text:
27 Crafts for Preschool: Activities for Preschool Children
Bonus: Get our newsletter & special offers for free. We will not share or sell your email address. View our Privacy Policy
Make Your Own Mini Book
We are adding the craft to your Crafts.
The craft was added to your Crafts.
Click here to view your Crafts.
When you have a creative thought or an inkling to draw, make sure you have something handy to write in. That's why the Make Your Own Mini Book craft is so great. This paper craft for kids gives you pages and pages to fill with your thoughts and drawings. The best part is that you made it yourself. Have a blast painting and decorating the outside to look exactly the way you want with this painting craft for kids. Don't waste time and money buying notebooks at the store. All you have to do is settle down at home and get to work.
Materials: Paper Crafts, Painting & Coloring
Age Group: Preschool & Kindergarten, Elementary School, Pre-Teens
Time to complete: In an evening
Tags / Related Topics
Images from other crafters
Your Recently Viewed Projects
Something worth saving?
- SAVE your favorite projects
- ADD personal reminder notes
- QUICKLY reference your projects
Free eBooks
Our Newest Projects & Articles
- Superhero Free Printable Valentines
- 5 Superhero Craft Ideas and Comic Book Crafts
- Lovely Origami Butterflies
- Rainbow Loom Patterns: 15 Rainbow Loom Charms to Make
- 14 Crafts for Boys: Sports Crafts for Kids, Superhero Crafts, and More
- Snowflake Rainbow Loom Charms
- Lovely Lily Rainbow Loom Charms
- Super Simple Rainbow Loom Bow
- Rainbow Loom Pokeball
- 3D Ladybug Rainbow Loom Charm


text:
Home > Author
Kim McCloskey
StewardshipSimplot Hatfield: Investing In Environmental Stewardship
March 27, 2012
When considering the need for environmental responsibility in ag retail, Terry Swinson, operations manager for Simplot Grower Solutions in Hatfield, … Read More
StewardshipCPS Lake Odessa: Commitment To Improved Stewardship
March 14, 2012
Like many agricultural businesses across the nation, the land Crop Production Services (CPS), Lake Odessa, MI, sits on has housed … Read More
StewardshipCPS Clarkson: Ambassador For Retail Stewardship
February 28, 2012
Growing up on a farm gives one a unique perspective on environmental stewardship, says Daron Bell, manager of Crop Production … Read More
StewardshipMaxYield Cooperative: Environmental Stewardship Through Precision Ag
February 21, 2012
Tom Winkel began his career in ag retail in 1977, an age in agriculture, he explains, that was very different … Read More
StewardshipCeres Solutions: Adopting An Environmentally Conscious Mindset
February 8, 2012
What a difference a few decades makes when it comes to environmental respect, says Joe Hawkins, manager for Ceres Solutions … Read More
StewardshipIllini FS: Building Trust In The Community
February 8, 2012
"Outreach" is defined as the act of extending services and benefits to a wider section of the population, something that … Read More
StewardshipWilbur-Ellis Caldwell: Protecting People And The Planet
February 2, 2012
For the team at Wilbur-Ellis in Caldwell, ID, environmental stewardship is a way to care for current and future generations. … Read More
StewardshipSimplot Edison: Creating Sustainable Work Practices
January 24, 2012
For some companies, environmental stewardship represents an attempt to make amends for America's historic track record in this arena. "Unfortunately, … Read More
StewardshipCPS Wynne: Protecting The Environment
January 19, 2012
From a young age, children at the dinner table are taught to count their blessings before they eat. According to … Read More
Stewardship2011 Environmental Respect Awards: The State Winners
July 12, 2011
These 17 retailers are recognized for their commitment to agricultural environmentalism: Protecting Our Blessings Crop Production Services, Wynne, AR From … Read More
Latest News
Crop InputsABM Patents Microbial R&D Technique
November 25, 2014
Focused Microbial Diversity (FMD) is a newly patented technique employed by Advanced Biological Marketing (ABM) to research and develop microbials that will be used in ABM products Read More
Crop InputsStorage Options Help Grain Growers Go To Market
November 24, 2014
While on-farm storage in a traditional upright storage bin is one possibility for storing grain, it may not be for everyone. Read More
Eric SfiligojGiving Thanks For Another Great Year
November 24, 2014
As Thanksgiving Day 2014 arrives, agriculture has plenty to be thankful for. Read More
Crop InputsSyngenta Cost-Cutting Program To Affect 1,800 Jobs
November 24, 2014
The company's Accelerating Operational Leverage program will result in job reductions and relocations totaling around 1,800 across the company, the majority of which will occur in 2015. Read More
EquipmentAGCO Announces Operator Of The Year Finalists
November 20, 2014
Four custom applicators have been selected by AGCO Application Equipment as finalists for 2014 Operator of the Year, an honor that recognizes them as being among the top professionals in their industry throughout North America. Read More
MicronutrientsH.J. Baker Expands Tiger-Sul Business
November 20, 2014
H.J. Baker has created and filled two strategic positions in business development and sales within its Crop Performance Division. Read More
FertilizerGeneral Mills Honors United Suppliers For Nitrogen Opti…
November 19, 2014
