found 1 items matching your search. FeaturedUzuri DesignsWe specialize in Print Design, Web Design, and High quality printing. Uzuri is the Kiswahili word for beauty, and Uzuri Designs delivers beautiful, eye catching material for all of your marketing need 1 Page 1 of 1


text:
We will help you find a local arabic teacher in Isle Of Colonsay and across the UK. Local arabic tuition is an excellent way to boost confidence as well as increasing grades and doesn't have to be dull! Please use our service to search for Isle Of Colonsay arabic tutors and select which best meets your requirements.
First Tutors is the only place to find the top Isle Of Colonsay arabic tutors for your needs, helping you find a home Isle Of Colonsay arabic teacher for any subject including GCSE English, A-Level Maths or even university Economics. We also help with language tuition, so if you want to learn German, take a Arabic course or prepare yourself for French life, we can help - and all in Isle Of Colonsay! We order arabic Isle Of Colonsay tutors in your area by arabic lesson costs and proximity to you.
If you are a Isle Of Colonsay arabic tutor wanting to offer your home arabic tuition services in Isle Of Colonsay or anywhere else across the UK, please register with First Tutors or find out more about what we offer here. Home arabic tuition is a great way to re-affirm your own arabic knowledge while helping someone else, all in the Isle Of Colonsay area!


text:
Vashikaran guru ji - Pt Raman Sharma astrologer famous for love marriage specialist, best astrologer in India and call us Ph 9872334718
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
Love is a most valuable prize of a person's life. There are many hurdle as well in some love stories that they need to face rejection of their love marriage by family, a family dispute or any incident due to love issues. A love marriage specialist is the best option to avoid all these issues that badly affect your family.
Vashikaran GuruJi has professional experience in love marriage problem solutions. He has solved countless cases during last 10 years. He is an expert in inter-caste lover marriage, inter-religion love marriage, family fight that not allow for your love marriage etc. With vashikaran techniques, he resolve the issues and convince lover's parents for their love marriage.
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Tumblr: https://www.tumblr.com/blog/deliciouskittymentality
Google Plus: https://plus.google.com/b/109869715069774576323/


text:
Connecting Expats In Delhi
Views: 460
Send me your photos...
I'm interested to in this! I'm fluent in english and native in romanian language. I live in Czech Republic now, but I want to move in India somewhere.
I can work in VP marketing or in Travel desk.
my email id - email@example.com.
Please send your CV, photos and other details, which you would like to share.
hello Nataly!
Did you receive my CV? I sent it to you 3 days ago. Looking forwards to hear from you as soon as possible because, as I told you, I want to move in India, anywhere.
Hey Nataly, do you look for people who want to work in FUJO. I have graduated in tourism and hospitality area. I am from Bulgaria and planning to come for second time in India for like 5 months...
Thank you in advance!
dimitar/jimmy :)
where are you located?
Shahpur Jat, near Kaus Haus metro station...
r there any positions still available?
i'm an economics, marketing and management graduate from south africa with a 3 year track record in marketing, advertsing and event co-ordination... i live in south delhi and would b really interested in working in this type of industry...
anyz hope to hear from you soon
Hi Nataly
I represent a design & strategy company based out of New Delhi
We are looking out to tie-up with advertising agencies in other towns so that we can work together on assignments which they outsource to design company's
Our strength is in a few fields:
1) Strategy
2) Graphic Design
3) product Design & Packaging
4) Online - Social Media management
5) Web Design
6) Corporate Audio Visuals
some samples of our work:
I hope to hear back from you, maybe we can share experiences and work together from our cities


text:
Long Description: Prepare for Action, Action Music, Action Impending Buildup, pending, buyout music, royalty free music downloads and royalty-free production music
Keywords: Repetitive strings introduce the atmosphere, with a subtle horn melody underpinning and providing movement. Ends with a piano and flute against strings coda phrase. alertness, anticipation, arrangement, background, base, basis, build-up, construction, dry run, education, establishment, evolution, expectation, fitting, foresight, formation, foundation, gestation, getting ready, groundwork, homework, incubation, lead time, making ready, manufacture, measure, plan, precaution, preparedness, preparing, provision, putting in order, qualification, readying, rehearsal, rundown, safeguard, schoolwork, study, substructure, training, tryout,


text:
Although the stereotypical view of the average American teenager is that she is glued to the phone, not all adolescent communications are verbal. Your teen may communicate with you or her peers, nonverbally. While nonverbal communication may seem more subtle than saying what she means, understanding how your teen communicates is critical to understanding what she is thinking and feeling.
Use and Cues
If you think the diatribe your teen spouts while debating the merits of staying out late is the only way she communicates with you, you aren't looking at her nonverbal language. Nonverbal communication includes actions, facial expressions, gestures, body postures and eye contact. Just because your teen isn't talking to you doesn't mean that she isn't providing cues that she is angry, sad or happy via nonverbal communication. Looking for these cues and teaching her how to look for them in other people can make the difference between effective communication and running into roadblocks.
Help your teen learn it isn't what he says that makes an impression, but what he does. Your teen's actions are a nonverbal way for him to communicate his emotions and thoughts to others. Behavioral changes, such as drastic mood swings, may provide tell-tale signs that something is wrong. Looking for these behaviors is helpful when your teen refuses to pipe up about what is going on in her life. If your even-tempered teen starts crying or storms off in a huff when you ask about her day at school, she is telling you nonverbally that she is stressed about something in her life.
Body Language
Learning how to communicate nonverbally by reading other people's body language is a key part of your teen's developing emotional literacy, according to KidsHealth. Not only should your teen learn that other people give off nonverbal signals by how they hold themselves, she should know that she is doing the same thing. If your teen comes home with her head bent down, tell her that you can read her emotional state as sad or depressed.
Facial Expressions
Like body language, facial expressions are a readable way for your teen to communicate what he is feeling or thinking. While your teen may have the ability to recognize anger, frustration sadness or happiness on someone else' face, he may not realize how easy it is for others to read these emotions on his face. For example, your teen walks in to a job interview and has a blank stare or bored expression on his face while he is meeting with his prospective boss. It is unlikely that he will be on the top of the list to get the job. Help your teen become more aware of his nonverbal communications by asking him to look in the mirror when he makes a bored, mad or sad face. This can help him avoid seeming unpleasant to others.


text:
General Question
What do you observe or find most important in first impressions?
Asked by CuriousLoner (1809) August 29th, 2012
When first meeting a person for whatever reason, what do you notice first?
Looks, tone of voice, clothing, posture, how they greet you?
Do you consider this person's location of living,lifestyle,status etc. Before meeting them for the first time? Are you willing to just make a impression based of meeting them or will other things always factor in?
And if you do say you would judge them based off just the person for being them, do you really mean it? Is it even possible?
Any sub-concision things that you take maybe?
Observing members: 0 Composing members: 0
12 Answers
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text:
Speech class topics list including twenty five of the best from art awards and air travel to fashion models and Hippie temporary community gatherings for education assignments needs and communication orals in:
|a. middle school;
b. high school;
c. college;
d. university.
1. Art awards and academy prizes. And note that there is more to see and discover than just summing up the Academy of Motion Picture Arts and Sciences categories.
2. Body art face painting and airbrushed tattoos that lasts for only a couple of weeks / months.
3. Movies: the characters, the spinning of the story lines, and splendid script writers who are honored with best scenario awards – perfect examples enough.
4. Museums, fairs, expositions, souvenirs, memorabilia, collectibles all what is informative in ….
5. Music: bands, singers, stars, the lyrics you will never forget and why, internet music radio stations and their specialized music genres.
6. Behind the scenes of a news network station.
7. Safety and security in air travel – take the persuasive approach on big issues or the three already basic outlined speech class projects.
8. Gardening: famous old palace and castle gardens in France.
9. Historical reenacting and living history.
10. Building and maintaining highways and roads – make it convincing as in these speaking plans. Mix in the high school speech topic you choose a sense of urgency.
11. World Records: strange, funny or amazing records in the Guinness World Records Book. E.g. Having the Longest Ears on a Living Dog 🙂 Really, it's in the famous book edition! Ideal for public speaking training purposes.
12. Theme Parks: review your favorite one after a nice day.
13. How to Set Up a Weblog and Earn Money and why it does not work like that for most of us. It is tragic for a lot of hardworking and typing bloggers who want to change the world with writing on the big themes ideas, but that's the way it is …
14. Aviation pioneers and their aircrafts. Consider the first notable woman Amelia Earhart.
15. Fashion Models: the difference between top and supermodels.
16. Presidential Campaigns: pick out one campaign for speech class of view my larger collection anyway.
17. Jewelry: the gold- and silversmith and the craftmanship from element to the ring or bracelet you wear.
18. How original Aboriginal Australian boomerangs are made to fly back and return to it's sender …
19. Cheerleading uniforms and supplies.
20. Musicals on Broadway and West End.
21. Famous tunes and songs in commercial breaks. You can find them online around the globe. What is the story, the feeling behind it?
22. Hippie temporary community gatherings nowadays – you bet serious teachers above fifty years know what the seventies really were … 🙂
23. Witches or other mythical figures.
More Speech Class Topics
High School Speech Topic
3 Ways to Find What You Like
24. How a book becomes a best seller. Put in some persuasion thoughts and approach the subject from the needs of writers and publishers.
25. Preserved dried flower wreaths and plants.
Nice for ever lasting bouquets and not only for Christmas time:


