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The Senior Nutrition Program of Catholic Charities of Fairfield County, Inc. is a non-profit program that provides nutritious meals, nutrition education and socialization to the elderly population in Southwestern Connecticut.
Catholic Charities has administered this program for the past 37 years and we serve more than 100 congregate meals and 250 home delivered meals to the elderly on a daily basis.
Congregate meals are served at 10 meal sites daily to individuals who are 60 years of age or older:
Stamford Sites
Stamford Senior Center, 888 Washington Blvd. (203) 977-5151
Jewish Community Center, 1035 Newfield Avenue (203) 322-7900
St. John's Episcopal Church – The Over 60 Club, 628 Main Street (203) 705-8679
Norwalk Sites
Norwalk Senior Center, 11 Allen Road (203) 847-3115
South Norwalk Senior Center, 92 Cedar Street (203) 299-1500
Broad River Homes, 108 New Canaan Avenue (203) 846-3700
Westport Site
The Westport Center for Senior Activities, 21 Imperial Avenue (203) 341-5099
Greenwich Site
Hill House, 8 Riverside Avenue (203) 637-3177 (seven days a week)
Dinner Sites
The Marvin, 60 Gregory Blvd., Norwalk, (203) 854-4660
Irving Freese Apartments, 57 Ward Street, Norwalk (203) 241-2422
Meal recipients must be 60 years of age or older and homebound. Donations are suggested for both of these services and one can register or refer someone to our program by phoning our office at (203) 324-6175 or emailing firstname.lastname@example.com.
Catholic Charities' Senior Nutrition Program is funded in part by the federal Older Americans Act through the Southwestern Connecticut Agency on Aging. Individuals age 60 or over are eligible for services under the approved project. For more information, you can also visit www.SWCAA.org.
Who to Contact
Maureen Neubrger, Director
Contact Info
30 Myano Lane Suite 14
Stamford, CT 06902
(203) 324-6175
Fax: (203) 323-1108
Program Hours
Monday-Friday 9:30am – 4:00pm
Client Stories
James started out as a congregate program recipient, then became the President of the Senior Nutrition Program Advisory Council for numerous terms. He has utilized a consortium of our services over the years and currently is Home Delivered meal recipient. Here is his story.
"I am an 83 year old male who is currently home-bound and 100% disabled . Without Senior Nutrition Program and home care I could not manage. My doctors do not want me near a stove! The Senior Nutrition meals are well balanced and I am not able to count calories. I could not manage without Catholic Charities Senior Nutrition Program. The program delivers the meals in all kind of weather, snow, sleet, rain or hail. God Bless the Senior Nutrition Program."


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subset: commoncrawl


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Black Mirror Will Make You Fear That Webcam on Your Computer
The stunning season three trailer covers secret spying, PTSD, and online identities.
With the popularity of Mr. Robot and Westworld, techno sci-fi dramas have been some of the most compelling new arrivals to television in the last year. It seems, after six years of nerding out to Game of Thrones, wider TV audiences are no longer ashamed to embrace the sci-fi/fantasy genres. And that's good news, because as we've seen with the two aforementioned shows, science fiction gives us a unique opportunity to explore themes within our rapidly technological world.
Few shows do this better than Black Mirror, the acclaimed BBC sci-fi anthology that truly captures the anxieties of our computerized world. The show, created by Charlie Brooker, originally appeared on the BBC in 2011 before it became an international hit when it was later added to Netflix. In its first short season, Black Mirror explored chilling near-future scenarios, including one where the British prime minister is muscled into having sex with a pig by terrorists. It's a scenario that bizarrely enough became near-reality when the news of David Cameron's own pig scandal broke last year. If anything, the greatest compliment (and/or fear) of any sci-fi author is to have their predictions come true.
And in the trailer for the long-awaited third season, Black Mirror teases various themes, including how we find self-worth in our online personas, dealing with PTSD, and our fears of the little webcam on our computer. Maybe that circle at the top of our computer will be watching us as we watch Black Mirror when it debuts on October 21.


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Ocean Nurseries November 05 2014
As the weather gets chilly we can't help, but reminisce about our summer at the beach. If you love the beach you may be considering an ocean nursery for your baby. Whether you decide to go with a traditional nautical navy nursery or something more tropical such as this seafoam beach nursery, there are so many magical ocean accessories out there to complete your decor.


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Koolteecha Technology
Monday, August 25, 2014 - A Day
1. DCI - CNN Student News
** Click here to watch the video **
** Click here to complete and submit your assignment **
2. Typing Web - Login Video (if you haven't already seen it, or to refresh your memory)
** Login and begin Lesson 1 - The Home Row Keys - Work for at least 15 minutes
** If your login isn't working today, you can still begin Lesson 1 without logging in. ** We'll figure it out before our next class. :)
3. How to Use the Snipping Tool
** Click here to watch the video **
** Click here to access a digital copy of the handout you received in class today **
Practice a little bit on your own in class today....login to Edmodo this evening for an extra credit opportunity!
4. GCFLearnFree - Computer Basics - Lesson One
** Watch the video** (or you can click on it from the webpage above)
** Read through the lesson carefully, and fill in your Interactive Notes as you go. This
isn't homework...we'll continue working on Tuesday.
You'll need to REFRESH your Tackk to see this link:
NEW LINK! Finished with your Lesson One Interactive Notes? Did you fill in ALL of the blanks?
Practice what you know at QUIZLET!


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African Petroleum Corporation announces an update to its prospective oil resources at its 90% owned and operated Senegal Offshore Sud Profond and Rufisque Offshore Profond licence blocks in Senegal and its 100% owned and operated A1 and A4 offshore blocks in The Gambia.
The Company engaged the independent petroleum consultant, ERC Equipoise Ltd, to prepare an updated assessment of prospective oil resources attributable to the Company's Gambian Licences, and an initial assessment of the Senegal Licences.
The two discoveries made by Cairn Energy at SNE-1 and FAN-1 in Senegal have had a positive impact on the chance of success for prospects within African Petroleum's portfolio.
African Petroleum Corporation Limited
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beatles »
6 Greatest Publicity Stunts in Music History
With social media a key component in any musician's publicity-filled arsenal in the 21st century, a lot of emphasis is placed on ensuring no artist succumbs to the many dangers of the internet. However,Read More »
The Paper Suns brighten your afternoon
Psychedelic and heartfelt, though still enjoying the sun, indie rock trio The Paper Suns have a 3-track album entitled "Over Water" available now at their Bandcamp page. It always brightens my day when IRead More »
NO impress Bono with "Meet Me After Dark"
You know that you're on the right path when you get a call from Bono saying that he's a fan of your music. Los Angeles-based pop rock 4-piece NO have had this experience, andRead More »


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The Flash Season 2: Exploring How Heroes Should Face Tragedy
Spoilers ahead for The Flash season 2!
At the beginning of the second season for the hit CW show The Flash, Barry Allen stands alone in a deteriorating S.T.A.R. Labs. His team has disintegrated and he works alone to help repair Central City after it was nearly destroyed by a black hole (see the end of season 1). In saving the city, two dear friends were lost in selfless sacrifice and their deaths are something Barry can't forgive himself for.
As the season came to its end, and after undergoing even more tragedy in fighting the evil Zoom, a distraught Barry Allen ran back in time to the night of his mom's murder and prevented it from happening. He had suffered enough pain.
For the fans, this was an exciting twist since it meant the famous Flashpoint storyline from the comics would play out on the show. But from a character standpoint, this was a surprising move on Barry's part. Only a few episodes before, Barry, while trapped in the Speed Force, learned he needed to accept his mom's death. But with his dad's murder at the hands of Zoom, the heroic and hopeful Flash cracked and tried to undo his tragic past.
Many fans noted how dark the season was in general, afraid the show was losing its cheery tone. But I believe through all the heartache Flash and his team suffer through, the showrunners had a point they wanted us to learn from Mr. Allen: to be a great hero, you must accept tragedy. Barry didn't do this, and I think we're supposed to acknowledge his poor decision.
As the Flash, Barry has a unique ability in toying with time to fix mistakes. It's a wish I'm sure we've all had before. If only I can go back to such-and-such year and say this to so-and-so and avoid what happened instead! But as we've seen on The Flash already, playing with time may correct one mistake and create a host of other headaches.
I imagine we'll see this amplified in the coming season. Perhaps the world is falling apart and facing destruction; maybe a different type of tragedy strikes Barry's life; perhaps old friends are now distant strangers. The possibilities are endless, but it's likely, no matter what happens, Barry's decision to radically alter time will bring more hurt than healing.
We can't time-travel (yet), so we don't have the luxury of playing with the past. That doesn't stop us completely, though. We all have tragedies that have happened in our lives, whether personally, in the family, or as a nation. The tendency is to mourn for a short while and then move on as quickly as possible. We bury our past/our pain beneath entertainment, work, or any other excuse we find. Everyone hits a point where they can take no more.
As understandable as that is, it's not healthy. What makes the Flash a superhero is the tragedy he's endured. What makes us better humans is facing pain and standing up to misfortune. In the case of Barry Allen at the end of season 2 of The Flash, we would do better to learn from his mistake rather than follow his example.
What did you think of The Flash Season 2? Let me know in the comments below!