United Suppliers winning proposal detailed SUSTAIN, a consulting network that provides customized products and services for farmers using a needs-based system approach. Read More
EmployeesOhio AgriBusiness Association Awards $25,000 In Scholar…
November 19, 2014
Each year, the Ohio AgriBusiness Association Educational Trust scholarship program awards scholarship dollars to students enrolled in an agriculture-related field attending several state colleges. Read More
ManagementServi-Tech Names New CEO
November 17, 2014
Servi-Tech has named Greg Ruehle its new president and CEO. Read More
CropLife 100Pinnacle Ag Acquires Colorado Aerial Application Outlet
November 17, 2014
Ft. Lupton, CO-based Reck Aviation — a full-service chemical application company providing aerial crop applications of fertilizers and crop protection products — will operate as part of Pinnacle's AgOne Application Services brand. Read More
Eric SfiligojMcDonald's Message: Biotech Crops Scarier Than Cancer
November 17, 2014
Despite their potential health benefits, one of the world's largest potato users will pass on a new biotech offering. Read More
MicronutrientsWinField Releases 2014 NutriSolutions Results
November 14, 2014
A number of significant regional and national crop deficiency trends emerged from the 2014 WinField NutriSolutions tissue sampling program. Read More
FertilizerH.J. Baker Opens Chinese Production Lines
November 14, 2014
The occasion was the official launch of the Tiger-Sul sulphur Bentonite production line of two much anticipated fertilizer products in China, T90CR sulphur fertilizer and TZinc micronutrient enhanced sulphur fertilizer. Read More
ManagementOhio Certified Crop Adviser Program Accepting Nominatio…
November 14, 2014
The award recognizes an individual who delivers exceptional customer service for farmer clients in nutrient management, soil and water management, integrated pest management and crop production in Ohio. Read More
HerbicidesSyngenta Announces Acuron Trial Plot Results
November 14, 2014
Acuron was tested at 167 trial locations across 35 states. Trials included 95 Syngenta locations, 54 university locations and 18 distributor locations. Read More
Crop InputsVerdesian Expands Sales Force
November 13, 2014
The new sales representatives will work with growers, retail partners and distributors to oversee technical training and product education. Read More
EquipmentAGCO Raises $100K For Wounded Warrior Project
November 13, 2014
AGCO Corp. partnered with local AGCO dealers across the U.S. and Canada to raise nearly $100,000 in support of wounded service veterans. Read More
HerbicidesDow AgroSciences Announces Launch Of Enlist Duo Herbici…
November 12, 2014
It will be launched in conjunction with a stewarded introduction of Enlist corn, and seed production of Enlist soybeans in 2015. Read More

subset: commoncrawl


text:
NOAO Library
I want to
Search the Online Catalog
Access Electronic Journals
Find Informative External Links
Use Library Services
Learn about Library Collections
Explore NOAO's History
Catch up on News Items
Connect to the UA Libraries
Check NOAO Publications Lists
See What's on the Website
Look at Library Map
NOAO-S (CTIO) Library
Librarian & Citation Analyst
Hours: 9 a.m. - 2 p.m.
About the CTIO (NOAO-South) Libraries
The physical collection consists of books, journals, and observatory publications in astronomy, physics, mathematics, science, and technology. The libraries have complete runs of the major astronomical journals (some dating from the 19th century), including ApJ, AJ, MNRAS, and A&A.
The journals and observatory publications are located at Cerro Tololo; the books and the last two years of journals are located mostly in La Serena. The CTIO libraries share the same library catalog with the NOAO library in Tucson.
New issues of journals are no longer received in paper format (with a few exceptions). These issues are available electronically through subscriptions available from the library webpage and online library catalog. The electronic journal subscriptions at CTIO, Tucson, and Gemini are different (not shared).
The library maintains the journal subscriptions, circulates materials, obtains articles, and provides reference assistance.
Page created 12/2/13; last update 12/3/13
NOAO Library
950 N. Cherry Ave.
Tucson, AZ 85719
P.O. Box 26732
Tucson, AZ 85726
Hours: Open 24 hours/day


text:
- Freeport murder suspect Damon Dixson taken into custody in Rockford
- Local gas station employee arrested for selling liquor to minor
- Renewable Fuel Standard delay 'a mixed blessing,' Bustos says
- Rockford delegation presents inaugural 'Rockford Award' to Norwegian Air
- Education in Illinois making slow progress, according to report
- Illinois GOP Congressional delegation: Obama's immigration plan undermines rule of law
- Suspect, 17, charged in Halloween hit-and-run in Roscoe
- Saint Anthony College of Nursing president to retire
- Man found guilty in deadly August 2013 crash at Mulford and Garrett Lane
- 'The Price is Right Live!' at Coronado March 1; tickets on sale Nov. 21
To the Editor: Time to get serious about juvenile crime
When I was inducted in June as president of the Illinois State Bar Association, I made a commitment to work closely with our association members to reform our woefully inadequate juvenile justice system. Sadly, our state spends far more annually to incarcerate youth (a staggering $100 million) than it does on youth prevention and intervention programming (a mere $3 million).