text:
'Hocus Pocus' instrumental in Nike ad
One of the biggest stars of the World Cup hasn't been a player, but a commercial. Nike's much-ballyhooed "Write the Future" ad, playing throughout the tournament, was directed by Alejandro Inarritu (Babel, 21 Grams).
The official three-minute-long version on YouTube uploaded by Nike now has nearly 18 million views.
The commercial's soundtrack is perhaps the driving force, impossible to shake. It seems like the perfect fit for the spot, a composition made to order. Instead, it's a 1970s hit by Dutch band Focus, called "Hocus Pocus."
Focus lead singer Thijs van Leer (pronounced like "Tice"), reached by phone in the Netherlands, said the commercial was a surprise, approved by the song's publisher and shown to him when it was nearly at a completed state.
"It's coming from heaven," said van Leer, now 62. "The last thing you would expect, you know? The last thing on my mind."
In 1973, the song reached No. 9 in the United States and No. 20 on the British charts, a big achievement for an instrumental song. The World Cup exposure led to its re-entrance to the UK charts this month after 37 years. The song landed at No. 57, two spots below a Black Eyed Peas song.
Clocking in at nearly 7 minutes long, "Hocus Pocus" features van Leer yodeling, playing the organ, singing falsetto, and playing a wicked rock flute. The twists and turns in it were perfect for the divergent storylines in the commercial, said Mark Bernath, the co-creative director of the commercial from the advertising agency Wieden+Kennedy Amsterdam. A former drummer from New York, he just happened to be hanging out with van Leer hoping to jam a little bit, calling the song and the commercial a "serendipitous marriage of pictures and music."
"We were hoping to come upon something that feels like a piece of magic," Bernath said. "When you have a song called 'Hocus Pocus' you're halfway there. It's a combination of musical ideas that seems like they couldn't possibly go together. It's constantly suprising. It just works.
The lunacy of the song isn't lost on van Leer, who says it was created during an impromptu jam session in the rehearsal studio. Normally, van Leer composed songs behind a piano at home. This was just off the cuff. The guitar player played a riff, the drummer played a short solo.
"Then I began to yodel behind my Hammond organ," he said. "I did that for the first time in my life." And is that an accordion solo in the song?
"Harmonium, actually," he said with a laugh. "It's even worse."
So it seems a perfect fit for a commercial meant to entertain, but also to make people laugh.
"The song has many gags, many jokes, you could say," van Leer said. "The film is an amazing film, full of spirit and humor. The song is filled with humor, so it was like a marriage."
Focus, which has kept touring with different lineups throughout the years, is actually back in the studio currently, re-recording its biggest hits and then working on a new album, Focus 10.
But for now, van Leer, is still touring, saying he's proud of the new life for his impromptu hit nearly 40 years ago.
"It's amazing how it spread the world again, this song. So much attention on this song. I'm still amazed and very thankful."
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As promised, I'm back to talk a bit more about my countdown deals promotion with amazon.com. Here's a very brief recap of this post. First, only KDP Select members may attempt a countdown deals promotion. All previous contracts and sales with other e-book providers must be cleared off the books before you enroll in KDP Select. You may start a countdown deals promotion thirty days after you enroll in KDP. You can't change the price of your novel for a certain period of time before / after the promotion.
The story:
On December 9th, my countdown deals promotion launched. See the post above for the general play by play of the deal.
On December 10th & 11th, the price increased from $0.99 to $1.99. Something interesting happened. My book managed to make it onto five top 100 lists. At it's peak, here are the categories and the rankings:
In the first 33 hours in the $0.99 bracket, I sold 60 copies of the novel. In the second 33 hours in the $1.99 bracket, I sold 41 copies of the novel. At the time of this posting, I have sold 1 novel at the $2.99 bracket.
It took approximately 50 sales to breach the 4,000 overall rank on amazon, and the remainder of the sales has kept it fluctuating between 4,500 and 3,390 overall ranking.
Here is the sour truth of things: Prior to the countdown promotion, I had sold a grand total of 25 books via amazon. (My indiegogo campaign 'sold' an additional 37 copies of the novel.)
In short, the countdown deal promotion has made a significantly huge impact on my fledgling writing career. This is entirely thanks to the Google+, twitter, and facebook communities, as well as thanks to the power of family and friends.
I definitely couldn't have gotten even this far without your help.
Now, a little sidetracking to discuss the problems with this promotion.
First, because I'm an unknown, new author, I don't have the selling power of others. I don't have a fan base. I have to entirely rely on people seeing the cover, checking out the description, and then deciding to check out a sample. That's a really hard sale. A really hard sale.
Because so many people bought the book as gifts for others, the book has gotten a great deal of exposure I otherwise couldn't get.
I'm steadfast against free book promotions. Why? I worked many long and hard hours on this book. It's my career. It's my life. I give away a lot of my time as it is. I need to pay the bills. Free promotions don't help me pay the bills. At least with $0.99 and higher countdown deal promotions, I can at least try to pay my way in my household and catch up on holiday season bills. This lets me reach fans who aren't certain about my books because of its normal price.
I priced Storm Without End at $5.99 to reflect the length. It's almost 100,000 words, compared to The Eye of God ($4.99) which is 80,000 words. I will mark Storm Without End and The Eye of God both down a dollar or two when the second books of their series launch. I feel this will give new readers a chance to get older books at a more reasonable price. (And I will also run countdown promotions in conjunction with novel releases so new readers can get more books for a better price.)
Second, I'm an independent author. There is definitely a negative connotation with being an independent. There is a belief that because I'm an independent, I'm automatically not good enough to be a traditionally-published author. Now, I don't know if this is true or not. Sure, I submitted my books. Sure, I got rejected — on requests for fulls after partials.
I've also learned a lot since then. You can read this post for more on why I am so very grateful to have been rejected. But, as with all things, reality and finances got in the way. I had to choose: Go independent or give up my writing career completely.
I chose going independent. I am a writer. That is what I do, and that is what I'll always do. But, because I don't have a publishing house backing me, I have a lot of hurdles I must overcome. I don't mind it, but it is a major complication in the whole launching a career thing.
The Issue with Gifting
Back to the quirks of the countdown deals promotion: When I launched this promo, I suggested that people give my novel to their friends and family as a cheap Christmas present. A lot of people liked this idea, and that's what they did. When you deal with rankings with amazon, gifting only counts to your ranking when the receiver accepts the book.
Almost 40 hours after the end of the $0.99 promotion, there are still people accepting the gifts. This is great! But, it means you will have no idea what your actual numbers will be (or how your rank will be impacted) because of how gifting works. This is just something you should be aware of.
The Lure of the Stats Page
Don't ask me to make sense of the stats page. I really can't figure out how it works. Sometimes it is real time. Sometimes it isn't. Sometimes, it can take a day or more for a sale to be noted. Sometimes seconds. I'm convinced that sometimes, it just doesn't get added at all.
I can't give you tips on how to avoid obsessively checking out the sales/stats page in the kdp site. I haven't succeeded at avoiding staring at it in hopes my novel will somehow be a runaway best hit. (And the realist in me says that I've probably hit my peak and will be extremely, extremely lucky if I ever even get remotely close to the 3,000s ever again.)
It is what it is. (You mean poopyhead stats page!)
I will leave you with this as food for thought for now.
There will be more, probably at around the 100 hour marker in the countdown deals promotion. It'll probably involve me crying into a frosty glass of something alcoholic as my chance for fame crumbles to dust. Or me breaking out the port because the unthinkable success runaway I don't think could ever happen to me happened. Either way, I suspect there will be alcohol involved.