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A practical way to contribute to the work is to give regularly monthly, quarterly or yearly by Standing Order. Please complete the Standing Order Form and return it to YOUR bank.
Make a Gift Aid Declaration
If you are a UK taxpayer you can boost your donation by 25p of Gift Aid for every £1 you donate. Gift Aid can be reclaimed by the Church from the tax you pay for the current tax year. If you want us to reclaim the tax please download and complete the Gift Aid Declaration, reading the notes before you sign and return to us. The Declaration covers gifts made in any form: standing order, cheque or cash so long as they can be identified as made by you. You need only complete the declaration once but remember to tell us if you no longer pay sufficient tax as you will be responsible for paying any difference between what we claim and what you have paid.
The Declaration form is available Gift Aid Form to download as a PDF file. On it you will find further details about Gift Aid Giving. Please print it, complete and sign the form before returning it to:


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|Hello and thank you for visiting. You've landed on the homepage of Altacircle.com a blog focused mostly on digital marketing. I'm John and I've had the good luck or bad luck to work in interactive marketing since 1995.
Marketing is constantly changing and as they continue to put microchips in more places, more opportunites arise to do amazing things.
Enjoy and feel free to comment, disagree or even rant — it is all fun and a good use of time.
Latest Post
Mirum Launch Cited in DCG Report
Mirum's Launch Cited as an Example of Organizational Change Best Practice
Launched in January 2015, Mirum was the culmination of three years of complex planning and relationship building to unite 11 J. Walter Thompson Company digital agencies located across the globe. Mirum has since expanded to 47 offices in 24 countries, with over 2,400 professionals in 2016.
The story of Mirum's founding parallels the dramatic consolidation of digital agencies over the past several years, with many mergers and acquisitions implemented to meet the needs of global clients and build the global reach of agencies. Unfortunately, as the report points out, many studies show that 70-90% of mergers and acquisitions fail because organizations don't follow best practice in managing the organizational change.
The report, "Organizational Change Management: An (Emerging) Core Competency for Customer Experience Management" by Connie Moore, Senior Vice President of Research at Digital Clarity Group, focuses on what executives should do to ensure success with specific recommendations and case studies.


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Our next Alpha Courses will be starting sometime in January 2018. Check back for more details coming soon!
The Alpha Course is an opportunity to explore the meaning of life in an informal, fun and friendly environment. There are a series of films looking at topics including 'Who is Jesus?' and 'Why and how do I pray?'. Each session begins with food and each film is followed by discussion in small groups. There will also be a weekend day (on-site) as part of the course, which gives room for loads more discussion and a chance to get to know one another even better.
Alpha is for everyone; no question is out of bounds and you are free to discuss as much or as little as you wish. We don't assume any background knowledge of or belief in Christianity and everyone is welcome.
"The Alpha Course, particularly the Alpha day, was incredible and so special. I met with God and my life has been impacted in a profound way!" Former Alpha Guest
''I got involved in Alpha just by taking the course, but soon enough it became such a big part of my life, so I went on as a volunteer. You can always see God at work there. If you want miracles - you go to Alpha.'' Former Alpha Guest


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When you check out wome of the crap they spend our tax dolars on ya have to wonder if anyone has a clue?
You folks in Iowa cold??
He'll be heading to FL in a couple of months with our daughters, who take him there once a year, but not until after the chance of snow is over. I'm not kidding.


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Third place angler Brandon Lester is catching fish. He's very confident and says today's sunny conditions will help add to his weight. Remember that leader David Kilgore said the same thing. Clearly, they both are on to something.
Look for my story about Brandon tonight here on bassmaster.com.
Further down the leader board in 15th place is Shaw Grigsby. He's catching fish too and offered this thought of the day.
The sunny, calm conditions will hamper the umbrella rig bite.
There's a lot of A-Rig slinging going on so it'll be interesting so see how that prediction turns out.
As for Shaw he said the best practice days for him on the days with the worst weather. Might be time for a change in plans for Shaw.


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The CWU Graduate Students Association (GSA) provides representation and the promotion of graduate students' interests on this campus. The GSA's mission is to unite students across disciplines by focusing on a common professional identity, promoting student success, offering social gatherings, and encouraging philanthropic outreach. We also realize that the nature of graduate study requires that a governing body that does not unduly burden the individual graduate student. In our relationship with the Graduate School, we affirm with the administration that effective graduate education operates from fair and responsible representation of all graduate students; this body provides a student voice to departments and the Graduate School. If you are interested in finding out more, please email Alexandra (Allie) Sheldon, President of the GSA at firstname.lastname@example.com.
The club has the following officer open positions - please consider volunteering!
Graduate Council Reps (1 from each college)
More information:


text: Penguins are flightless marine birds from South Pole. Although all penguin species are native to the southern hemisphere, they are not found only in cold climates, such as Antarctica. In fact, only a few species of penguin live so far south. Several species are found in the temperate zone, and one species, the Galápagos Penguin, lives near the equator.


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Boston Scientific on Friday announced CE Mark approval and has launched its new spinal cord stimulator system in Europe. The Precision Spectra Spinal Cord Stimulator (SCS) System is the world's first and only SCS system with 32 contacts and 32 dedicated power sources, according to Boston Scientific. It is designed to provide relief to a wide range of patients who suffer from chronic pain.
The first commercial implant of the Precision Spectra System was performed last month at the Basildon and Thurrock University Hospitals in the United Kingdom.
Chronic pain affects one in five adults in Europe – about 95 million people 15 to 64 years of age, Boston Scientific says.
Spinal cord stimulators deliver electrical pulses from an implantable pulse generator to leads with stimulating contacts. These electrical pulses mask pain signals traveling up the spinal cord to the brain. Until now, Boston Scientific said, such systems have offered a maximum of 16 contacts and two lead ports, with each lead port allowing the placement of one lead. By providing 32 contacts and four lead ports—twice that of any other SCS system—the Precision Spectra System offers more coverage of the spinal cord for the management of chronic pain. Additional lead ports also give physicians more flexibility to treat their patients' pain at time of implant and more flexibility to adapt to changing pain patterns in the future.
Officials at Boston Scientific say they plan to invest and expand their offerings in neuromodulation and for the treatment of chronic pain in a big way. The Natick, Mass.-based company with about 5,000 Minnesota employees has recently begun other clinical trials and has launched several new products in Europe.
The Precision Spectra SCS System is currently under review by the U.S. Food and Drug Administration, and is not available for sale in the United States.
For more information on SCS, visit www.ControlYourPain.com.
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The University of Iowa is one of four sites to host small clinical trial of new technology to treat tinnitus.


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Dr. Steven Jackson has received the 2012 Joseph A. Greene Excellence in Teaching Award from the College of Business at the University of Southern Mississippi. Jackson is associate professor in the School of Accountancy. He was a member of the Gulf Coast faculty from 1992-1996 and returned to his current position at the Gulf Park campus in Long Beach in 2007.
Jackson's research interests are in accounting education and behavioral issues in ethics and auditing. He has published articles in such refereed journals as Journal of Accounting and Finance Research, Journal of Economic and Social Measurement and Journal of Accountancy. Jackson served as the editor of the journal Advances in Accounting, Finance and Economics from 2007 to 2009. He is the co-author of "Managerial Accounting: A Focus on Ethical Decision Making."
He is also the co-author of the managerial accounting textbook "Managerial ACCT2," now in its second edition.


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As you've probably heard, today (October 28) Rockstar announced new content being added for the next generation of Grand Theft Auto V, with the PS4 and XBOX One releases slated for a November 18 release, while PC is delayed until January 27, 2015. Today several new vehicles, side missions and weapons were announced. Before I add the information, can I request the community DOES NOT ADD FALSE OR SPECULATIVE INFORMATION to the articles below;
The Dodo Seaplane has had a screenshot released showing it's amazing detail as this classic returns from the 3D Universe.
The Dukes makes a re-appearance from Grand Theft Auto IV as the new armoured car, the Duke O'Death.
The Stallion returns as a stock muscle car that can be used in races to be unlocked, as well as a repainted stock Gauntlet.
The Atomic Blimp has a new variant, the Xero Blimp, which is faster and more maneuverable.
Take to the waters in a new side mission, the Wildlife Photography Challenge which can be done to unlock the new and larger Kraken Sub.
As Michael, follow a trail of clues to unravel a grisly murder mystery. Put the clues together and you'll unlock two LA Noire inspired Filters for Snapmatic.
Ever thought the Picador was a little small for your liking? Well tear up the streets in the new Picador inspired Marshall monster truck.
Deal incredible damage with the new Rail Gun or Hatchet available soon at Ammu-Nation.