As we look toward the coming year, I propose a resolution to get serious about the problem of juvenile crime.
Illinois has two model programs that could be expanded. The Redeploy Illinois initiative receives state funds to provide comprehensive services to delinquent youth in their local communities. In the 23 counties that have Redeploy sites, it is working successfully, especially in St. Clair County where it has dramatically lowered the percentage of youths who are incarcerated. A relatively small increase in Redeploy funding could have a big impact.
The other state program is the Mental Health Juvenile Justice Initiative, funded by the National Institute of Mental Health and administered by the Illinois Department of Human Services. Some 66 percent of youths in the juvenile justice system have a diagnosable psychiatric condition. Based on the program's initial success—it grew from four to 34 counties—it should be expanded even further.
Shifting financial resources to community-based programming will be a real gift, not only to our youth but to our communities. It will produce better outcomes for youth in conflict, ultimately reduce the tax burden of incarcerating youth, and make our communities safer. It can be a win-win for everyone.
Mark D. Hassakis
President, Illinois State Bar Association, 2010-11
Mt. Vernon, Ill.
From the Dec. 8-14, 2010 issue


text:
Skywriters Write Out the Value of Pi to 1,000 Places
Newest 1 Comment
For their next trick, they will print out the first 1,000,000 prime numbers.
Abusive comment hidden. (Show it anyway.)
FREE SHIPPING SALE - All T-Shirt Orders Get Free Worldwide Shipping
That's right: free shipping whether you're in the USA or anywhere else in the world!
Success! Your email has been sent!


text:
iPhone - iPod
Logo Quiz Answers for iPhone - iPodHome / iPhone - iPod /
Game Questions & Answers (Q&A) service lets you ask questions about video games for game consoles or PC games. So ask your Logo Quiz question for iPhone - iPod and get answers from real gamers or answer questions and share your insights and experience with the rest of the gaming community.
- Developer: June Software Private Limited
- Publisher: June Software Private Limited
- Genre: Miscellaneous Trivia / Game Show
- Release Date: Mar 25, 2009
- ESRB: Not Set
Question & Answers
Help i'm stuck! NBC logo
Hi, I have the logo quiz for blackberry and have a few questions. 1. The NBC logo is given, but it won't take NBC or National Broadcasting Company as an answer. 2. There's a swirly type logo, looks like a mosque that's fallen over, it's yellow, purple and pink. The pink thingy looks like a teardrop on its side. Can somebody please help?
Answer from: Ivan's Angel
For NBC put in CNBC. The swirly type thing is Thai Airways
Answer this QuestionAll Questions Ask a Question
Unanswered Questions
What's the brand
From: Shelly 5
From: Shelly 5
Total 0 Answers
Posted on: 11-01-2014
Posted on: 11-01-2014
4 letter co name ends in c
From: Crib kid
From: Crib kid
Total 0 Answers
Posted on: 08-28-2014
Posted on: 08-28-2014
Total 0 Answers
Posted on: 04-24-2014
Posted on: 04-24-2014
Blue rectangular trimmed in white
From: Sam
From: Sam
Total 0 Answers
Posted on: 06-03-2013
Posted on: 06-03-2013
Videos You May Like...
View All Videos
Logo Quiz - Logo Quiz Level 2 Answers
- Aug 24, 2014, Views: 445
Game Related Options
New Released Games
- Kara Kara Onnanoko Ga!, (iPhone-iPod)
- Mona Pet, (iPhone-iPod)
- Light Speed (2014), (iPhone-iPod)
- Happy Race, (iPhone-iPod)
- Gridiron Thunder, (iPhone-iPod)
- Squeazy, (iPhone-iPod)
- Numberize Game, (iPhone-iPod)
- Gravity Break, (iPhone-iPod)
- ~777+, (iPhone-iPod)
- Wicked Lair, (iPhone-iPod)
Popular iPhone - iPod Games
- Virtual Families 2: Our Dream House, (iPhone-iPod)
- Angry Birds Transformers, (iPhone-iPod)
- The Secret Society: Hidden Mystery, (iPhone-iPod)
- Kim Kardashian: Hollywood, (iPhone-iPod)
- WordBrain, (iPhone-iPod)
- The Sims: FreePlay, (iPhone-iPod)
- The Simpsons: Tapped Out, (iPhone-iPod)
- Episode: Choose Your Story, (iPhone-iPod)
- My Singing Monsters, (iPhone-iPod)
- Card Wars - Adventure Time, (iPhone-iPod)
Popular Videos
- Kim Kardashian: Hollywood - Gameplay, (iPhone-iPod)
- Episode: Choose Your Story - Gameplay, (iPhone-iPod)
- How To Train Your Dragon 2 - Ready To Fly Trailer, (Wii)
- The Settlers - Kingdoms Of Anteria - Announcement Trailer, (PC)
- Entwined - Quick Look Trailer, (PS3)
- Blue Estate - Official Launch Trailer, (PS4)
- Goat Commando 3D Simulator - Gameplay, (iPhone-iPod)
- Munin - Official Launch Trailer, (iPad)


text:
37% of U.S. mobile users favor m-commerce over stores
And 42% of U.S. smartphone and tablet owners say they will do more shopping on their mobile devices this holiday season than they did ...