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Minnesota Vikings general manager Rick Spielman said Tuesday that he will not trade quarterback Christian Ponder.
Ponder, selected with the 12th pick in the 2011 draft, is one of two quarterbacks on the Vikings' roster, currently expected to back up Matt Cassel.
"That would leave us with one quarterback. I would not do that," Spielman said. "We're going to need two quarterbacks. Three."
Ponder's disappointing performance the last three seasons is the main reason the Vikings could potentially draft the next quarterback of the future this week.
Spielman won't be afraid to draft a QB with the No. 8 pick despite Ponder not working out as the team had hoped.
"Christian's still a very young quarterback. He's shown flashes, but he just hasn't been as consistent as you want yet," Spielman said. "You can't be afraid. If there's a guy there, regardless of position, that you have strong feelings for, whether it's at 8 or 40, or wherever it is, or 16, then you take him. The one thing you cannot be afraid (of) in this job is to take chances or take risks on guys you feel strongly about. When you don't do that, I don't know how you get better."
The Vikings decided not to pick up the fifth-year option on Ponder that would have paid him $9.686 million in 2015.


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Just with to write an update as it has been a while since I've last had time to share the exciting activities we're doing.
First of all, a convention is coming up in California on the 24th May.
For those of you who are interested in meeting some kiggers, some of the members from KCS will be there.
Drop by the forum thread and find out details on how to find them~^__^
Anime North is another convention that MissKiki is taking part in in Canada.
If you are in that vicinity, be sure to turn up okie~
Check out the discussion here~
Hotaru has been taking a few shoots in a spankingly new looking place~
Take a look and give her your comments here:
We have so many new members lately, from all over the globe
Drop in and say hi if you haven't.
And check out who is close to you, you may even motivate one another to pursue your kigurumi cosplay dream~^__^
Now for the really really exciting news~^__^
I have thought of a way to make a moving mouth kigurumi mask~!!!And I can't wait to start building it~
I have made the new shells for this and also bought the material I need.
I think by next week, you should be able to see some physical progress~
Keep a lookout there to see the next technological advancement of kigurumi cosplay~^___^
And last but not the least… heh heh heh…
I shall not reveal too much as the official teaser is coming soon…
Let's just say be prepare to… PVP~^__^


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Jay Sean's Neon is on the blink
Jay Sean's much-delayed new LP is safe and sound – but where are the ripped-up bhangra beats?
Jay Sean: Think twice before you approach a celebrity you really admire
Jay Sean, born Kamaljit Singh Jhooti, 29, from Hounslow, has had hits including Eyes On You and Down, which reached No.1 in the US singles chart last year. He performs at the Mobo Awards on Wednesday and will release a new album in December.
Jay Sean to collaborate with Girls Aloud Nadine
Music news: Jay Sean has revealed he is working on tracks with Keisha Buchanan and Nadine Coyle.
Jay Sean reveals pop collaborations
Jay Sean has revealed he is working on tracks with Keisha Buchanan and Nadine Coyle.
Jay Sean: 'They wanted me to be James Blunt'
Jay Sean, 27, was planning to be a doctor before he became the r'n'b singing sensation we see before us today, enjoying chart success with Top 10 hits Eyes On You and Stolen. He was in Miami to work with Lil Wayne and his new single, Tonight, is out now.


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Results 1 to 6 of 6
Thread: mysql newb question (easy)
06-19-04, 12:30 #1Registered User
- Join Date
- May 2004
- Posts
- 13
Unanswered: mysql newb question (easy)
I'm a newb to mysql, just installed it a few days ago and I just can't seem to login correctly. to get privileges.
when i open up
the current user is "ODBC@localhost" and i have no privilege to use the "mysql" database
however when i go to
run : cmd : and type in "c:/mysql/bin/mysql -u root"
the current user is "root@localhost" and i do have permission to use the mysql database.
how can i have it so that when i open c:/mysql/bin/mysql to have permissions?
I know it has something to do with GRANT but i haven't been able to get it right.
or how can i have it so that when i go to c:/mysql/bin/mysql
just have the root@localhost open automatically instead of
what the hell is ODBC@localhost anyway?
06-19-04, 12:57 #2Registered User
- Join Date
- Jun 2004
- Posts
- 7
GRANT ALL PRIVILEGES ON database.* TO user@localhost IDENTIFIED BY 'password'
where database.* is whatever your db is called, *.* does everything.
make sure user@localhost is not in quotes. Most likely there is no password, and you can skip everything after the user@localhost.
Oh, and the odbc@locahost must be your default user, usually I've just seen it as '@localhost' or something like that.
Hope this helps.
06-19-04, 13:04 #3Registered User
- Join Date
- May 2004
- Posts
- 13
"GRANT ALL PRIVILEGES ON *.* TO user@localhost"
i type that exact phrase and it return a line with "->"
and i have no idea what to type
i just closed it again and fired up under user@localhost and my i got error 1044 saying access denied to "use mysql;"
06-20-04, 00:22 #4Registered User
- Join Date
- May 2004
- Posts
- 13
can anyone else help this is frustrating, i've been on mysql.com all day learning but still no progress
learned a bunch of other stuff though
06-20-04, 14:45 #5Registered User
- Join Date
- Jun 2004
- Posts
- 7
the -> line means that it is waiting for more SQL query or it's waiting for the terminal character ';'
put a ; at the end of that and it should work
Also, when you log into mysql, do the mysql -u root and enter no password, that should be the default.
06-21-04, 23:59 #6Registered User
- Join Date
- May 2004
- Posts
- 13
thanks for the tip,
is there anyway i can have it so when i double click on the mysql.exe it automatically login's to root
instead of having to go to run and typing in
c:/mysql/bin/mysql -u root


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Network Teams Institute May 15-16, 2013 Math Assessment Session. Shifts in Assessments. Six Shifts in Mathematics Assessments. Let's Paginate!. Skip all the blue sheets and the table of contents Start with "About the Regents Examination…" Carefully number all other pages 1-48
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
Six Shifts in Mathematics Assessments
Skip all the blue sheets and the table of contents
Start with "About the Regents Examination…"
Carefully number all other pages 1-48
This will take you approximately 2 minutes, 19 seconds
Peter Swerdzewski: Fellow for Assessment
Zach Warner
Turn to pages 29 and 35; you'll find two Sets of Items.
Complete at least 5 items on each set. Be sure that they are corresponding items.
Check your thinking against the answer keys and rationales
Determine the three key differences between the two sets.
Reading Time
About the Regents Examination in Algebra 1 (Common Core)
Page 2
Test Blueprint: Regents Examination in Algebra I (Common Core)
Page 4
Thoughts and Reactions to this document?
Instructional Implications of this document?
Is there any news on page 7?
What is the instructional impact of page 8?
7.RP.A.3: Use proportional relationships to solve multistep ratio and percent problems.
Complete items 1,2,4,10 yourself.
Check your thinking against the rationale.
Discuss your observations about the 3 items including their similarities and differences.
Examine the standards at play. What is accomplished by measuring 7RP3 in multiple ways?
How does document and its implications reflect the ways you have taught in the past?
How does the document and its implications reveal a coming change in your practice?
What's news?
Name 3 implications for instruction.
Name 3 implications for teacher created assessment and/ or common assessments.
What action steps or must-do's come to mind when reviewing these pages?
What, of these slides and conversations would you want to duplicate for PD in your context?