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Hydraulic Clutch Slave Cylinder; 83-86 Jeep CJ
Hydraulic Clutch Slave Cylinder 1983-1986 Jeep CJ By Omix-ADA
Pricing: $70.99
Are your clutch master or slave cylinders leaking? Over time the seals will deteriorate. It's always a good idea to replace both the clutch master and slave cylinder at the same time. Usually if one is bad, the other is close behind. Save yourself a roadside or trail breakdown in the future. Get high quality clutch parts for your Jeep from Omix-ADA.
- 16909.02
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Shipping Information
- Item Requires Shipping
- 1.5 lbs.
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I love cold smoked sausage. We use curing salt and mix, grind, and stuff all our own venison sausage. We have always taken our sausage to a local farmer but now have built our own smoke house. It is 4' x 4' and 8' high. It has a fire box off to the side and the smoke is piped in. I have also added a "turkey fryer" inside the smoke house with a 100 lb lp tank, so if I need to add heat I can at any time. I would love to here how everyone else cold smokes (how many days, how much smoke, temperature, etc) I will send pictures of the smoke house soon.
Cold Smoking venison sausage
SmokingMeatForums.com Top Picks
Show us some pics of your build if you don't mind.
This is a picture of my cold smoker. Over to the right of the smoker is where the fire box is located, as you can see good old WI winter already. LOL
- 20,254 Posts. Joined 11/2010
- Location: Omak,Washington,U.S.A.
- Points: 1067
- Select All Posts By This User
Very nice smoker..... Got pics of it in operation and meat hanging... Maybe you noticed.... WE LOVE PICS...(Q-Views).....
- 248 Posts. Joined 6/2013
- Location: Phoenix AZ
- Points: 17
- Select All Posts By This User
Looks great and perfect weather for cold smoking :) Fill her up and make us hungry! Yea, we all love pictures.
Well cross your fingers for pictures in operation. Our hunting season begins this Saturday in WI so hopefully my boys and I will bring home the "bacon" I have been smoking it empty for the last two weeks to get it somewhat "seasoned before we hopefully hang sausage in it. Does anyone have any advise on time to smoke sausage. My plan right now is to smoke it all day (around 70 degrees or less) and let it go out at night to let the smoke cure through. I plan to do this for about 7 days. Kielbasa will be for about 12 to 14 hours then done. What does everone think about that idea?
- 12,899 Posts. Joined 10/2012
- Location: live north of fort smith arkansas between alma and mountainburg.
- Points: 345
- Select All Posts By This User
Nice looking smoker. I am not sure what kind of sausage your making. About what size casing are you using? 7 days seams like a little much. While I am saying that I give my bacon about 40 hours, 10 hours at night then in the fridge for the day and repeat.
Happy smoken.
Dave, Our sausage sticks are the 2 1/2 inch casings. I think around 3 lbs a piece. The Kielbasa is about a pound per ring.
- 6,911 Posts. Joined 4/2010
- Location: Raleigh,NC
- Points: 430
- Select All Posts By This User
I form a pellicle on the casing and cold smoke for 4 to 6 hrs. Good enough smoke for me. I like a light smoke flavor of apple wood to accompany the sausage
- 283 Posts. Joined 11/2013
- Location: Brandon, MB
- Points: 53
- Select All Posts By This User
Yeah, I don't think I would go much longer than 8 hours for your first batch. Give it a try after letting it rest in the fridge overnight, and if you want more smoke, do another 8 hours. I only cold smoke my hams for about 16 hours, and they are plenty smoky for me.
Interesting? I have smoked sausage in a Bradley smoker a few times. I could get about 15 sticks in it and would smoke it for about 2 days straight. Then I would hang it in my garage (which is about 50 degrees for 5 or 6 days to let the smoke pull through it. I hope we get enough meat this year so I can try a stick a day LOL.
What is a pellicle?
- 20,254 Posts. Joined 11/2010
- Location: Omak,Washington,U.S.A.
- Points: 1067
- Select All Posts By This User
- 5,759 Posts. Joined 11/2011
- Location: N. Central Idaho
- Points: 543
- Select All Posts By This User
I remember as a kid we did smoked sausage rings, whole ring fit in a cast iron skillet when we cooked it, I think all they used was salt peter, salt and pepper and garlic, I would like to get that recipe it was a good one. Nice build on the smoker
Good luck and let us know
A full smoker is a happy smoker
Anyone have an idea where I can find a door for a fire box on my smoke house? I would say about 12" x 12" or so.
That sounds like a job for a metal fabricator? Good luck on the hunt and nice smokehouse!
Great hunting season ended up with 4 deer for my 2 sons and I. Now time to make the sausage and start smoking. Very excited to try out the new smoker!
Nice hunt and keep us posted with some pics please !
Well we made sausage, hot snack sticks, venison bacon, and chilli cheese wieners Sunday. Got all the hots sticks and bacon smoked and now I have around 300lbs of sausage in the smoker. I will continue to smoke that for at least a week. Then once it is done smoking I will hang it in my garage for a week or so to let the smoke draw thru. The smoker stays at about 50 degrees. I will post some pictures when my son gets home from school today. Simply because I don't know how to do it. LOL
- 3 Posts. Joined 11/2015
- Points: 10
- Select All Posts By This User
I use the 1 1/2" x 12" fibrous casings ( holds 1 lb). I have only cold smoked the past two years, and have never allowed the heat to go over 80 degrees. I smoke the meat 24 /7 for 60 - 72 hours, then let it hang or refrigerate for a couple of weeks. I was very skeptical of not bringing the heat to a higher temperature (165 to kill bacteria), but i was told numerous times from different people that the cure and smoke will take care of that. I am a big fan of cold smoked sausage vs regular summer sausage and everyone who eats this sausage absolutely falls in love with it.
I guess I'm doing it right, no one has gotten sick yet :-)
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Each month, Rated People connects over 50,000 homeowners with quality tradesmen - simply enter your job details in the form above to get started.
We'll match your job to the relevant tradesmen in your area - those interested will pay to see your contact details and call you to quote.
As each tradesman expresses interest in quoting on your job, we'll send you their profile and ratings to help you choose the right one for you.
After the work is complete, return to the site and rate your tradesman based on their quality of work, reliability and value for money.
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Ferris State University Selects Innotas PPM to Streamline Its IT Project Management ProcessesInnotas' IT Management Solution Provides Comprehensive PPM and Reporting Capabilities to FSU (January 09, 2012)
SAN FRANCISCO, CA -- (Marketwire) -- 01/09/12 -- Innotas, a leading provider of Cloud Solutions for IT Management, announced today that Ferris State University (FSU) has implemented Innotas' Project Portfolio Management (PPM) solution to manage its IT projects. Innotas PPM has streamlined FSU's IT project request processes and achieved significant efficiencies in its IT operations, along with enabling the quickly-growing university to better prioritize IT projects and resources to align with its strategic direction. FSU has more than 15,000 students across 19 satellite locations in Michigan.
"With all our project information in one place, managers now know who's working on what in which project, enabling senior management more visibility," said Jim Cook, IT Project Manager at Ferris State University. "Additionally, it is a huge benefit to be able to produce reports on-demand for senior management about what we are working on, instead of having to go through emails to find the necessary information."
FSU's IT department is charged with supporting, developing, and managing the IT infrastructure to meet the needs of the school, faculty, and students. To meet the demanding growth of its student population and locations, FSU's IT organization of 65 full time employees juggles requests for new strategic IT projects, while maintaining ongoing IT efforts.
Prior to implementing the Innotas system, the university had not appointed a single individual in charge of all its IT projects, nor was there a single repository for all project information and details. In addition to the need for a central IT management system, FSU's IT department had no easy way to report on project costs and resources.
According to Cook, FSU selected Innotas' cloud-based solution because of its comprehensive PPM functionality, including:
- The resource management module that delivers accurate and real-time views of available resource by type and role to staff requests.
- The project workbench for managing and tracking all tasks, milestones and changes to the projects.
- The request management module for enhancing stage gate approvals and standard project requests forms were also very important features for FSU.
- The dashboards that show both summary and detail data in a visual, easy to read format were key to the FSU selection decision.
With Innotas PPM, FSU's requirements and project requests processes have improved, reducing the time it takes to gather information. The Innotas solution has also helped the IT team become more productive, allowing them to focus on project execution by reducing multiple meetings to a single monthly gathering. With improved clarity and visibility in the deployment of both resources and project roll-outs, the IT department has experienced improved communication between the IT team and the many departments and customers it serves at FSU.
"A challenge such as what Ferris State was up against is the perfect showcase for Innotas' capabilities," said Kevin Kern, President and CEO of Innotas. "We've seen firsthand that our solution has given the FSU IT department increased confidence in its budgeting and forecasting, providing specific costs and timeframes associated with each IT project. It's made their life easier, which is our overall goal."
About Innotas
Innotas, a leading provider of Cloud-based solutions for IT Management, delivers a seamless way to manage Resources, Applications, and Projects across all IT. Innotas' solutions include Project Portfolio Management (PPM), Application Portfolio Management (APM), and Innotas Integration Platform. Headquartered in San Francisco, Innotas has hundreds of customers nationwide, including Bank of the West, Tiffany & Co., Crocs, Crayola, City of Memphis, RelayHealth, and World Vision. For more information, visit www.innotas.com or contact 866-692-7362.
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Further, it reported that this 4 member Commission had disposed off five cases in a day, summoned few Deputy Directors of School Education (in connection with information sought on misappropriation of fund under SSA) to appear before it and warned all State Information Officers/PIO's/APIO's to update themselves with the knowledge of RTI Act and furnish the information sought by the public; non-compliance of which would attract penal action as per provision of the Act.
Does this mean, corruption in the state will be checked and corrupt officials booked and there will be transparent dealings? Are we heading towards corruption free society? Or the Commission would also fall into the gallows of corruption?