37% of U.S. mobile users favor m-commerce over stores
And 42% of U.S. smartphone and tablet owners say they will do more shopping on their mobile devices this holiday season than they did ...
Hold the phone: 66% of time spent with e-retail is on mobile
So, clearly, it's not a passing fad. Clever use of mobile apps by retailers such as Target and Wal-Mart is driving more consumers to ...
Sign up for IRNewsLink and get daily reports delivery to your inbox. Sign Up Now
Sponsored by 2013 Social Media 300
1 - 10 of 196 Results
The social network relaunches its Atlas ad-serving and measurement platform, which it says lets advertisers better track a consumer's path to purchase.
Posted 09/29/2014Zak StamborPost a comment
The move comes as more marketers get into the deep water with detailed web data.
Posted 03/21/2014Allison EnrightPost a comment
The social network plans to buy Microsoft Corp.'s Atlas Advertiser Suite.
Posted 02/28/2013Zak StamborPost a comment
Advertisers can get a more accurate read on whether consumers see ads.
Posted 04/18/2012Allison EnrightPost a comment
David Rosenblatt was the CEO of ad firm DoubleClick before its purchase by Google.
Posted 11/07/2011Allison EnrightPost a comment
Related Content:1stdibs
Jeff Clayton joins the company from database marketing firm Merkle.
Posted 07/25/2011Zak StamborPost a comment
Joe Gagnon will lead e-Dialog, while John Healy becomes CEO of MBS Insight.
Posted 04/18/2011Thad RueterPost a comment
The company will use the financing for product development and geographical expansion.
Posted 03/14/2011Thad RueterPost a comment
Daily deal site Groupon has reportedly rejected an offer from Google Inc.
Posted 12/06/2010Zak StamborPost a comment
An investment firm report suggests the deal is done.
Posted 11/29/2010Zak Stambor1 comments


text:
|I love drawing fanart, but I'm not very talented as an artist so I mostly draw for fun.
Skywarp/Thundercracker Fics and ContestFirst a question: if i make a GroupContest, would you rather prefer Summer Theme or Autumn/halloween Winter/Christmas/NewYear ?Skywarp/Thundercracker Fics and Contest by SkyWxThunderCxSLASH
Ok here some SKY/TC
Title: Cripple
Rated: PG-13/R
Warnings: Sparksex; light profanity
Pairings: Skywarp/Thundercracker
Summary: TFA 'verse. "Now...!" He couldn't believe he was about to say this. Never once in his life did he want to speak these words to anyone or anything, but... Optics squeezed shut, he exclaimed, "Give it to me!"
Title: Repair (Afterglow)
Rated: G
Warnings: CUDDLES. ;w;
Pairings: Starscream/Thundercracker/Skywarp
Summary: Skywarp is badly damaged, Thundercracker has a bad way of showing his concern and Starsream is a dick. All until repairs need to be made.
Title: Sadism and Masochism
Rating: MA
Warnings: Wee, mech sex! Also, very mild profanity. Mostly mech sex, though.
Pairing: Thundercracker/Skywarp/Thundercracker
Summary: Thundercracker and
|I love drawing fanart, but I'm not very talented as an artist so I mostly draw for fun.


text:
Team USA accomplished its goal, winning the gold medal at the FIBA World Cup by routing Serbia, 129-92, Sunday in Madrid.
Well here we are; September has arrived. You know what that means: it's almost time for training camp to begin.
As the "Ice Bucket Challenge" continues to take over the social media world in an effort to raise money and awareness for ALS research, Blackhawks winger Marian Hossa became the latest Chicago ath
After coasting through their first four games in Las Vegas, the Bulls fell to the Kings, 80-61, in the quarterfinal round of the NBA's summer-league tournament, ending their run in the deser
Led by former University of New Mexico teammates Tony Snell and Cameron Bairstow, the Bulls triumphed over the 76ers, 79-68, Thursday night to remain perfect in Summer League play.


text:
It is what it says on the tin - Holiday Inn Express. Hotel is clean and staff friendly. It is a bit out of the way though and in an unusual location. It is very out of the way and choice of eating out is very limited. For our purposes though, it did the job.
- Reservation Options:
- TripAdvisor is proud to partner with Booking.com, Travelocity, IHG, Priceline, Agoda, Despegar.com, Odigeo, Hotels.com, TripOnline SA and Tingo so you can book your Holiday Inn Express Alcobendas reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Holiday Inn Express Alcobendas Hotel Alcobendas
- Holiday Inn Alcobendas
- Alcobendas Holiday Inn


text:
Trinity Baptist Church - Welcome to Trinity! | 4815 Six ...
Welcome toTrinity! I am so glad you have chosen to visit our church's web site. Trinity Baptist Church is a vibrant and growing family of faith.
Ridge Road Baptist Church - Raleigh, NC
©2011 Ridge Road Baptist Church. All rights reserved. A Cooperative Baptist Fellowship Congregation
Raleigh, North Carolina (NC) profile: population, maps ...