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Keep the Internet neutral, but keep neutrality on the Internet (where it belongs)
by Ian Scales
"Heavy pressure is apparently being applied (although we can only speculate on this, due to the opaque way the European process is progressed) by countries such as Sweden and Germany to get rid of 138 and observers, such as Monica Horten (www.iptegrity.com), fear that it may be on its way out for the final reading."
The Copyright Enforcement Enigma 'accurate and absorbing account of the story of the Telecoms Package' -Journal of International Commercial Law and Technology
'...a must read for those interested in knowing in depth about copyright enforcement and Internet.' -Journal of Intellectual Property Rights.
Read more
Iptegrity.com is the website of Dr Monica Horten. She is a trainer & consultant on Internet governance policy, published author& Visiting Fellow at the London School of Economics & Political Science. She served as an independent expert on the Council of Europe Committee on Internet freedom. She has worked on CoE, EU and UNDP funded projects in eastern Europe and beyond. She was shortlisted for The Guardian Open Internet Poll 2012. Iptegrity offers expert insights into Internet policy (and now Brexit). Iptegrity has a core readership in the Brussels policy community, and has been cited in the media. Please acknowledge Iptegrity when you cite or link. For more, see IP politics with integrity
Iptegrity.com is made available free of charge for non-commercial use, Please link-back & attribute Monica Horten. Thank you for respecting this.
Contact me to use iptegrity content for commercial purposes


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This was our house in Africa, good solid brick and mortar.
This is our house in the USA... the buying power is much less here! The house is nice, but it's wooden and old.
This was our pool in Africa..
This is our pool here... actually we don't have one, we have to pay $5 for a swim in the town pool.
This was our tennis court in Africa...
We don't have a tennis court here, the kids play at school.
This was our car in Africa... except this is not the exact car, we had one exactly like it!
This is our car here... Hard to say which we prefer, I love JEEP, but the Chevy is so BIG ! It is Texas after all.
This was our pet in Africa, standing in our orchard of 40 fully grown fruit trees...
This is our pet here in the USA, and one of our baby trees in our orchard of 4.
This is the beach in Africa
This is the beach here...
These are our mountains in Africa
These are the hills here... although to be fair... The Rockies would leave our little African mountains in the shadows.
If you woke up to a noise in Africa it could be
If you wake up to a noise in the USA it could be
And now you know why we moved to the USA.


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Medical Specialty >> Psychiatry
Doctors Lounge - Psychiatry AnswersBack to Psychiatry Answers List
If you think you may have a medical emergency, call your doctor or 911 immediately. Doctors Lounge (www.doctorslounge.com) does not recommend or endorse any specific tests, physicians, products, procedures, opinions, or other information that may be mentioned on the Site.
DISCLAIMER: The information provided on www.doctorslounge.com is designed to support, not replace, the relationship that exists between a patient/site visitor and his/her physician. Please read our 'Terms and Conditions of Use' carefully before using this site.
Date of last update: 8/24/2017.
Forum Name: Psychiatric Topics
Question: Panic Disorder: Can I be helped?
|Krash - Sun Dec 02, 2007 9:43 pm
Share |
I was wondering if you could give me some advice on antidepressants. I am almost 24 years old and have experienced panic attacks now for almost 6 years.I have always had anxiety since I can remember. In the last few years my nurse practioner has had me try several antidepressants. I first started on paxil and it made my mouth jittery and lost my appetite. I then tried zoloft and it did the same thing. I then was on effexor xr for a year. I quit taking it because if I missed one day of it I got very dizzy and unbalanced. I did not want to make it become something I had to have so I slowly got myself off of it. I have panic disorder and so March I asked her about Lexapro and she prescribed it for me. I was taking the 10 miligram dose once a day. I only took three days worth before I stopped it. About an hour after I took the pill on the third day I was lying in bed and started having severe panic attack. I suffered these attacks all day everyday for 17 days. I was vomiting and felt like I had ice cold water running through my entire body. I felt like no one could help me that if they did try to that it would kill me because my body was so stressed out I didn't think it could take anything else. After almost loosing my job at work and staying with a friend I eventually went to the er where they just thought it was my stomache because I told them it fell like my stomach was falling all the time. They had me drink a numbing medicine which I just knew was going to make me quit breathing because I already felt like I couldn't. Even after not taking the medicine it seemed that my body was still having effects from it. I am very scaired to take any medicine. I had trouble breathing and felt like I was dying from that. It was the sickest I had ever been and I felt like I was trying to run from it. I need to take some kind of medicine to get me over these attacks I believe but just not sure what there is now. I feel the doctors in my rural (one doctor one nurse practioner) area don't know how to handle me anymore and the nearest town for another doctor is 30 miles away and since I cant even go 5 miles before getting sick I don't know if I have any other options left. I have tried talking to pyschologists but they seem to not help. I have panic for going out of town. I have not left our little town for a year now and I just would like some advice from a trained doctor in this field.
Thank You Katrina
forgot to mention that I am 90 pounds and like 5 foot 1 if it makes any difference to my story.
|Dr. Chan Lowe - Wed Jan 30, 2008 11:33 pm
Share |
Hi Katrina,
It sounds like you are really struggling with your symptoms. There are various other medications for anxiety. It seems that the SSRI class of antidepressants are giving you too many side effects. You may want to consider using a different class of antidepressant. Occasionally, a medicine called buproprione (Welbutrin) can be very effective for anxiety. Your doctor can determine if this may be a good medicine for you.
Keep in mind also, that basically all antidepressants take a while to reach full effect, often three to four weeks. You may also want to talk with your doctor about temporarily using a medicine to help calm you down, such as valium. These types of medicines do not treat the anxiety, only the symptoms so they are not good choices for long term management but it can help to lessen symptoms until the other medicine can begin to control the anxiety.
If at all possible, I would strongly recommend that you see a psychiatrist. This way you can get expert recommendations about managing your anxiety disorder. Hang in there. It can seem like an eternity trying to get through this but I assure you you can overcome your anxiety. It is often also very helpful just to talk with someone, not necessarily a doctor. Even just having a good friend to talk with can be very helpful.
Best wishes.
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Free Copy of Your Guide to Lowering Your Blood Pressure with DASH!
Get with the plan that is clinically proven to significantly reduce blood pressure! This updated booklet contains a week's worth of sample menus and recipes recalculated using 2005 nutrient content data. The "Dietary Approaches to Stop Hypertension" eating plan features plenty of fruits, vegetables, whole grains, and other foods that are heart healthy and lower in salt/sodium. Also contains additional information on weight loss and physical activity.
This post may contain affiliate links, please see my disclosure policy here.


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GraphicRiver 1st birthday 760819
Stock Vector \ Characters \ Animals
Birthday \ Illustration \ ToyCartoon \ Celebrating \ Cartoon \ Bottle \ Holding
Stock Vector - 1st birthday 760819 by fractalgr @ GraphicRiver
Stock Vector \ Characters \ Animals
Cartoon illustration of teddy bear celebrating 1st birthday.
Layered: Yes; Graphics Files Included: JPG Image, Vector EPS; Minimum Adobe CS Version: CS.
Acorn Woodcut; Oak Tree Sketch; Rubber Stamps; Christmas and New Year; Barbecue Silhouettes; Fish Design; Sharks and Hooks.
Keywords: animal, baby, balloon, bear, birthday, bottle, cartoon, cute, hat, holding, illustration, mascot, milk, party, teddy, toy.
Download Link


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Blog Categories: Politics | News | Music | Do This! | Chow | Media | Cinema | Education | Arts and Culture | Fun in General | TV | Sports | more categories»
We have some bummer news, White Lies fans. "We're sorry to announce that due to circumstances our date in Tucson has been cancelled," the gothic band stated on their Facebook page. Refunds are available at the point of purchase, according to the Facebook status. No reschedule date has been set at this time.
Showing 1-2 of 2
. November 9-19 in the Center for the Arts Black Box Theatre (Thu.-Sat. at 7:30 p.m., Sun.… More