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CD38 contains an ADP ribosylcyclase domain that mediates intracellular Ca2+ signaling by the production of cyclic ADP-ribose (cADPR), but the mechanisms by which the agonists activate this enzyme remain unclear. The present study tested a hypothesis that a special lipid-raft (LR) form, ceramide-enriched lipid platform, contributes to CD38 activation to produce cADPR in response to muscarinic type 1 (M1) receptor stimulation in bovine coronary arterial myocytes (CAMs). By confocal microscopic analysis, oxotremorine (Oxo), an M1 receptor agonist, was found to increase LR clustering on the membrane with the formation of a complex of CD38 and LR components such as GM1, acid sphingomyelinase (ASMase), and ceramide, a typical ceramide-enriched macrodomain. At 80 μM, Oxo increased LR clustering by 78.8%, which was abolished by LR disruptors, methyl-β-cyclodextrin (MCD), or filipin. With the use of a fluorescence resonance energy transfer (FRET) technique, 15.5 ± 1.9% energy transfer rate (vs. 5.3 ± 0.9% of control) between CD38 and LR component, ganglioside M1 was detected, further confirming the proximity of both molecules. In the presence of MCD or filipin, there were no FRET signals detected. In floated detergent-resistant membrane fractions, CD38 significantly increased in LR fractions of CAMs treated by Oxo. Moreover, MCD or filipin attenuated Oxo-induced production of cADPR via CD38. Functionally, Oxo-induced intracellular Ca2+ release and coronary artery constriction via cADPR were also blocked by LR disruption or ASMase inhibition. These results provide the first evidence that the formation of ceramide-enriched lipid macrodomains is crucial for Oxo-induced activation of CD38 to produce cADPR in CAMs, and these lipid macrodomains mediate transmembrane signaling of M1 receptor activation to produce second messenger cADPR.
- lipid microdomains
- sphingolipid
- lipid mediator
- smooth muscle
- calcium signaling
cyclic adp-ribose (cADPR), an alternative Ca2+ mobilizing second messenger to inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], has been indicated to participate in the regulation of many cell functions or physiological processes, including insulin secretion, egg fertilization, cell proliferation, nitric oxide-induced Ca2+ movement, lymphocyte activation, and neural activity (13, 37). Recent studies in our laboratory and by others have reported that in vascular smooth muscle, cADPR contributes to the Ca2+ release from the sarcoplasmic reticulum (SR) induced by Ins(1,4,5)P3-independent agonists, such as acetylcholine, oxotremorine (Oxo), 5-hydroxytryptamine, angiotensin II, and endothelin, leading to the contraction of arterial smooth muscle via ryanodine receptor (RyR)-mediated intracellular Ca2+ release (5, 11, 26). This cADPR/RyR-mediated Ca2+ signaling is now recognized as a fundamental mechanism regulating vascular function. CD38 is one of the important enzymes responsible for the production and metabolism of cADPR in vascular cells, and therefore CD38/cADPR pathway is considered as an important signaling pathway in the regulation of vasomotor responses (32). It is believed that the activation of this enzyme participates, at least in part, in Oxo-induced vasoconstriction and other receptor-mediated agonist responses (21, 38). However, the precise mechanism by which CD38 is activated by various agonists still remains unclear. Since this enzyme has been shown to localize outside cells constitutively in many studies, it is very puzzling how this enzyme could produce an intracellular second messenger to exert its signaling action.
Recently, lipid-raft (LR) clustering on the cell membrane is emerging as a novel mechanism mediating the transmembrane signaling in response to various stimuli in a variety of cell types, including lymphocytes, endothelial cells, and neurons (15, 16, 41). It has been shown that clustered membrane LRs form membrane signaling platforms, in particular, the ceramide-enriched platforms or macrodomains. These lipid platforms may recruit or aggregate various signaling molecules such as trimeric G proteins, small G proteins, sphingomyelin (SM), tyrosine kinases, NADPH oxidase subunits, phosphatases, and many others, resulting in the activation of different signaling pathways. Recent studies have reported that LRs are closely associated with the CD38 pathway in various lymphocytes and these LRs may be responsible for endocytosis of CD38 and the activation of certain signaling pathways (35, 46). However, the identity of these LRs remains unclear, and it is also unknown whether LRs during its clustering are involved in cADPR-mediated Ca2+ signaling. In previous studies, a rapid activation and translocation of acid sphingomyelinase (ASMase) into LRs were observed in response to various stimuli such as FasL, TNF-α, and endostatin (29, 33, 41). Moreover, our previous work also demonstrated that lysosomal ASMase contributes to the formation of LR redox signaling platforms by hydrolyzing SM to produce ceramide, which produces ceramide-enriched macrodomains or LR platforms, ultimately resulting in endothelial dysfunction (18). Based on these observations, the present study was designed to test the hypothesis that ceramide-enriched LR platforms may mediate an agonist-induced transmembrane signaling of CD38 and thereby participate in Oxo-induced CD38 activation to produce cADPR in coronary arterial myocytes (CAMs). To test this hypothesis, we first characterized the formation of ceramide-enriched LR clusters in CAMs in response to Oxo. We then examined the role of this LR clustering in aggregation and activation of CD38 in the membrane of these cells and explored the related molecular mechanisms mediating Oxo-induced ceramide production and LR platforms formation via ASMase/ceramide pathway. Furthermore, we determined whether this ceramide-enriched signaling platform with activated CD38 contributes to Ca2+ release in CAMs, ultimately leading to the constriction of coronary arteries.
Bovine hearts were purchased from a local slaughterhouse and were used to isolate coronary artery smooth muscle cells. No living animals were used for the present study, and therefore there was no concern over vertebrate animal use.
Cell culture.
Bovine CAMs were cultured as described previously (3, 42–45). Briefly, coronary arteries dissected from bovine hearts were first rinsed with 5% FBS in medium 199 containing 25 mM HEPES with 1% penicillin, 0.3% gentamycin, and 0.3% nystatin and then cut into segments, and the lumen was filled with 0.4% collagenase in medium 199. After 30 min of incubation at 37°C, the arteries were flushed with medium 199. The strips of denuded arteries were placed into gelatin-coated flasks with medium 199 containing 10% FBS with 1% l-glutamine, 0.1% tylosin, and 1% penicillin-streptomycin. CAMs migrated to the flasks within 3–5 days. Once cell growth was established, the arteries were removed and growing CAMs were cultured in medium 199 containing 20% FBS. The identification of CAMs was based on positive staining by an anti-α-actin antibody. Cells of passages 2–4 were used in the present study.
Confocal analysis of LR clusters in CAMs.
Individual LRs are too small (∼50 nm in diameter) to be resolved by standard light microscopy; however, if LR components are cross-linked in living cells, clustered raft protein and lipid components can be visualized by fluorescence microscopy. For microscopic detection of LR platforms, CAMs were grown on glass coverslips and then treated with different doses of Oxo (20–120 μM; Sigma, St. Louis, MO) for 15 min to induce clustering of LRs. In additional groups of cells, LR disruptors filipin (1 μg; Sigma) or methyl-β-cyclodextrin (MCD; 100 μM; Sigma) or an ASMase inhibitor desipramine (Des, 10 μM; Sigma) were added and incubated for 20 min before Oxo stimulation.
These cells were then washed in cold PBS and fixed for 10 min in 4% paraformaldehyde and blocked with 1% BSA in TBS for 30 min. GM1 gangliosides enriched in LRs were stained by Alexa488-labeled cholera toxin B (Alexa488-CTXB, 1 μg/ml) (Molecular Probes, Eugene, OR). The stained cells were then extensively washed, fixed in 4% paraformaldehyde for another 10 min, and mounted on glass slide with Vectashield mounting media (Vector, Burlingame, CA). The staining of LRs was visualized using an Olympus scanning confocal microscope (Olympus, Tokyo, Japan) at excitation/emission of 495/519 nm. The patch or macrodomain formation of Alexa488-labeled CTX, namely, gangliosides complex, represents the clusters of LR. Clustering was defined as one or several intense spots or patches, rather than diffuse fluorescence on the cell surface, whereas a vast majority of unstimulated cells displayed a homogenous or diffuse distribution of fluorescence throughout the membrane. In each experiment, the presence or absence of clustering in 200 cells was independently scored by unwitting researchers after the criteria for positive spots of fluorescence were specified. Cells that displayed a homogenous distribution of fluorescence were indicated negative. Results were given as the percentage of cells showing one or more clusters after indicated treatments.
Colocalization of LR clusters and CD38, ASMase, or ceramide in CAMs.
For dual-staining detection of the colocalization of LRs and ceramide or ASMase, CAMs were first incubated with Alexa488-labeled CTX as described in Confocal analysis of LR clusters in CAMs and then with mouse anti-ceramide IgM antibody (1:200; Alexis Biochemicals, San Diego, CA) or goat anti-ASMase antibodies (1:200; Santa Cruz, Santa Cruz, CA) separately in different groups of cells, which was followed by Cy3-conjugated anti-mouse or Texas red-labeled anti-goat (Molecular Probes, Eugene, OR) secondary antibody as needed, respectively.
Fluorescence resonance energy transfer measurement.
To further determine the molecular complex in LR clusters, CAMs were stained with tetramethylrhodamine isothiocyanate (TRITC) -labeled CTXB and FITC-labeled anti-CD38 antibody and then visualized under a confocal microscope. An acceptor bleaching protocol was employed to measure the fluorescence resonance energy transfer (FRET) efficiency (31, 36). After the prebleaching images were normally taken, the laser intensity at an excitation wavelength of the acceptor (TRITC) was increased from 50 to 98 W/cm2 and continued to excite the sample cells for 2 min to bleach the acceptor fluorescence. After the excitation intensity was adjusted back to 50 W/cm2, the postbleaching image was then taken. The FRET images were obtained by the subtraction of the prebleaching image from the postbleaching image and shown in blue color. After the measurement of FITC fluorescence intensity in images acquired at pre- and postbleaching as well as the FRET images, a FRET efficiency was calculated through the following formula: E = (FITCpost − FITCpre)/FITCpost × 100% as described previously (14), where FITC fluorescence intensity was obtained from labeling of CD38.
Assay of ASMase activity.
The activity of ASMase in CAMs was determined as described in our previous studies (39). Briefly, homogenates from CAMs were centrifuged at 1,000 g for 10 min to remove cell nuclei, and 50 μl of the supernatant homogenate were used for enzyme activity analysis. The ASMase activity was measured using radiolabeled substrate, [N-methyl-14C]-SM (Perkin-Elmer Life Sciences). The assay mixture contained 100 nmol of SM (1,154 dpm/nmol) in 100 mM sodium acetate (pH 5.0), 0.1% Triton X-100, and 0.1 mM EDTA. After incubation at 37°C for 1 h, the reaction was stopped by adding 1.5 ml of chloroform:methanol (2:1 vol/vol), followed by an addition of 0.2 ml of water. A portion of the aqueous phase was transferred to scintillation vials and counted in a liquid scintillation counter for radioactivity of the reaction product, [14C]choline phosphate.
Floatation of membrane LR fractions.
To isolate LR microdomain fractions from cell membrane, CAMs were lysed in 1.5 ml morpholinoethanesulfonic acid-buffered sample buffer containing (in μmol/l) 25 morpholinoethanesulfonic acid, 150 NaCl, 1 EDTA, 1 PMSF, and 1 Na3VO4 and a mixture of "complete" protease inhibitors and 1% Triton X-100 (pH 6.5). Cell extracts were homogenized by five passages through a 25-gauge needle. Homogenates were adjusted with 60% OptiPrep Density Gradient medium to 40% and overlaid with discontinuous 30% to 5% OptiPrep Density Gradient medium. Samples were centrifuged at 32,000 rpm for 30 h at 4°C using a SW32.1 rotor. Fractions were collected from top to bottom. For immunoblot analysis of LR-associated proteins, these fractions were precipitated by mixing with equal volume of 30% trichloroacetic acid and 30 min of incubation on ice. Precipitated proteins were spun down by centrifugation at 13,000 rpm at 4°C for 15 min. The protein pellet was carefully washed with cold acetone twice, air dried, and then resuspended in 1 mol/l Tris·HCl (pH 8.0), which was ready for immunoblot analysis.
Western blot analysis.
For immunodetection of LR-associated proteins, 50 μl of resuspended proteins were subjected to SDS-PAGE, transferred onto a nitrocellulose membrane, and blocked as described previously (41). The membrane was probed with primary monoclonal antibodies of anti-flotillin-1 (1:1,000, a LR marker proteins; BD Biosciences, San Jose, CA), anti-CD38 (1:1,000, BD Biosciences), or anti-ASMase (1:1,000, Santa Cruz) overnight at 4°C, followed by an incubation with horseradish peroxidase-labeled anti-mouse or anti-goat IgG (1:5,000). The immunoreactive bands were detected by chemiluminescence methods according to the manufacturer's instruction and visualized on Kodak Omat film. The intensity of the immunoreactive bands was quantified by densitometry.
HPLC analysis of ADP-ribosyl cyclase activity in CAMs.
To determine ADP-ribosyl cyclase activity, homogenates from CAMs (100 μg protein) were incubated with 100 μM β-nicotinamide guanine dinucleotide (β-NGD+) at 37°C for 90 min. β-NGD+ was used as a substrate to determine ADP-ribosyl cyclase activity because this enzyme is able to convert NGD into cyclic GDP-ribose (cGDPR) at the same reaction efficiency as working to convert β-NAD+ into cADPR, but cGDPR unlike cADPR may not be hydrolyzed by cADPR hydrolase. Therefore, the use of β-NGD+ will maximally detect the production of this enzyme by the blockade of a further hydrolysis of its products. After the reaction, the mixtures were centrifuged at 4°C through an Amicon microultrafilter at 13,800 g to remove proteins, and then cGDPR was analyzed by HPLC with a fluorescence detector (Agilent 1100 series HPLC system and G1321aspectrofluorometer). The excitation wavelength of 300 nm and the emission wavelength of 410 nm were used to detect cGDPR produced via cADP-ribosyl cyclase. All HPLC data were collected and analyzed by an Agilent Chemstation. Nucleotides were resolved on a 3-μm Supelcosil LC-18 column (4.6 × 150 mm) with a 5-μm Supelcosil LC-18 guard column (4.6 × 20 mm, Supelco, Bellefonte, BA). The injection volume was 20 μl. The mobile phase consisted of 150 mM ammonium acetate (pH 5.5) containing 5% methanol (solvent A) and 50% methanol (solvent B). The solvent system was a linear gradient of 5% solvent B in A to 30% solvent B in A over 1 min, held for 25 min, and then increased to 50% solvent B over 1 min. The flow rate was 0.8 ml/min. Peak identities were confirmed by the comigration with known standards. Quantitative measurements were performed by the comparison of a known concentration of standards.
Measurement of intracellular Ca2+ concentration in CAMs.
Determination of intracellular Ca2+ concentration in CAMs was performed to determine the contribution of LR-CD38 platforms to cADPR-mediated Ca2+ signaling. Briefly, CAMs were cultured on a glass coverslip to reach ∼60–70% confluence, and the culture medium was then removed. The cells were next loaded with fura-2 acetoxymethyl ester (fura-2 AM, 5 μM; Molecular Probes) at 37°C for 30 min and incubated for 20 min to allow a complete hydrolysis of intracellular fura-2 AM to fura-2 in a Ca2+-free Hanks' buffered saline solution containing (in mM) 137 NaCl, 5.4 KCl, 4.2 NaHCO3, 3 Na2HPO4, 0.4 KH2PO4, 0.5 MgCl2, 0.8 MgSO4, 10 glucose, and 10 HEPES (pH 7.4). The coverslip was mounted horizontally on a Nikon Diaphoto TMD inverted microscope (Nikon, Japan). Oxo (80 μM) was added to the bath solution to induce Ca2+ release. This dose of Oxo was used in our several previous studies where muscarinic type 1 (M1) receptor was activated and cADPR and related Ca2+ release were produced in CAMs (11, 38). To examine the specificity of Oxo and the role of LRs in Oxo-induced Ca2+ release, the cells were pretreated for 15 min with 8-bromo-cADPR (8-Br-cADPR, a cADPR antagonist at 30 μM), LR disruptors, filipin (1 μg) or MCD (100 μM), or an ASMase inhibitor, Des (10 μM), and then Oxo-induced Ca2+ release was redetermined. All these experiments were conducted in Ca2+-free Hanks' buffer. A SPOT digital camera (SPOT RT Monochrome; Diagnostic Instruments) was used to record fura-2 fluorescence images. Metafluor imaging and analysis software (Universal Imaging) was used to acquire, digitize, and store the images.
Vascular reactivity in perfused small coronary arteries in vitro.
Vascular reactivity in isolated perfused and pressurized small bovine coronary artery was measured as described previously (11). The intramural coronary arteries (<300 μm in diameter) were dissected from fresh cow hearts and assembled between two glass tips. After a 60-min equilibration period under the transmural pressure of 60 mmHg, Oxo (80 μM) was added to the bath solution to induce vasoconstriction. When Oxo-induced contraction reached a stable plateau, the internal diameters of the arteries were measured and recorded. To examine the contribution of ASM/ceramide and LR/CD38 pathway to Oxo-induced vasoconstriction, the arteries were pretreated for 20 min with one of the following compounds: nicotinamide (Nicot; 6 mM; an inhibitor of ADP-ribosyl cyclase), 8-Br-cADPR (30 μM; a cADPR antagonist), LR disruptors, MCD (100 μM), and filipin (1 μg), or an ASMase inhibitor, Des (10 μM). Oxo (80 μM) was added and the vasoconstrictor response was observed as described in the protocol without these components. The contractile responses to Oxo were expressed as the percent reduction in vascular internal diameters.
Data are presented as means ± SE. Significant differences between and within multiple groups were examined using ANOVA for repeated measures, followed by Duncan's multiple-range test. Student's t-test was used to determine the significance of difference in two groups of experiments. P < 0.05 was considered statistically significant.
Cofocal microscopy of Oxo-induced LR clustering in CAMs.
Figure 1A presents the typical fluorescent confocal microscopic images depicting Alexa488-CTX-labeled patches on the cell membrane of CAMs. Under the resting condition (control), there was only a diffuse fluorescent staining on the cell membrane, indicating a possible distribution of a single LR. When CAMs were incubated with Oxo, some large fluorescent dots or patches were detected on the cell membrane, indicating LR patches or macrodomains. Figure 1B summarized the effects of different doses of Oxo on the LR clustering by counting these LR clusters or patches. It was found that under control condition, CAMs displayed a small percentage with LR clustering (19.6 ± 3.9%). After these cells were stimulated with Oxo, LR-clustered positive cells increased significantly in a dose-dependent manner. At 80 μM, Oxo increased LR clustering by 65.7 ± 9.4% (P < 0.05, n = 5). This Oxo-induced LR clustering effect was abolished by LR disruptors, MCD and filipin, respectively. Des, a selective inhibitor of ASMase, also inhibited this Oxo-induced LR clustering.
Oxo-induced FRET between CD38 and LR component, GM1.
FRET was detected by FITC-labeled anti-CD38 antibody and TRITC-labeled CTX. As shown in Fig. 2A, top, the green image showed the FITC-labeled anti-CD38 antibody, whereas the red image showed a labeling of GM1 by TRITC-CTX. The overlaid image mainly showed colocalization, but a subtraction of pre- from postimage on FITC image represents FRET in that we used an acceptor (TRITC) bleaching protocol. In this protocol, TRITC was bleached, and therefore the normally weakened FITC signal due to energy transferring into neighbor molecules could be enhanced. The increase in the FITC-labeled CD38 signal indicates the increase of FRET efficiency, which was derived from the blockade of acceptation of photons by TRITC (bleached). Under control conditions, when two images were overlaid, there was no yellow spots or patches detected. When CAMs were stimulated by Oxo, yellow spots were seen, which represent a colocalization of GM1 and CD38 (right). The green fluorescence intensity of FITC-anti-CD38 antibody (middle) increased after the acceptor of TRITC labeling CTX was bleached during Oxo stimulation, whereas the red fluorescence intensity of TRITC labeling CTX almost disappeared due to bleaching. Under this condition, there was no yellow spot detectable in the overlaid image. Figure 2A, bottom, showed a subtracted image between the pre- and postacceptor bleaching, and the blue color intensity represented increased FITC fluorescence due to FRET. Figure 2B showed the summarized results of FRET analysis. When compared with control (5.3 ± 0.9%), the efficiency of FRET in the Oxo-treated group of CAMs increased significantly to 15.5 ± 1.9% (P < 0.01, n = 5). When a same molecule of the cells (CD38) was stained by FITC-labeled primary antibody followed by TRITC-labeled secondary antibody and subjected to FRET protocol, a maximal FRET efficiency was 25% (data not shown). Pretreatment with LR disruptors, MCD or filipin, decreased the FRET efficiency remarkably. In addition, FRET efficiency was also inhibited by Des, an ASMase inhibitor (Fig. 2B).