View more photos (we have 62 pictures of Raleigh, NC available) Submit your own pictures of this city and show them to the World
Find Doctors in Raleigh, NC | 1,563 Raleigh Doctors
Find 1,563 local doctors practicing in Raleigh, NC. Read 3,002 ratings and reviews to help find the best doctor in your area.
Raleigh, North Carolina Home Health Care Agencies: Private ...
Directory of Raleigh North Carolina Private Home Care Providers and Home Health Agencies sources iclude: North Carolina Department of Human Resources, search engine ...
Raleigh, North Carolina - Wikipedia, the free encyclopedia
Earlier capitals . Bath the oldest town in North Carolina and was the first nominal capital from 1705 until 1722, when Edenton took over the role, but the colony had ...
Home Health & Hospice Care | Amedisys, Inc.
Amedisys may be the right choice for your loved one. Hear real testimonials from our team members and patients about how Amedisys Home Health Care has made a ...
Churches in Raleigh by Denomination, NC North Carolina ...
Find Raleigh, North Carolina NC churches in our online church directory with address, phone number, website, denomination, map and more details. Visit Church Angel ...
Gentiva Health Services - Gentiva - Great Healthcare Has ...
We're glad you stopped by. As America's home health and hospice leader, the Gentiva family of companies has been bringing great healthcare home for four decades.
Wellness - Health Information | Directory | Community
Wellness.com does not provide medical advice, diagnosis or treatment nor do we verify or endorse any specific business or professional listed on the site.


text:
Culture Fest 2014 Welcome To The Lotus Garden
Utica's annual Culture Fest is a collection of international food, music and crafts that makes Utica an international melting pot.
Pitcher Street to Be Closed Starting Monday, 4/21
The State Department of Transportation is announcing that Pitcher Street will be closed between Dwyer Ave and Broad Street beginning Monday April 24.
Central Valley School District Teacher Placed On Administrative Leave
A Central Valley School District middle school teacher has been placed on administrative leave.
DOT Travel Advisory for Routes 5/8/12 Beginning April 1, 2014 [UPDATE]
UPDATE: 03/29/2014 9:20am The Department of Transportation says that the contractor has added the French Road ramp to NY 12 northbound to the advisory. It will be closed as well beginning Tuesday morning.
Buffalo Bills Owner Ralph Wilson Dies at 95 [VIDEO]
Ralph Wilson, the owner and founder of the NFL's Buffalo Bills has died at the age of 95.
Have You Seen Madison Yesford? [UPDATE]
UPDATE: 03/24/2014 5:05pm Oneida County Sheriffs and Utica Police say Madison Yesford is now in custody.
State Police Begin DWI Check Points For St. Patrick's Day Weekend
If you're planning on enjoying the St. Patrick's Day festivities this weekend, make sure you have a designated driver.
Valley Shooting One Year Later – A Community Remembers [AUDIO]
One year ago the Villages of Herkimer and Mohawk suffered a tragedy as Kurt Myers allegedly shot and killed 4 people leading to a near 19 hour standoff that ended with him also shooting and killing an FBI K-9 before being killed by police.
St. Patrick's Day Parade Food Drive
This year's St. Patrick's Day parade will be held this Saturday March 15.
'Fight For Our Valley Schools' Rally Held
Herkimer College hosted the "Fight for Our Valley Schools" rally.
Brindisi Helps Pass Legislation to Delay And Reform Common Core
Assemblyman Anthony Brindisi announced the State Assembly has passed legislation he sponsored that would prevent certain Common Core test scores from becoming part of students' permanent records.
Fay Street Warehouse Demolition Meeting
The Fay Street Warehouse is finally coming down this Sunday March 2nd.


text:
Cookies are small text files stored on your computer. They allow us to distinguish you from other users
when you visit this site and to provide helpful features such as your recent searches. Cookies themselves
do not contain any personal information, and Infotel does not use cookies to collect personal information.
Book online or call +44 (0)1775 843417 at standard rates.
Penthouse Zeestijl de Luxe is located in the centre of Domburg and just 210 metres from the Zeeland beaches. It features full kitchen facilities, a private parking garage and a private balcony. The whole apartment is decorated with parquet flooring, warm colours and modern furnishings. Zeestijl de Luxe's seating area is set up with 2 sofas around the flat-screen TV with DVD player. Guests can make use of the modern kitchen including a gas hob, microwave/oven and dishwasher. There is a dining area with all the necessary kitchenware and cutlery provided. A variety of hiking and cycling trails is available in the nearby area of Domburg. The Domburgsche Golf Club is just 400 metres away.
Our secure connection ensures your details are protected.
Your details are only used to guarantee your booking and nothing else.
You will pay absolutely no booking fees - You pay the hotel direct and
we won't charge you a single penny.
Would you like to know more?
You can find further information in our Privacy Statement
and Terms & Conditions
These are general hotel policies for
Penthouse Zeestijl de Luxe.
As they may vary per room type; please also check the room description.
Telephone +44 (0)1775 843417 at standard rates
Infotel™ is a division of Infotel Solutions Ltd.
Copyright © 2014. All rights reserved.