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Cornerstone Blog« Back the Blog Home
What is The Patient Protection and Affordable Care Act?
August 08, 2011 | Leave a Comment
The Patient Protection and Affordable Care Act (PPACA) is a federal statute that was signed into law by President Barack Obama on March 23, 2010. This act along with the Health Care and Education Reconciliation Act of 2010 (signed into law on March 30, 2010) make up the health care reform of 2010. Together the laws focus on reform of the private health insurance market, providing better coverage for those with pre-existing conditions, making healthcare more affordable for those that financially qualify, improving prescription drug coverage in Medicare and extending the life of the Medicare by approximately 12 years.
What is included in the law?
The law includes numerous health-related provisions that take effect over the next several years, beginning last year in 2010. Some of the major provisions include:
- Guaranteed issue and community rating will be implemented nationally so that insurers must offer the same premium to all applicants of the same age, sex, and geographical location regardless of pre-existing conditions.
- Health insurance exchanges will form in each state, offering a marketplace where individuals and small businesses can compare policies and premiums, and buy insurance.
- Firms employing 50 or more people but not offering health insurance will pay a "shared responsibility payment" if the government has had to subsidize an employee's health care.
- Non exempt persons not securing minimum essential health insurance coverage are also fined under the shared responsibility rules. This requirement to maintain insurance or pay a fine is often referred to as the individual mandate, though being insured is not actually mandated by law.
- Improved benefits for Medicare prescription drug coverage are to be implemented.
- Medicaid eligibility is expanded to include all individuals and families with incomes up to 133% of the poverty level.
- Low income persons and families above the Medicaid level and up to 400% of the poverty level will receive subsidies on a sliding scale if they choose to purchase insurance via an exchange.
- Very small businesses will be able to get subsidies if they purchase insurance through an exchange.
- Establishment of a national voluntary insurance program for purchasing community living assistance services and support.
- The law will introduce minimum standards for health insurance policies and remove all annual and lifetime coverage caps.
- Policies issued before the law came into effect are "grandfathered" and are mostly not affected by the new rules.
How will it be funded?
The Act's provisions are designed to be funded by a variety of taxes and offsets. Major sources of new revenue include Medicare tax on incomes for individuals earning over $200,000 and joint filers over $250,000, an annual fee on insurance providers, and a 40% tax on "Cadillac" insurance policies. There are also taxes on pharmaceutical companies, high-cost diagnostic equipment makers, and a federal sales tax on indoor tanning services.
How are legal challenges impacting the law?
Organizations and lawmakers who opposed the passage of the bill have taken legal action against it and several court challenges are currently at various stages. The targets of the threatened lawsuits are several key provisions of the bill, but most focus on the "individual mandate". A total of 28 states have filed joint or individual lawsuits (including 26 states engaged in a joint action) to overturn the individual mandate portions of the law, arguing that fining individuals for failing to buy insurance is not within the scope of Congress's taxing powers. Members of several state legislatures are attempting to counteract and prevent elements of the bill within their states, and legislators in 29 states have introduced measures to amend their constitutions to nullify portions of the health care reform law. It is likely that the constitutionality of the bill will be challenged before the Supreme Court sometime within the next year or so.
Posted in Benefits, Legislative Alerts | Tagged Health Care and Education Reconciliation Act of 2010, health care reform of 2010, Patient Protection and Affordable Care Act, PPACA


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Written on: 17/05/2012
Have been a member of Vitality for a while and the difference it has made to my life is amazing. Upon joining I received a full induction as I wasn't confident with all the equipment available. As part of my membership I was also given a personal fitness programme to follow and an hours free personal training to get me started and help me achieve my goal. As well as the gym being fantastic the classes were great too, over 20 to choose from and all free.
The gym has also been massively extended and includes new equipment plus a second studio. I only pay £18.99 for a full membership but i am aware of an off peak membership that is slightly cheaper. I would recommend this gym to everyone as it is friendly and open to all.


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Quick Details
- Place of Origin: Guangdong, China (Mainland)
- Brand Name: SZOMK
- Model Number: AK40023
- Protection Level: IP54
- Type: Junction Box, iron
- External Size: 300*250*150mm
- Material: iron enclosure
- Color: white color with black are available
- Mini order: 1pc
- Delivery time: 3-15 days after payment
- Net WT: 5kg
- Payment: Paypal & TT & Western Union & Escrow
- Customization: silkscreen & sticker & cutout & color
- Package: Carton and plastic foam and tape packaging
Packaging & Delivery
|Packaging Details
|plastic enclosure for electronic devices is packaged with standard export carton .
|Delivery Time
|Shipped in 5 days after payment
Specifications of Iron enclosure
Iron enclosure
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21 April to 7 May 2017
126 Artist-Run Gallery, 15 St Bridget's Place, Galway
'brainchild' is the 126 Artist-Run Gallery's 10th Annual members' show, which will be held in both 126 Gallery in Galway and The Western Carolina University (WCU) Fine Art Museum, USA. This exhibition is particularly exciting as we will also offer one of our members a month-long residency opportunity at WCU in October of this year.
For any further information contact firstname.lastname@example.com


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Wednesday, May 24
Midweek Magnolias DT Challenge #165 Dies or Chipboard
10 comments:
It is such a fabulous card Suzi dear!!! Those flowers are yummy, as is the fabulous paper you used and the gorgeous colouring of this cutie :) xxxReplyDelete
What a beautiful card! I love the way you have coloured Tilda and the layout using those balloon dies :DReplyDelete
Thank you for joining us at Crafty Purple Frog and good luck.
Huge hugs
Amy xx
It's very pretty card! Beautiful flowers and cute Tilda. i like this balloon dies! Thank you for joining with Paradise of Magnolia. Hugs, mlarisa.ReplyDelete
Thanks for entering our Floral Profusion challenge at Crafty Cardmakers. If you would be so kind as to link directly to our challenge post, as requested in our rules, and not to the front page of our blog then your card would automatically be added via the linky. It would have explained this when you tried to enter it.ReplyDelete
I will publish it this time manually but would really appreciate that you add the link in the future please.
Sorry for the nag!
What a beautiful card. I love the soft blues, and the expert way you have coloured Tilda. There are more than enough gorgeous flowers to match our challenge theme and that little balloon die is just too cute.
I look forward to seeing you again.
Lin xx
Wonderful card, Suzi!!!ReplyDelete
Hugs, Candy
Gorgeous make, love the soft colours & the added detailsReplyDelete
Thank you for sharing with us at 'Crafty Purple Frog, this month. Good Luck!
Hugs Shell xx
What an absolutely delightful card. Thanks for joining in at Crafty Cardmakers and good luck with the challenge, Angela xReplyDelete
Thank you for sharing your card and joining in my challenge at Crafty Cardmakers this time.ReplyDelete
so very pretty - I love this! Thanks for joining us at Countryview Challenges and good luck!ReplyDelete
This is a beautiful card, so many fabulous details.ReplyDelete
Thank you for joining us at Crafty Purple Frog.
Pauline - Crafting with Cotnob
Thank you so much for taking the time to visit and leave me some love, I may not be able to thank you all personally but it means the world to me reading your lovely comments.


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During the last SIDELab meeting, I've never heard the word 'model' mentioned so many times and with difference meanings. In order to expand the scope of the conversation about models, we had to start creating new words to describe the kind of model that we were talking about—like baby models, meta-models, and modified models. One of the problems that we—as a society—face is that there are so many models available to understand a problem and design a solution, that it is hard to choose the most appropriate model to design the most effective solution to a problem. The essential question we seemed to be asking (according to this author) is what is the best way to go about designing a solution?
The initial purpose of SIDELab was to discover, learn and apply the various models associated with Systems, Integrative, Design and Evaluative Thinking. While each method of thinking has its own 'best' model to conceptualize and design a solution to a problem, we decided early on in SIDELab that each method of thinking and associated models works best in varying situations and contexts.
For example, in Integrative Thinking, the methodology is a way to think through problems that have solutions that are in tension with each other and asks the problem-solver to create a better model that combines the best of both models. But what if there are hundreds of solutions that aren't in tension with each other? Can you unify all those solutions into one that contains the best elements of each? Or in Systems Thinking, there is a model to understand the feedback loops in a system. But what if there are so many feedback loops, that trying to understand them only exacerbates the problem to a point where a solution seems almost impossible to design.
One of the models that we are currently working with is called, Solution Search, which is a model that comes out of the SIDELab discussions. But considering that we are not even able to decide on a single model to use to find a solution, maybe we should take a step back. I think that a much more apt name would be Model Search—but without the drama and pageantry that goes along with its TV counterpart. In this model—or meta-model—we would use a process to decide on a specific model to use which fits the problem and its associated context so as to maximize the time and energy spent designing a solution. For example, if we attempt to solve the problem that there is excessive garbage produced as result of food vendors in Toronto's PATH system (an underground walkway in Toronto that links 30 kilometres of shopping, services and entertainment) we may use an Integrative approach because there are two clear solutions (re-usable dishes and cutlery or a better recycling procedure), rather than understanding the hundreds of feedback loops that occur in high traffic retail spaces.
One of the greatest assets that SIDELab is its rich diversity in professional and academic experience. Each person that attends our meetings is bringing with them a set of models and tools with anecdotes and case studies that help illuminate the problem and solution. As a result, one of the greatest challenges that this group faces is how to reconcile all these models and ways of thinking into a common language and process that attempts to maximize our energy spent designing solutions to complex, or wicked, problems.
We may get there…stay tuned.
Written by Nick Petten.