CD38 aggregation in isolated LR fractions.
By Western blot analysis, the expression of flotillin-1, a LR specific marker, was primarily found in the fractions between the 5% and 30% gradients, which was referred to LR fractions previously (27). As shown in Fig. 3, the ratio of CD38 levels in LR fractions to those in other fractions were summarized, because their clustering in LR fractions of cell membrane, rather than increase in expression, is comparable. CD38 could be detected in most of the membrane fractions from CAMs under control conditions. There was a marked increase in CD38 protein in LR fractions when these cells were stimulated by Oxo. However, when the cells were pretreated by MCD, filipin, or Des, a Oxo-induced increase in CD38 in LR fractions was significantly blocked.
Contribution of ceramide and ASMase to Oxo-induced LR clustering.
To examine whether ASMase/ceramide is involved in Oxo-induced LR clustering and whether Oxo-induced LR clustering forms ceramide-enriched macrodomains, we stained CAMs with Alexa488-CTX and Texas red-conjugated anti-ASMase antibody or Cy3-labeled ceramide. As shown in Fig. 4, Oxo stimulation caused an aggregation of both ceramide (Fig. 4A) and ASMase (Fig. 4B) in LR clusters (colocalized with CTX-labeled GM1), which were exhibited by yellow dots or patches (right). When these cells were pretreated by either MCD or filipin, both CTX clusters (Alexa488 green fluorescence) and aggregated ASMase (Texas red fluorescence) or ceramide (Cy3 fluorescence) were no longer observed. Correspondingly, in isolated LR fractions, ASMase was found to be increased in CAMs treated with Oxo, whereas in other fractions, ASMase contents remained unchanged or reduced. However, in the presence of LR disruptors, MCD or ASMase inhibitor, Des, this redistribution of ASMase induced by Oxo was abolished (Fig. 3).
To further confirm whether ASMase is involved in Oxo-induced LR clustering, we directly measured ASMase activity in CAMs. As shown in Fig. 5A, Oxo treatment of CAMs for 15 min markedly increased ASMase activity in CAMs, which was substantially inhibited by ASMase inhibition with Des. However, LR disruptors, MCD or filipin, only had minimal inhibitory effect on Oxo-induced enhancement of ASMase activity, suggesting that ASMase activation may precede LR clustering.
Role of ceramide-enriched lipid platforms with CD38 in Oxo-induced increase in ADP-ribosylcyclase activity in CAMs.
To explore the functional significance of ceramide-enriched lipid platforms with CD38 in CAMs, we directly observed the Oxo-induced increase in CD38 activity by HPLC in these cells before and after a pretreatment with LR disruptors, MCD or ASMase inhibitor, Des. As shown in Fig. 5B, the conversion rate of β-NGD to cGDPR was 43.1 ± 1.9 under control conditions. When cells were incubated with Oxo, the rate was increased significantly (57.6 ± 3.9). Pretreatment of CAMs with ADP-ribosylcylase inhibitor, Nicot, significantly inhibited Oxo-induced production of cADPR. MCD and filipin also inhibited the effects of Oxo significantly.
Association of ceramide-enriched lipid-CD38 platforms with Oxo-induced intracellular Ca2+ release in CAMs.
To further determine the role of ceramide-enriched lipid-CD38 platforms in the regulation of intracellular Ca2+ release, we examined Oxo-induced intracellular Ca2+ release in CAMs under control conditions and when they were treated with LR disruptors, MCD or filipin, and ASMase inhibitor, Des. In Fig. 6A, typical microscopic digitalized signal plots of Ca2+ release within CAMs bathed in Ca2+-free solution are presented. It is clear that in the presence of 8-Br-cADPR (a cADPR antagonist), Des, MCD or filipin, Oxo-induced Ca2+ release in CAMs was significantly reduced compared with Oxo stimulation alone. Calculated Ca2+ concentrations under different conditions are presented in Fig. 6B. In response to Oxo stimulation, intracellular Ca2+ concentration increased from 149.8 ± 32.7 to 495.0 ± 130.6 nM in CAMs. This Oxo-induced increase in intracellular Ca2+ levels could be attenuated or blocked by inhibition of LR clustering or formation of ceramide-enriched platforms either with LR disruptors or ASMase inhibitor.
Role of ceramide-enriched lipid platforms with CD38 in Oxo-induced vasoconstriction.
Consistent with our previous reports (11, 43), we found that in the presence of Nicot or 8-Br-cADPR, the Oxo-induced contraction of small coronary arteries was significantly attenuated. The maximal contraction decreased from 47.2 ± 6.8% to 21.1 ± 4.2% or 27.3 ± 4.8%, respectively (n = 5). In the presence of MCD or filipin, the Oxo-induced contraction was also significantly reduced to a very similar extent to that induced by Nicot or 8-Br-cADPR. When ASMase inhibitor, Des, was used to treat the coronary arteries, the maximal contraction induced by Oxo decreased from 47.2 ± 6.8% to 26.5 ± 3.9% (n = 5) (Fig. 6C).
The main findings of the present study are that 1) Oxo induced LR clustering in CAMs as shown by the formation of larger membrane LR patches as detected by confocal microscopy; 2) Oxo induced an aggregation of CD38 in LR clusters with enriched ceramide, which resulted in the increased activity of CD38 as ADP-ribosylcyclase, and disruption of LRs inhibited the effects of Oxo on this enzyme activity; and 3) blockade of ASMase/ceramide inhibited Oxo-induced LR clustering and consequent CD38 activation and cADPR production, whereby an Oxo-induced increase in intracellular Ca2+ release and vasoconstriction was significantly attenuated. These results support our hypothesis that ceramide-enriched LR platforms mediate Oxo-induced transmembrane signaling of CD38 and thereby activate its ADP-ribosylcyclase to produce cADPR in CAMs.
Despite the increasing evidence that cADPR signaling becomes an essential mechanism in the regulation of the intracellular Ca2+ level in a variety of mammalian cells (6, 19), so far it remains unknown what mechanism mediates cADPR production in response to various agonists or stimuli and how a transmembrane signaling upon agonist stimulation occurs to activate cADPR producing enzyme and leads to responses of intracellular effectors. Moreover, there is evidence that CD38, a major enzyme producing cADPR in mammalian cells or tissues, is mainly present on the surface of cells serving as an ectoenzyme (1). It is imperative to know how an ectoenzyme could produce a second messenger to mediate intracellular Ca2+ signaling as an important function of cADPR. In the present study, we tested a novel hypothesis that LRs may form a signaling platform in vascular smooth muscle cells where CD38 can be aggregated and activated to produce cADPR when these cells are stimulated by agonists. This LRs-mediated transmembrane signaling may represent a novel mechanism initiating cADRP production and subsequent signaling cascade. The rationale of this hypothesis is based on the fact that the LRs may serve as anchoring platforms for organizing components of the signal transduction systems (8, 12, 30) and thereby participate in cellular signaling. In this regard, these LRs are known as specialized membrane microdomains rich of cholesterol and sphingolipids, which act as organizing centers by sequestering specific sets of proteins for different cellular activities such as membrane molecular trafficking and signal transduction (2). In vascular smooth muscle cells, LRs have been demonstrated to play an important role in several cell signaling pathways, such as agonist-induced Ca2+ release, phosphorylation of EGF receptor, and activation of RhoA (7, 17, 24). Using confocal microscopic analysis of GM1 labeling by CTX as a marker of LR clusters, we found that under control conditions, LRs were diffusely distributed throughout the membrane of CAMs and that stimulation of these cells with Oxo resulted in a dose-dependent formation of CTX-positive fluorescent patches, which represented LR clusters or macrodomains. This suggests that LR clustering occurs in arterial smooth muscle cells in response to agonists such as Oxo. Given its well-known signaling role, this LR clustering may be of importance in mediating signal transduction or functional response in these vascular smooth muscles. Indeed, we demonstrated that this LR clustering was coupled to CD38 aggregation, as shown in confocal microscopic colocalization analysis, FRET detection, and membrane flotation of LR fractions. These LR-CD38 complex or membrane platforms may result in CD38 translocation and activation by agonists such as Oxo. To our knowledge, these results for the first time provide direct evidence that LRs are able to be clustered with aggregation of CD38 in vascular smooth muscle cells. In previous studies, this LR-mediated CD38 clustering was also reported in T-cells or B-cells, which participates in the regulation of T-cell or B-cell function by facilitating critical associations or interactions with other signaling molecules such as myosin heavy chain class II, CD9, N-Ras, Akt, Erk, Lck and CD3-ζ (4, 25, 46, 48). However, the regulation of lymphocyte function by LR clustering has not been linked to its function of cADPR production and consequent Ca2+ mobilization.
In the present study, we provided further evidence that the aggregation of CD38 via LR clustering is involved in the activation of this enzyme to produce cADPR. By HPLC analysis of cGDPR production as an ADP-ribosyl cyclase activity, Oxo was found to significantly increase the activity of CD38 in CAMs, which was blocked by LR disruptors MCD and filipin. This suggests that LR clustering in these cells importantly contribute to the activation of CD38 as a cADPR-producing enzyme. To further determine the role of LR clustering in cADPR-mediated signaling, we addressed whether the activation of CD38 via LR clustering is attributed to Oxo-induced Ca2+ release response in CAMs. It has been reported that CD38 is a crucial pathway mediating Ca2+ release, and our previous work also demonstrated that cADPR-mediated Ca2+ mobilization represents an important signaling pathway participating in the Ca2+ regulation in vascular smooth muscle cells and in the vasoconstrictor response of coronary arteries when M1 ACh receptor is activated (40). Consistent with these previous studies, the present study found that Oxo induced Ca2+ release from the SR in CAMs buffered in Ca2+-free solution and that 8-Br-cADPR, an antagonist of cADPR, could block the action of Oxo. Interestingly, in the presence of LR disruptors, MCD or filipin, Oxo-induced Ca2+ release was substantially attenuated. In another series of experiments, we also examined the contribution of this LR clustering with CD38 to the vasoconstrictor response to Oxo. It was found that Oxo induced significant vasoconstriction with a maximal response of 47.2 ± 6.8% in perfused and pressurized small coronary arteries, which was significantly blocked by the LR disruptor, MCD or filipin. Taken together, these results indicate that LRs and their clustering importantly participate in the regulation of vascular function associated with CD38 activation and cADPR-mediated Ca2+ release in coronary arteries. To our knowledge, this represents the first report elucidating the functional association of LR clustering with CD38 activity to produce cADPR in the regulation of intracellular Ca2+ level of arterial myocytes and vasomotor response in coronary arteries. Previous studies also demonstrated the formation of LR-CD38 complex in lymphocytes in response to different stimuli (4, 25, 46). However, these studies did not demonstrate the correlation between the production of cADPR and agonist receptor functions when LR clustering occurred, but they assumed that CD38 may serve as a pleiotropic molecule in LR clusters, whose behavior or function is independent from its enzymatic activity in these cells (46). In some other studies, CD38 in LRs has been demonstrated as an interacting membrane molecule to facilitate the formation of different signaling complex in lymphocytes, which activates several different phosphorylation pathways to regulate cell proliferation or apoptosis (7, 48).
It should be noted that although Oxo or M1 agonist may not be a major regulator of coronary vascular tone under physiological conditions, the use of this compound represents a prototype of agonists that stimulates cADPR production and induces vasoconstriction of bovine coronary arteries because bovine coronary arteries did not respond to angiotensin II or norepinephrine to produce vasoconstriction based on our experience working on this type of arteries for more than 10 years. Another comparable vasoconstrictor in these arteries is to thromboxane A2 mimetic, U-46619, a commonly used agonist in these bovine arteries, but this compound produces vasoconstriction through Ins(1,4,5)P3 independent of LRs. With respect to agonists or stimuli that use cADPR as second messengers, recent studies have demonstrated that there are Oxo in smooth muscle cells, cholecystokinin in pancreatic acinar cells, glucose in pancreatic β-cells, physiological contact or fertilization in the eggs, histamine and oxytoxin in myometrial cells, and thrombin in platelets. Based our results in using Oxo, an agonist producing comparable vasoconstriction to Ins(1,4,5)P3 stimulator, we believe the LR-mediated activation of ADP-ribosylcyclase demonstrated in the present study is mainly attributed to this type of agonists. This LR-signaling mechanism may not be used by other Ca2+ regulatory pathways.
To explore the mechanism by which Oxo induces the formation of LR clusters with CD38 in CAMs, we tested a hypothesis that ASMase or ceramide plays a critical role in Oxo-induced LR clustering and thereby contributes to corresponding functional changes in CAMs or coronary arterial smooth muscle. There is considerable evidence that rapid activation and translocation of ASMase into cell membrane trigger or facilitate the formation of LR clusters in response to different stimuli (22). Recent studies in our laboratory have shown that lysosome-associated vesicle transportation or trafficking of ASMase from intracellular compartments to cell membrane is a prerequisite for ASMase activation in the process of LR clustering in endothelial cells (18). This ASMase translocated into cell membrane hydrolyzes SM to produce ceramide and leads to the formation of ceramide-enriched macrodomains or LR platforms, which cluster or recruit some receptors or signaling molecules (22). The present study provided several lines of evidence that this translocated ASMase and consequently produced ceramide are present in LR clusters with CD38 in CAMs and that ceramide production from ASMase importantly contributes to the formation and function of LR clusters with CD38. First, under stimulation of Oxo, ASMase and ceramide were found to be aggregated in LR clusters with CD38, as shown by the colocalization of GM1 labeled by Alexa-CTX and ASMase, and ceramide labeled by Texas red-conjugated anti-ASMase antibody and Cy3-conjugated anti-ceramide antibody, respectively. Second, by membrane floatation, ASMase was found to be enriched in LR fractions and ASMase activity significantly increased in these fractions. The ASMase inhibitor, Des, could block these effects, but LR disruptors did not have obvious effects on Oxo-induced ASMase activation. These results suggest that ASMase enrichment and activation are involved in Oxo-induced LR clustering, and even ASMase activation precedes the LR clustering because the LR disruptors could not block the increased ASMase activity stimulated by Oxo. Third, a biochemical analysis demonstrated that ASMase inhibition by Des significantly blocked ADP-ribosylcyclase activity of CD38 in CAMs, indicating that ASMase importantly contributes to CD38 activation to produce cADPR. Fourth, functional studies by the measuring intracellular Ca2+ level in CAMs and the vasoconstrictor response of coronary arteries to Oxo provided evidence that ASMase inhibition attenuated the effect of Oxo-induced Ca2+ activation and vasoconstriction in these arteries. All these results support the view that ASMase and its product ceramide pathway play a crucial role in Oxo-induced LR clustering and consequently CD38 activation and that the formation of these ceramide-enriched lipid platforms importantly participate in the CD38-cADPR-mediated signaling upon activation of M1 receptor in CAMs.
Oxo or some other agonists may produce Ca2+ release and consequent vasoconstriction through several pathways such as activation of plasma membrane Ca2+ channels and Ins(1,4,5)P3 production and resulting intracellular Ca2+ release. However, these two pathways will be blocked by using a Ca2+-free bath solution in our Ca2+ assay and vasoreactivity studies as a routine in such studies. Under this condition, Ca2+ influx is blocked and therefore its role in mediating Oxo action was minimized. In addition, it was reported that when cells are bathed in Ca2+ free solution, PLC failed to respond to any stimulus to produce Ins(1,4,5)P3 (34). Therefore, there was only a minimal action of Ins(1,4,5)P3 in such preparations. However, our results did not exclude the action of Ca2+ influx or Ins(1,4,5)P3 in the action of Oxo or other agonists under other conditions.
There was a concern regarding alternative pathways for cADPR production. It has been demonstrated that a soluble protein, ADP-ribosyl cyclase and its membrane-bound homologous CD38 and CD157, are involved in the production of cADPR with the substrate NAD+ by a cyclizing reaction (9, 23), which has been shown in a variety of cells and tissues such as sea urchin eggs, pancreatic acinar cells, human T lymphocytes, rat brain, and rat smooth muscle cells. Over the last 10 years, we have demonstrated that this cADPR-mediated Ca2+ signaling is present in cardiovascular tissues such as the myocardium, renal microvessel, and coronary arteries (10, 32, 38). We also demonstrated that ADP-ribosylcyclases activity is primarily derived from CD38 (a GPI-anchored protein) in coronary arterial smooth muscle. In addition, other studies reported that CD38 internalization is of importance in mediating cADP-ribose production in cell cytosol (14, 47). Therefore, based on current understanding, the high level of ADP-ribosylcyclase activity to convert NAD+ to cADPR may be associated with the internalization of a membrane-bound enzyme, although there may be some unidentified pathways. One such unidentified pathway was reported recently in myometrial cells, which possesses ADP-ribosylcyclase activity to produce cADPR and NAADP independent of CD38 and CD157 (28). In addition, CD38-mediated cADPR has been reported to be enhanced by protein kinase A and cAMP-regulated guanine nucleotide exchange factor II (20). It seems that different type of cells may use different enzymatic pathways to produce cADPR. This CD38-independent pathway for cADPR production has not yet been demonstrated in vascular smooth muscle cells. However, our results in the present studies did not exclude the role of this CD38-independent pathway in mediating vascular reactivity under other conditions or when these cells are stimulated by other agonists.
In summary, the present study demonstrated that Oxo stimulated LR clustering in CAMs and that a membrane signaling complex or platforms could be formed by LR clustering. This LR clustering or formation of signaling platforms importantly participates in Ca2+ transient response of CAMs to Oxo and corresponding coronary arterial constriction. The Oxo-induced formation of LR signaling platform with CD38 as a complex was dependent on the enrichment and activation of ASMase and consequent production of ceramide. Therefore, it is the ceramide-enriched macrodomain or lipid platforms that mediate the action of Oxo in mediating CD38 translocation and activation, thereby producing cADPR to induce Ca2+ release from the SR of CAMs. This LR clustering and consequent formation of signaling platforms may represent an important mechanism that is implicated in CD38/cADPR-mediated signaling in coronary arterial smooth muscle in response to vasoactive agonists such as Oxo.
This study was supported by National Institutes of Health Grants HL-57244, HL-75316, and DK-54927.
↵* S.-J. Jia and S. Jin contribute equally to this work.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2008 by the American Physiological Society