Registered Office: Infotel Solutions Ltd, Boston Road, Gosberton, Spalding, Lincs UK PE11 4NR For further information visit www.infotelsolutions.com


text:
Synovial mesenchymal stem cells (MSCs) have high proliferative and chondrogenic potentials, and MSCs transplanted into the articular cartilage defect produce abundant extracellular matrix. Because of similarities between the articular cartilage and the intervertebral disc cartilage, synovial MSCs are a potential cell source for disc regeneration. Here, we examined the effect of intradiscal transplantation of synovial MSCs after aspiration of nucleus pulposus in rabbits.
The nucleus pulposus tissues of rabbit's intervertebral discs were aspirated to induce disc degeneration, and allogenic synovial MSCs were transplanted. At 2, 4, 6, 8, 16, 24 weeks postoperatively, we evaluated with imaging analyses such as X-ray and magnetic resonance imaging (MRI), and histological analysis. To investigate interaction between synovial MSCs and nucleus pulposus cells, human synovial MSCs and rat nucleus pulposus cells were co-cultured, and species specific microarray were performed.
The existence of transplanted cells labeled with DiI or derived from green fluorescent protein (GFP)-expressing transgenic rabbits was confirmed up until 24 weeks. X-ray analyses demonstrated that intervertebral disc height in the MSC group remained higher than that in the degeneration group. T2 weighted MR imaging showed higher signal intensity of nucleus pulposus in the MSC group. Immunohistological analyses revealed higher expression of type II collagen around nucleus pulposus cells in the MSC group compared with even that of the normal group. In co-culture of rat nucleus pulposus cells and human synovial MSCs, species specific microarray revealed that gene profiles of nucleus pulposus were altered markedly with suppression of genes relating matrix degradative enzymes and inflammatory cytokines.
Synovial MSCs injected into the nucleus pulposus space promoted synthesis of the remaining nucleus pulposus cells to type II collagen and inhibition of expressions of degradative enzymes and inflammatory cytokines, resulting in maintaining the structure of the intervertebral disc being maintained.
Intervertebral discs lie between adjacent vertebrae in the spine and are composed of three major structures called nucleus pulposus, annulus fibrosus, and cartilage end plates [1]. The nucleus pulposus of normal disc includes sparse chondrocytes surrounded by extracellular matrix which mainly consist of type II collagen and proteoglycan. It functions as a shock absorber against mechanical load due to its highly hydrophilic structure. Intervertebral disc degeneration accompanies aging, and it causes low back pain [2,3]. To regenerate intervertebral discs, various approaches applying cytokines [4,5], gene transfection [6], and nucleus pulposus cells [7] have been attempted in animal models. Some reports have demonstrated that transplantation of bone marrow mesenchymal stem cells (MSCs) delayed degeneration of the nucleus pulposus [8-10].
An increasing number of reports have shown that MSCs can be isolated from other various mesenchymal tissues other than bone marrow, and that their similarities as MSCs and the specificities dependent of their MSC source are emerging [11-13]. Our comparative in vivo study showed that bone marrow MSCs and synovial MSCs produced a higher amount of cartilage matrix than adipose MSCs and muscle MSCs after transplantation into articular cartilage defect of the knee in rabbits [14]. We also demonstrated that synovial MSCs expanded faster than bone marrow MSCs when cultured with 10% human autologous serum [15]. Synovial MSCs and bone marrow MSCs have a similar chondrogenic potential, but synovial MSCs are more useful from the standpoint of yield when cultured with human autologous serum.
Histologically and biochemically, some similarities exist between the nucleus pulposus and the articular cartilage. In this study, we investigated whether intradiscal transplantation of synovial MSCs delayed disc degeneration in a rabbit model. MSCs labeled with DiI or derived from green fluorescent protein (GFP) expressing transgenic rabbit [16] were used for tracking of transplanted cells. Furthermore, human synovial MSCs and rat nucleus pulposus cells were co-cultured in vitro, and their interaction was clarified by a species specific microarray system. Finally, we demonstrated the effectiveness and limitations of this method and advocated a possible mechanism to prevent intervertebral disc degeneration in a rabbit model.
Materials and methods
Cell isolation and culture
This study was approved by the Animal Experimentation Committee of Tokyo Medical and Dental University. Wild type Japanese white rabbits and GFP transgenic rabbits [16] (Kitayama Labes Co., Ltd., Nagano, Japan) were anesthetized with an intramuscular injection of 25 mg/kg ketamine hydrochloride and 150 μg/kg medetomidine hydrochloride. Synovium was harvested aseptically from knee joints of the rabbits, and bone marrow was obtained from their femurs by flushing with Hanks' balanced salt solution (Invitrogen, Carlsbad, CA, USA).
The harvested synovium was digested in a 3 mg/ml collagenase type V (Sigma-Aldrich Co., St. Louis, MO, USA) in α-minimal essential medium (αMEM) (Invitrogen) for three hours at 37°C. The digested tissues were filtered through a cell strainer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) with 70-μm pore size. The obtained cells were seeded at 5 × 104 cells/cm2 in 145-cm2 culture dishes (Nalge Nunc International, Rochester, NY, USA) and cultured with complete medium, αMEM containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin, and 250 ng/ml amphotericin B. The medium was replaced to remove nonadherent cells two days later. After being cultured for seven days, the cells were harvested with 0.25% trypsin-EDTA (Invitrogen) and cryopreserved at 1 × 106 cells/ml in αMEM with 5% dimethylsulfoxide (Wako, Osaka, Japan) and 10% FBS.