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The cytoskeleton participates in many aspects of transporter protein regulation. In this study, by using yeast two-hybrid screening, we identified the cytoskeletal protein actin as a binding partner with the UT-A1 urea transporter. This suggests that actin plays a role in regulating UT-A1 activity. Actin specifically binds to the carboxyl terminus of UT-A1. A serial mutation study shows that actin binding to UT-A1's carboxyl terminus was abolished when serine 918 was mutated to alanine. In polarized UT-A1-MDCK cells, cortical filamentous (F) actin colocalizes with UT-A1 at the apical membrane and the subapical cytoplasm. In the cell surface, both actin and UT-A1 are distributed in the lipid raft microdomains. Disruption of the F-actin cytoskeleton by latrunculin B resulted in UT-A1 accumulation in the cell membrane as measured by biotinylation. This effect was mainly due to inhibition of UT-A1 endocytosis in both clathrin and caveolin-mediated endocytic pathways. In contrast, actin depolymerization facilitated forskolin-stimulated UT-A1 trafficking to the cell surface. Functionally, depolymerization of actin by latrunculin B significantly increased UT-A1 urea transport activity in an oocyte expression system. Our study shows that cortical F-actin not only serves as a structural protein, but directly interacts with UT-A1 and plays an important role in controlling UT-A1 cell surface expression by affecting both endocytosis and trafficking, therefore regulating UT-A1 bioactivity.
- cytoskeletal protein
- latrunculin
- membrane protein
- urea transport
urea plays a critical role in the urine concentration mechanism in the kidney's inner medulla. The major mechanism for delivering urea to the inner medullary interstitium is urea reabsorption mediated by the UT-A1 urea transporter from the terminal inner medullary collecting duct (IMCD) (26, 34). A UT-A1/A3 knockout mouse has impaired urea clearance and urinary concentrating ability (10). In vivo, UT-A1 transport activity is mainly regulated by vasopressin. Vasopressin increases kidney urea transport activity by increasing UT-A1 in the apical plasma membrane (19) and UT-A1 phosphorylation (43).
Successful trafficking to and residing on the cell surface are the prerequisites for transport proteins to execute their proper functions. The regulatory mechanism for membrane protein trafficking to cell surface, as well as maintaining its membrane expression, is complex. It is under strict spatial and temporal regulation and is particularly controlled by some important accessory proteins. Direct protein-protein interactions by accessory proteins have been shown to determine the specificity of membrane protein in sorting, membrane trafficking, and retrieval (14, 27). During the past decade, much attention has been paid to those proteins. Several proteins, including SPA-1 (27), syntaxin-3 (24), SNAP23 (38), Rho GTPase (16, 25), dynein and dynactin (30), and actin (37, 42), are involved in regulating aquaporin 2 (AQP2) water channel trafficking and membrane expression. UT-A1 membrane trafficking, endocytosis, and degradation has been shown to be regulated by the SNARE-associated proteins snapin (25), dynamin (15), caveolin (9), and MDM2 (5).
The actin cytoskeleton is the basic component for the cell's structure, with actin being one of the most abundant cytosolic proteins. It is a highly conserved protein with three isotypes (α, β, γ > 90% aa homology). Actin is vital to the function of all eukaryotic cells. Not only a structural protein or a simple scaffold protein, actin is also required for mitosis, cytokinesis, cell motility, muscle contract, maintenance of cell shape, endocytosis, and secretion (1, 16). Actin exists in two forms, monomeric actin (G-actin) and filamentous actin (F-actin). F-actin is in dynamic equilibrium with soluble G-actin. The extent of F-actin polymerization/depolymerization changes according to the cell's physiological needs and its specialization.
A number of cytoskeletal proteins like ERM (ezrin/radixin/moesin), many isoforms of actin, cofilin, dynein, and tubulin have been identified in exosomes containing AQP2 (29). The epithelial sodium channel (ENaC) complex from bovine epithelia contains ankyrin, spectrin, and actin (36). These findings suggested that actin cytoskeleton and actin-associated proteins could directly interact with and actively participate in the regulation of these channels. In addition to AQP2 (28) and ENaC (21), the cystic fibrosis transmembrane conductance regulator CFTR (31), the Na-K-2Cl cotransporter NKCC1 (20), cardiac L-type Ca channels (33), the Na+/H+ exchanger NHE1 (41), and the glucose transporter GLUT4 (18) are also reported to associate with actin. Some of their interactions with actin could occur indirectly by actin-associated proteins, like NHE3 indirectly binding to the cytoskeleton via actin binding adaptor protein ezrin (41).
In this study, using a yeast two-hybrid assay, we identified actin as a UT-A1-binding protein. We further found that, in polarized epithelial cells, the cortical actin colocalizes with UT-A1 in the apical membrane and subapical cytoplasm and plays an important role in the regulation of UT-A1 cell surface expression and urea transport activity.
To generate the bait construct for the yeast two-hybrid assay, a fragment of 144 nt corresponding to the 48 aa COOH terminus of human urea transporter UT-A1 was amplified by PCR from pcDNA3-UT-A1 and subcloned in frame into the multiple cloning sites of Nde I and Sal I digested pGBKT7 vector (Clontech). The insert sequence and reading frame were confirmed by DNA sequencing.
The UT-A1 COOH-terminal amino acids in pGBK-C-UT-A1 were mutated to alanine with the QuickChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions.
Yeast two-hybrid assay.
The bait of C-UT-A1 in pGBKT7 was transformed into the yeast strain AH109 (BD Biosciences) using the lithium acetate method. A single positive AH109 clone (bait) was selected from SD/-Trp plate and mated with the human kidney cDNA pretransformed library in Y187 (BD Biosciences). The mating mixtures were plated onto quadruple minus plates lacking Trp, Leu, Ade, and His. After 7–10 days, positive colonies were collected and replated in quadruple minus plates containing X-α-Gal for the second screening. All positive colonies were then processed for plasmid DNA purification and sequence analysis.
In vitro translation and binding assay.
In vitro translation was carried out using the TNT T7-coupled rabbit reticulocyte lysate system (Promega). The bait gene in pGBKT7 was tagged with c-myc and contains a T7 promoter. The targeted gene actin (clones C30, C35) in pACT2 isolated from cDNA library screening was tagged with hemagglutinin (HA). A 50-μl reaction mixture was prepared by using 25 μl of TNT reticulocyte lysate, 2 μl of reaction buffer, 1 μl of 1 mM amino acid mixture (minus methionine), 2 μl of 10 mCi/ml [35S]methionine (Amersham), 1 μg of DNA template, and 1 μl of T7 RNA polymerase. After incubation for 30 min at 30°C, 10 μl of bait and 10 μl of target reaction were mixed and preincubated for 1 h, then 10 μl of myc antibody (BD Biosciences) was added. For monitoring the quality of the protein synthesis, 10 μl of bait or target reaction was incubated with c-myc or HA antibody. The combined mixtures were precipitated by protein-A beads. After washing, the beads were eluted by boiling in Laemmli sample buffer and the proteins were resolved by SDS-PAGE. The gel was dried and analyzed by autoradiography.
Cell culture and cell treatment.
A stably transfected MDCK cell expressing UT-A1 (12) was used in this study. The cells were grown in DMEM supplemented with 10% FCS, 25 mM HEPES, and antibiotics in a 5% CO2 incubator at 37°C. After becoming confluent, the cells were treated with the following compounds alone or in different combinations: 0.1 μM jasplakinolide (Molecular Probes), 1 μM latrunculin B (Molecular Probes), 5 μM methyl-β-cyclodextrin (MβCD, Sigma), or 50 μg/ml concanavalin A (Sigma), for different times as described in detail in the relevant experiments. Cells were then processed for cell surface biotinylation, lipid raft isolation, Western blot analysis, or immunofluorescence microscopy.
UT-A1-MDCK cells were grown on a Transwell insert filter (Corning) for 5–6 days to allow the cells to develop polarity. The cells were fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized with 0.1% Triton X-100 for 5 min, and then were exposed to primary antibody against UT-A1 (1:400) for 1 h, followed by staining with fluorescein isothiocyanate (FITC)-conjugated secondary goat anti-rabbit antibody (Sigma) for another 1 h. To stain the F-actin, the cells were further incubated with Texas Red-X phalloidin (Molecular Probes) (1:500) for 20 min. After PBS washing, the cells were mounted with Vectashield (Vector Laboratories) and analyzed by confocal laser scanning microscopy.