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Tag Archives: World of Warcraft
Four Evocative Landscapes in World of Warcraft's Draenor
So I began looking for beauty in Draenor and found it all around me, even in the highly hostile Tanaan Jungle. Of course, what I find beautiful in Draenor landscapes may not appeal to other people; after all, there is … Continue reading
War Begins Within (or Pandaria Nostalgia)
A conversation with guildmates turned nostalgic a few months ago. We were stalking rare monsters in the Zorammarsh, a gloomy swamp in Draenor's Tanaan Jungle. The planet Draenor is the setting of World of Warcraft's most recent expansion and is, … Continue reading
Posted in Virtual Life, World of Warcraft Tagged Draenor, Pandaria, war is hell, war within, World of Warcraft Leave a comment
Make Friends with Failure
When my World of Warcraft (WoW) raiding team decides to take on a new (for us) fight, we're aware that failure is all but inevitable the first time – perhaps many times thereafter. The boss (biggest baddie) in any specific … Continue reading
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Amused in a WoW Raid
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The I Ching and the Pandaren Starting Zone: PHI in WoW 7
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Stable Cottage is a Grade II listed, stone and pan tiled terraced cottage converted from a former barn in the hamlet of Houlsyke, two miles from Danby in the North Yorks Moors National Park. Ideal for families as it contains a double bedroom and a room with children's bunk beds as well as a family bathroom, this cottage can sleep four people in comfort. The cottage also contains a small fitted kitchen and a sitting room with a dining table and an electric fire. Outside the cottage is off road parking for 2 cars and an enclosed terraced garden (accessed by 8 steps) with a patio and garden furniture and a BBQ. Wonderfully located in the heart of the North Yorks Moors National Park, Stable Cottage is the perfect holiday cottage no matter what time of year you travel.
Two bedrooms: 1 x double, 1 x children's (2'6") bunks. Lower ground floor bathroom with bath, shower over, basin & WC. Small fitted kitchen. Sitting room with electric fire & dining area.
oil-filled radiators with electric fire in sitting room. Electric oven & hob, combi microwave/grill, fridge, TV with Freeview, DVD, radio, WiFi. All fuel & power inc. in rent. Bed linen & towels inc. in rent. Cot, highchair & stairgate on request. Off road parking for 2 cars. Enclosed terraced garden (accessed by 8 steps) with patio, furniture & BBQ. Sorry, no pets. Shop & pub 2 miles.
The village of Danby nestles in the beautiful Esk Valley in the heart of The North York Moors. The village has two pubs (both serving food), a bakery and tearooms. There is also an excellent National Park Visitor Centre at Danby Lodge - an historic building set in 13 acres of riverside pasture, with facilities which include exhibitions, nature trails and tearooms. Danby also boasts the ruins of a 14th Century castle and the only remaining working water mill on the River Esk. The scenic Esk Valley Railway line runs through the village on its way to Whitby. The moorland villages of Hutton-le-hole and Lastingham, the market town of Pickering, the City of York and the Yorkshire Coast villages of Staithes, Saltburn and Robin Hood's Bay are all within easy touring distance.
The North York Moors National Park covers 554 square miles, with landscape ranging from heather-clad moorland and deep secluded dales, to the cliffs and coves of the magnificent coastline making it a haven for walkers.