The harvested bone marrow was filtered through a cell strainer with 70-μm pore size and plated in 145-cm2 culture dishes with the medium described above and then incubated at 37°C with 5% humidified CO2. The medium was replaced the next day. After being cultured for 14 days, the cells were harvested with 0.25% trypsin-EDTA, replated in 145-cm2 culture dishes, and cultured as passage 1. Passage 1 cells were harvested and cryopreserved after the culture for 14 days.
The frozen cells from synovium and bone marrow were thawed, plated at 3 × 103 cells/cm2 in 145-cm2 culture dishes, and incubated for five days. Harvested cells derived from wild type rabbits with 0.25% trypsin-EDTA were resuspended at 1 × 106 cells/ml in αMEM, and a DiI (Molecular Probes, Eugene, OR, USA) fluorescent lipophilic tracer was added at 5 μl/ml in αMEM. After incubation for 20 minutes at 37°C with 5% humidified CO2, the cells were centrifuged at 450 g for five minutes and washed twice with PBS. The obtained cells were used for further analyses.
Colony-forming unit assay
One thousand cells were plated in 60-cm2 dishes, cultured in complete medium for 14 days, and stained with 0.5% Crystal Violet in methanol for five minutes.
In vitro differentiation assay
Five hundred cells from rabbit synovium were plated in 60-cm2 dishes and cultured in complete medium for 14 days. For adipogenesis, the medium was then switched to adipogenic medium that consisted of complete medium supplemented with 10-7 M dexamethasone, 0.5 mM isobutylmethylxanthine, and 100 μM indomethacin. After four days, the adipogenic cultures were stained with 0.3% Oil Red-O solution. For calcification, the medium was then switched to calcification medium that consisted of complete medium supplemented with 10-9 M dexamethasone, 10 mM β-glycerol phosphate, and 50 μg/ml ascorbate-2-phosphates for an additional six weeks. These dishes were stained with 0.5% Alizarin Red solution. For chondrogenesis, 2 × 105 cells were plated in a 15 ml polypropylene tube (BD Falcon, Bedford, MA, USA) and pelleted by centrifugation at 450 g for 10 minutes. The pellets were cultured for 21 days in chondrogenic medium which contained 1,000 ng/ml bone morphogenetic protein 7 (Stryker Biotech, Boston, MA, USA), 10 ng/ml transforming growth factor-β3 (R&D Systems Inc., Minneapolis, MN, USA), and 100 nM dexamethasone. For histological analysis, the pellets were embedded in paraffin, cut into 5-μm sections, and stained with 1% Toluidine Blue.
In vivo transplantation
Mature female Japanese white rabbits weighing an average of 3.0 kg were anesthetized as mentioned above. The anterior surface of the lumbar spine was exposed through the anterolateral approach, and then L3 to L4, L4 to L5, and L5 to L6 intervertebral discs were identified. Nucleus pulposus tissues at L3 to L4 and L5 to L6 discs were aspirated using a 21-gauge needle on a 10 ml syringe to induce disc degeneration [10]. Then, L3 to L4 discs were untreated and referred to as the "degeneration group." L5 to L6 discs were injected with 100 μl of 1 × 107 allogenic MSCs/ml in PBS using a 27-gauge needle immediately after the nucleus pulposus aspiration, and referred to as the "MSC group." L4 to L5 discs were approached but not treated and referred to as the "normal group." After the operation, all rabbits were allowed to move in a cage freely. At 2, 4, 6, 8, 16, 24 weeks postoperatively, the rabbits were sacrificed with an overdose of sodium pentobarbital, and the lumbar spine was harvested.
Imaging analysis
Radiographs were taken immediately after harvest of the lumbar spine using X-ray equipment (CMB-2; SOFTEX, Kanagawa, Japan). Intervertebral disc height and vertebral body height were measured, and the disc height index (DHI) [17] was calculated. Alterations in the DHI were normalized to the DHI before aspiration of the nucleus pulposus and are indicated as "%DHI".
MR imaging at 3.0T (Achieva; Philips Medical Systems, Andover, MA, USA) was used with an 8-cm diameter surface dual coil. T2-weighted turbo spin-echo images (TE 130 ms, TR 3,200 ms, FOV 140 mm, matrix 320 × 360, slice thickness of 3 mm) of the lumbar spine were obtained at each time point.
Histology and fluorescent microscopy
The intervertebral discs including the adjacent vertebral bodies were fixed in 4% paraformaldehyde, decalcified with 20% EDTA, dehydrated in a gradient series of ethanol, and embedded in paraffin. Midline sagittal sections of the intervertebral discs were stained with Hematoxylin and Eosin.
For fluorescent microscopy, the nucleus pulposus was harvested, fixed in 4% paraformaldehyde, and transferred to 20% sucrose solution. Specimens were flash-frozen and cut in a cryostat. Sections were mounted on a slide and observed under epifluorescent microscopy.