Lipid raft fractionation.
Lipid raft isolation was performed with a 5–40% sucrose discontinuous gradient as described previously (4, 15). UT-A1-MDCK cells were grown on 100-mm plates and lysed in ice-cold 0.5% Brij96-TNEV buffer (10 mM Tris·HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 2 mM Na vanadate, and protease inhibitor cocktail) for 30 min on ice. Lysates were centrifuged at 3,000 rpm for 5 min. Five hundred microliters of postnuclear supernatant were mixed with an equal volume of 80% sucrose in TNEV and transferred to 13 × 51-mm Beckman centrifuge tubes. Three milliliters of 35% sucrose in TNEV were layered carefully on top of the mixture followed by another 1 ml layer of 5% sucrose. The sucrose gradient was centrifuged in a SW 50.1 rotor (Beckman) at 34,000 rpm (∼110,000 g) for 18 h at 4°C. Equal sizes of fractions (∼400 μl) were collected from the top to bottom of the tube and analyzed by Western blotting with respective antibodies.
Biotinylation and Western blot analysis.
Cell surface biotinylation was performed as described before (3). After treatment, the cells were washed with ice-cold PBS and labeled twice with freshly prepared 1 mg/ml EZ-Link Sulfo-NHS-SS-Biotin (Pierce) in borate buffer for 20 min in a cold room with gentle shaking. The excess biotin was quenched with 0.1 M l-lysine (Sigma). After washing, the cells were solubilized in 700 μl of RIPA buffer (150 mM NaCl, 10 mM Tris·HCl, pH 7.5, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitors). Fifty microliters of the supernatant was saved for Western blotting as pre-bead total protein, whereas 600 μl of the supernatant was incubated with 25 μl of Immunopure immobilized streptavidin agarose beads (Pierce) overnight at 4°C with gentle shaking. The biotin-labeled proteins were eluted in 35 μl of Laemmli sample buffer and run on SDS-PAGE for Western blot analysis. The following antibodies were used for Western blotting: UT-A1 (4), β-actin (Sigma), tubulin (Sigma), and caveolin (BD Bioscience).
Oocyte experiments.
Xenopus laevis oocytes were prepared and maintained in OR3 medium as described previously (30). The capped cRNA of UT-A1 was transcribed in vitro from linearized cDNAs with T7 polymerase using the mMESSAGE mMACHINE T7 Ultra Kit (Ambion). Two nanograms of UT-A1 cRNA in a total volume of 23 nl water were injected into each oocyte. Three days later, healthy oocytes were selected for functional assays by measuring [14C]urea uptake for each individual cell. For the cell actin depolymerization tests, oocytes were incubated with 0.2 μM latrunculin in OR3 medium for 24 h.
Statistical analysis.
All values are expressed as means ± SD. Statistical analysis of the data was performed by one-way analysis of variance (ANOVA) followed by Tukey honestly significant difference (HSD) tests. Differences were considered significant at P < 0.05.
Identification of actin as a UT-A1 interaction partner.
The urea transporter UT-A1 is a large molecule with 12 transmembrane segments and cytoplasmic NH2 and COOH termini. To identify the UT-A1 binding proteins, we first used a 48-aa COOH-terminal fragment of UT-A1 (C-UT) as bait and screened a 5 × 107 colony-forming units of kidney cDNA library using a yeast two-hybrid assay. After the second round selection, we obtained 149 colonies. DNA sequence analysis revealed four of them are the actin genes.
The association of actin and UT-A1 was confirmed by an in vitro binding assay. We used a rabbit reticulocyte lysate translation system and labeled the newly synthesized proteins with [35S]methionine. The bait (C-UT) with c-myc tag and the gene products of interest (C30, C35) conjugated with HA were mixed and immunoprecipitated with c-myc antibody. Figure 1A shows that the UT-A1 COOH terminus binds to actin (C30 and C35).
Potential actin binding sites in the UT-A1 COOH terminus.
To determine the actin-binding site on the COOH terminus of UT-A1, we subjected this segment to scanning mutagenesis, converting some key amino acids to alanine. We evaluated the ability to bind actin by an in vitro binding assay. Figure 1B shows that the mutant S918 lost its ability to bind actin.
Cortical actin colocalizes with UT-A1 in polarized UT-A1-MDCK cells.
The possible interaction of UT-A1 in vivo with actin was assessed in UT-A1-MDCK cells. Since actin could interact nonspecifically with IgG (8, 21) and immunoprecipitation may yield a false positive result, we performed immunostaining to visualize the colocalization of UT-A1 and actin in MDCK cells. The UT-A1 MDCK cells were grown on the filter for 4–5 days. The UT-A1 was detected by UT-A1 antibody and visualized by FITC-conjugated secondary antibody. The F-actin was stained with Texas Red-X phalloidin. The UT-A1 (green color) is mainly localized in apical membrane and within the subapical cytoplasm of the MDCK cells. Actin is distributed in the cell periphery along the cell membrane. Figure 2A shows the representative scanning from apical, middle, and basolateral sections. Cortical actin in apical sections is observed to colocalize with UT-A1.
Both UT-A1 and actin are identified in cell membrane lipid raft fractions.
The cell membrane contains specialized microdomains referred to as lipid rafts, which are enriched in cholesterol and sphingolipids. UT-A1 is mainly associated with lipid rafts in IMCD (4, 9) and HEK293 cells (9, 15), as well as in UT-A1-MDCK cells (4). We examined whether actin is also present in lipid rafts. Lipid raft fractions were isolated from UT-A1-MDCK cells and blotted with UT-A1 and actin antibodies. Figure 2B shows the representative blots of more than three independent experiments. Fractions 2–4 are denoted as the lipid raft fractions as indicated by caveolin, a marker of membrane lipid rafts. Both UT-A1 and actin are identified in cell membrane lipid raft domains. As a control for actin, tubulin, the cytosolic protein, is not found in lipid rafts.
Depolymerization of actin cytoskeleton increases UT-A1 surface accumulation.
Jasplakinolide binds to and stabilizes actin filaments and promotes actin polymerization (41). In contrast, latrunculin B binds to G-actin monomers and prevents polymerization to F-actin (16). We manipulated the actin polymerization status using these two compounds and examined whether this affects UT-A1 expression in the cell membrane. We treated UT-A1-MDCK cells with 0.1 μM jasplakinolide or 1 μM latrunculin B for 1 h and then processed them for cell surface biotinylation. As seen in Fig. 3, latrunculin B treatment significantly increased the amount of membrane UT-A1. In contrast, jasplakinolide treatment did not affect UT-A1 cell surface expression.
Actin depolymerization promotes forskolin-stimulated UT-A1 membrane trafficking.
UT-A1 molecules are quickly trafficked to the cell surface upon exposure to vasopressin or the cAMP stimulator forskolin (FSK) (3, 19). We next investigated whether disruption of F-actin will affect FSK-induced UT-A1 membrane trafficking. UT-A1-MDCK cells were pretreated with latrunculin B or jasplakinolide for 40 min followed by addition of 10 μM FSK for another 20 min. Cell surface biotinylation experiments showed that FSK stimulated UT-A1 membrane trafficking, which is consistent with an earlier report (3). Actin depolymerization by latrunculin did not block FSK's effect (Fig. 4). On the contrary, cotreatment with latrunculin and FSK had a synergistic effect on UT-A1 membrane expression. Actin depolymerization facilitates FSK stimulated-UT-A1 trafficking to the surface.
Actin influences both caveolin- and clathrin- mediated endocytosis of UT-A1.
The caveolin- and clathrin-mediated endocytotic pathways are the two major routes for endocytosis in eukaryotic cells. To examine whether actin participates in one or both pathways, UT-A1-MDCK cells were treated with MβCD, which inhibits caveolin-dependent endocytosis, or concanavalin A, which inhibits clathrin-coated pit-mediated endocytosis, and together with or without latrunculin for 1 h. Cell plasma membrane UT-A1 accumulation was measured by biotinylation. In agreement with our previous study (15), inhibition of either the caveolin or clathrin-mediated pathway increased UT-A1 membrane accumulation. Latrunculin treatment caused more UT-A1 accumulation in the cell membrane when either pathway was blocked (Fig. 5). This suggests that disruption of actin has broad inhibitory effects and reduces both the caveolin- and clathrin-mediated UT-A1 endocytosis.