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Lufthansa has canceled 1,200 of its 1,800 flights Friday, September 7, 2012 following a strike call by its cabin crew protesting proposed pay cuts and changes to their employment contract.
The German carrier has canceled 48 flights for Thursday as well.
Lufthansa said the nationwide strike call prompted it to cancel the majority of flights.
Lufthansa flights from New York, Boston, Atlanta, San Francisco, Denver, Los Angeles, Chicago, Miami, Fort Worth-Dallas and Seattle are among those canceled.
Travelers from India should note that Lufthansa flights from Mumbai, New Delhi, Chennai and Bengaluru for September 7, 2012 have been canceled.
Media reports from Germany say thousands of passengers are already stranded in Frankfurt.
The airline told travelers to expect unscheduled cancellations and delays of Lufthansa flights.
Although flights of Austrian Airlines, Brussels Airlines, Germanwings and SWISS as well as LH codeshare flights are not expected to be hit by the strike, Lufthansa cautioned travelers that it was not sure of the impact of the strike.
Flights of other airlines are also likely to be impacted by the strike since a large number of Lufthansa aircraft are parked on the tarmac leaving less room for incoming or outgoing flights.
Even after the strike ends, travelers should be prepared for irregularities in flight operations over the weekend.
Lufthansa said it'd allow passengers whose flights have been cancelled to get a refund or rebook their flights free of charge.
To mollify irate travelers, the airline is offering all passengers with tickets issued on/before September 4, 2012 for Lufthansa operated flights (even if not cancelled) departing before Saturday, September 8, 2012, 11:59PM one rebooking onto alternate flights by Lufthansa, Austrian Airlines, Brussels Airlines Germanwings or Swiss free of charge.
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Young Tiger NTR's latest movie 'Rabhasa' is gearing up for a grand release on August 14th as promised by producer Bellamkonda Suresh. When everything is going smooth, it seems like police have played spoilsport for the movie's audio release that is expected to be launched on July 20th. Here goes the details.
Telangana state police have denied permission to NTR's Rabhasa to get its audio released on September 20th. The issue seems to be regarding security cover, as the newly carved out state will be celebrating its first ever Bonalu festival on the very same day. And this being Ramzan season too, Hyderabad police are blanketing the city with tight security to not let the secular cover blow off.
'Rabhasa' team is now looking for another possible date, probably September 26th will be the audio release day, reveals a source inside Bellamkonda's camp. The team is confident about the peppy album scored by SS Thaman, who is these days criticized for his lacklustre tunes.


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Question: You intend to purchase Marigo common stock at 50
You intend to purchase Marigo common stock at $ 50 per share, hold it 1 year, and then sell it after a dividend of $ 6 is paid. How much will the stock price have to appreciate for you to satisfy your required rate of return of 15 percent?
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Answer to relevant QuestionsHeader Motor Inc. paid a $ 3.50 dividend last year. At a constant growth rate of 5 percent, what is the value of the common stock if the investors require a 20 percent rate of return? The preferred stock of Armlo pays a $ 2.75 dividend. What is the value of the stock if your required return is 9 percent? You are considering the purchase of Davis stock at a market price of $ 36.72 per share. Assume the stock pays an annual dividend of $ 2.33. What would be your expected return? Should you purchase the stock if your required ...You are considering an investment in one of two preferred stocks, TCF Capital or TAYC Capital Trust. TCF Capital pays an annual dividend of $ 2.69, while TAYC Capital pays an annual dividend of $ 2.44. If your required ...What might we expect to see in practice in the relative costs of different sources of capital?
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I got some neat cutters from hobby lobby today, a dress shoe, comes in 3 sizes, a crown or tiara also in 3 sizes and they also have a party hat that could be made into an evening dress or wedding dress. these where the metal kind, not any particular brand. 75c and $1.
I am such a sucker for new cookie cutters and I always like a reason to go to Hobby Lobby. Thanks for the info.
I saw those cutters too! Some that I had not seen before.
I saw them too. I got the largest crown cutter. I had to had cut some big crowns a few months ago!!
I hadn't seen those yet. Guess where I'm going today!!!!!!!!!!!!!!!
Wish we had a HL closer to me!!
I second that! We don't have them in MA but they do in MI and I've been to it a couple of times. They blow Michaels and AC Moore away!
We just got an AC Moore here & I like it better than michaels & Jo anns
I was like a kid in a candy shop when I went to Hobby Lobby last week. I always take a stroll down the Wilton aisle to see if there is anything new....and there they were---new cutters!
You are right on mommarivera! A little run to Ocala, to go to HL, is about a 2-3 hr trip for me, from Bradenton. Must be 4-5 for you, depending on where you live in Ft Myers. I wrote to them, asking for a store in the Bradenton/Sarasota area. They actually wrote back, saying they need 150,000 customers in a reasonable trade area. That's OK we can meet that, but they also are operating within a 1,000 mile radius of Okalahoma City, OK, so that probably leaves us out, and you folks in MA too. If anyone wants the e-mail address of their contact person, let me know.
well here in upstate NY, there is no HL. Lately I haven't even wanted to use the 40% coupon in either of Michael or AC Moore. There wasn't anything I wanted. Bummer
I will go by there this week again. maybe I missed it how are they packaged?
no hobby lobby in Canada-do they have a website maybe i could have some shipped? i'm getting seriously frustrated trying to find the not so typical (but obviously used a lot by cookie decorators) cutters like onsies and tshirts and especially the wedding/prom dress!! i don't live anywhere near the states either so no roadtrip for me (big sigh)
I went last night and got all the shoe and the large present cutter
I went last night and got all the shoe and the large present cutter