Actin regulates UT-A1 urea transport activity.
Finally, we examined whether modulation of actin could directly regulate UT-A1 urea transport activity. Unfortunately, neither measuring urea transport activity by tubular perfusion of rat IMCDs (40) nor transepithelial flux in UT-A1-MDCK cells (12) was successful. Latrunculin treatment caused significant tubular leakage and damaged MDCK cell barrier integrity. We then sought to use the oocyte expression system. Incubation of UT-A1-injected oocytes with 0.2 μM latrunculin for 24 h significantly increased UT-A1 urea transport activity. Cytochalasin D, another inhibitor of actin polymerization (16), also increased UT-A1 urea flux activity (Fig. 6).
The importance of actin in regulating transporter proteins has been appreciated in many studies. The well-organized actin network, particularly cortical filamentous actin beneath the apical membrane, plays a critical role in transporter protein membrane expression in polarized epithelial cells (16). The major finding of this study is that actin directly interacts with the UT-A1 carboxyl terminus and that the cortical actin plays an important role in the regulation of UT-A1 cell membrane expression and bioactivity.
Intact actin filaments are required for the vesicular trafficking of some ion channels and transporter proteins to the membrane (6, 17, 21, 22) and/or structurally anchoring the protein within the plasma membrane (21). Inhibition of actin polymerization blocks sodium-glucose transporter 1 (SGLT1) apical surface localization stimulated by 8-bromo-cAMP and significantly inhibits SGLT1 activity (17). This indicates that intact actin filaments are necessary to translocate SGLT1 from cytosolic compartments to the plasma membrane (17). cAMP/protein kinase A (PKA) induces CFTR-associated ion channel activity only in the presence of an organized actin cytoskeleton (31). The vasopressin-stimulated Na+ channel (ENaC) activity is inhibited by disrupting the organization of actin filaments (30).
However, other studies show that actin polymerization functions as a barrier for transporter protein trafficking between the apical membrane and the intracellular compartments (21, 28). Actin and actin-binding proteins may cross-link into three-dimensional networks. The vesicles or the transporter molecules are held in place by the cortical actin cytoskeleton (28) and are therefore prevented from contacting the cell membrane and inserting into the cell surface. Thus, depolymerization of cortical F-actin is considered an important prerequisite for transporter protein membrane trafficking and proper activity. AQP2 is another important transporter expressed in the IMCD. Vasopressin-stimulated apical sorting of AQP2 was associated with depolymerization of the actin cytoskeleton (35) in the mammalian collecting duct and in the amphibian bladder (7), indicating that actin filaments might retard the fusion processes. In rat collecting duct epithelium, the total F-actin content decreases by 20–30% after stimulation with vasopressin (35). Depolymerization of actin by latrunculin A has also been found to transiently promote neurotransmitter release in synapses (25). Similar to the regulation of AQP2 by cAMP, UT-A1 is activated by stimulation of the cAMP pathway and activation of PKA (43). Activation of PKA leads to a change in the F-actin cytoskeleton (35). This change could cause the increase in UT-A1 membrane abundance and urea transport activity that is seen in the current study (Figs. 4 and 6).
The amount of transporter protein in the cell plasma membrane is determined by both protein membrane trafficking (exocytosis) and retrieval (endocytosis). Clathrin-mediated endocytosis is the major endocytic pathway of many mammalian cells. Evidence from a variety of cell types demonstrates that the actin cytoskeleton plays an essential and active role in many key endocytic stages (13, 16, 32, 42). Within 5 min, latrunculin causes the actin patch to disappear and endocytosis to be completely abrogated in yeast (32). In the very early stage of clathrin-mediated endocytosis, actin accumulates at sites of endocytosis. Then, endocytic proteins and adaptors, like clathrin, Sla1, Sla2, and End3, are recruited to the actin patch, followed by regulators of the Arp2/3 complex (11, 32). Actin is also required for the invagination during the late stages of endocytic vesicle movement (13). Although cell membrane UT-A1 mainly resides in lipid raft subdomains, its endocytosis occurs through both caveolin- and clathrin-mediated endocytic pathways (15). The current study shows that inhibition of either pathway by concanavalin A or MβCD only partially inhibits UT-A1 endocytosis, which causes UT-A1 membrane accumulation. Latrunculin inhibits both clathrin- and caveolin-mediated UT-A1 endocytosis. The role of actin in the clathrin-mediated pathway has been well studied (11, 16, 42), but the function of actin in the caveolin pathway is less explored. Our results show that actin may also be involved in UT-A1 endocytosis through the caveolin endocytic pathway. In support of this, both actin and UT-A1 are found localized in lipid raft domains in the cell membrane (Fig. 2B).
In our study, treatment with jasplakinolide, which binds to and stabilizes actin filaments, does not alter UT-A1 membrane abundance. One of the possible explanations could be that the normal amount of F-actin on the apical side is enough to fulfill the cell's function. An increase in actin filaments would have no additional effect. An elegant experiment by Apodaca et al. (1) showed that jasplakinolide treatment selectively stimulated basolateral endocytosis of the fluid-phase marker FITC-dextran and horseradish peroxidase, whereas apical endocytosis of these markers was unaffected (1). The reason is that most filamentous actin is on the apical side of polarized epithelial cells.
Although much effort has been made, the consensus actin binding motif is still not certain. A potential motif for actin binding has been suggested as L/I-X-D/E-X-X-L/I (where X = any residue) (2, 39). However, some actin binding proteins do not have this sequence. The mutation L1020A in Slo1 calcium-activated potassium channels caused a reduction in Slo1 binding to β-actin, but the D1022A and L1025A mutations exhibited only slightly reduced binding to actin (44). The AQP2 COOH terminus does not contain typical actin binding motifs (28). A conserved 14-amino acid region (E631–F644) in the carboxyl terminus of αENaC has been shown to contribute specifically to actin's modulation of ENaC (21). In our study, we found that the amino acid residue S918, probably together with I919, in the UT-A1 COOH terminus mediates the actin binding. It is interesting to note that in most studies F-actin binds to the carboxyl terminus of the target proteins, such as α-subunit of the ENaC (21), AQP2 (28), Slo1 channel (44), as well as UT-A1 in the current study.
In summary, actin concentrated at the apical cortex provides many functions in the apical domain of epithelial cells (16). Here we found that by directly binding to UT-A1, cortical actin regulates UT-A1 expression in the cell membrane by regulating both endocytosis and membrane trafficking. This may have important physiological significance. Alteration of the actin cytoskeleton modulates UT-A1 activity. It has been shown that actin is also phosphorylated by PKA and that the phosphorylation reduces the rate of actin polymerization (30). Vasopressin depolymerizes apical F-actin in rat inner medullary collecting duct and increases water channel-carrying vesicle fusion with the apical membrane (35). Taken together, we propose that the actin cytoskeleton, by changing its polymerization state, may play an important role in vasopressin-mediated UT-A1 activity in vivo by regulating UT-A1 endocytosis and trafficking.
This work was supported by National Institutes of Health Grants R01-DK087838 and R21-DK080431 (to G. Chen).
No conflicts of interest, financial or otherwise, are declared by the author(s).
G.X., H.S., and C.B.C. performed the experiments; G.X., H.S., O.F., and G.C. analyzed the data; G.X., H.S., O.F., and G.C. interpreted the results of the experiments; O.F. and G.C. drafted the manuscript; G.C. conception and design of the research; G.C. prepared the figures; G.C. edited and revised the manuscript; G.C. approved the final version of the manuscript.
- Copyright © 2012 the American Physiological Society