Paraffin-embedded sections were deparaffinized in xylene, rehydrated through graded alcohol, and immersed in PBS. The samples were pretreated with 0.4 mg/ml proteinase K (DAKO, Carpinteria, CA, USA) in Tris-HCl buffer for 15 minutes at room temperature for antigen retrieval. Any residual enzymatic activity was removed by washing with PBS, and nonspecific staining was blocked by preincubation with PBS containing 10% normal horse serum for 20 minutes at room temperature. Mouse monoclonal antibody against human type II collagen (1:1,000 dilution; Daiichi Fine Chemical, Toyama, Japan) was placed on each section for one hour at room temperature. After extensive washing with PBS, the sections were incubated in the secondary antibody of biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA, USA) for 30 minutes at room temperature. Immunostaining was detected with VECTSTAIN ABC reagent (Vector Laboratories), followed by DAB staining. Counter staining was performed with Mayer-Hematoxylin.
Co-culture experiments and RNA isolation
Human synovium was obtained during anterior cruciate ligament reconstruction surgery for ligament injury and digested with 3 mg/ml collagenase type V in αMEM for three hours at 37°C. The digested tissues were filtered through a 70-μm pore cell strainer. The obtained cells were incubated with complete medium as human synovial MSCs. Nucleus pulposus tissues were harvested from wild type Wister rat, minced, digested for 30 minutes at 37°C with 0.01% trypsin-EDTA, and filtered through a 70-μm pore cell strainer. Nucleated cells were seeded in 60-cm2 culture dishes (Nalge Nunc International) with αMEM containing 10% FBS.
For co-culture, both passage 2 human synovial MSCs and passage 0 rat nucleus pulposus cells were seeded together in 60-cm2 culture dishes at 3 × 103 cells/cm2, respectively. For the control, only human synovial MSCs or only rat nucleus pulposus cells were seeded at 6 × 103 cells/cm2. After seven days, total RNA was isolated from cultured cells with the RNeasy Total RNA Mini Kit (Qiagen, Valencia, CA, USA).
Oligonucleotide microarray
Three μg of total RNA from each sample was first reverse transcribed to synthesize the first-strand cDNA using a T7-Oligo(dT) promoter primer by the One-Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA, USA). Following RNase H-mediated second-strand cDNA synthesis, the double-stranded cDNA was purified and served as a template in the subsequent in vitro transcription (IVT) reaction. The IVT reaction was carried out in the presence of T7 RNA Polymerase and a biotinylated nucleotide analog/ribonucleotide mix for complementary RNA (cRNA) amplification and biotin labeling using a GeneChip IVT Labeling kit (Affymetrix, Santa Clara, CA, USA). The biotinylated cRNA targets were then cleaned up, fragmented, and hybridized to GeneChip® Rat Genome 230 2.0 probe arrays (Affymetrix) and/or GeneChip® Human U-133 plus 2.0 probe arrays according to the manufacturer's instructions [18].
Data analysis was performed with GeneSpring software version 7.2 (Agilent Technologies, Palo Alto, CA, USA). To normalize the variations in staining intensity among chips, the 'signal' values for all genes on a given chip were divided by the median value for expression of all genes on the chip. To eliminate genes containing only a background signal, genes were selected only if the raw values of the 'signal' were more than the lower limit of the confidence interval. Expression of the gene was judged to be 'present' by the GeneChip Operating Software version 1.4 (Affymetrix). The microarray data were deposited in the Gene Expression Omnibus [19], [GEO:GSE24612]. Genes filtered with this quality criteria were subjected to further analysis.
A hierarchical-clustering analysis was performed using a minimum distance value of 0.001, a separation ratio of 0.5 and the standard definition of the correlation distance. A dendrogram was obtained from hierarchically clustering analysis using average linkage and distance metric equal to one minus the Pearson correlation applied to the microarray data.
Statistical analysis
To assess differences, two-factor ANOVA and Tukey-Kramer post-hoc tests were used. P-values less than 0.05 were considered statistically significant.
Characteristics of synovial cells as MSCs
A GFP rabbit showed green under its skin, especially in its muscles and bones under fluorescence (Figure 1a). Colony forming cells derived from GFP rabbit synovium demonstrated green under fluorescence (Figure 1b). These cells differentiated into chondrocytes and adipocytes, and were calcified when cultured in the appropriate differentiation medium (Figure 1c). As MSCs are defined by adherence to plastic and trilineage differentiation [20], our results indicate that the rabbit synovium-derived cells had characteristics of MSCs.
Figure 1. Cells from rabbit synovium have characteristics of MSCs. (a) Right hindlimb of GFP transgenic rabbit. (b) Colony forming cells derived from GFP transgenic rabbit synovium. (c) Differentiation potentials.
Existence of transplanted MSCs
DiI labeled synovial MSCs could be detected in the nucleus pulposus one day after intradiscal injection of the cells into the normal disc (Figure 2a). After aspiration of nucleus pulposus and injection of labeled synovial MSCs, DiI or GFP positive cells could be observed at 2, 8, and 24 weeks (Figure 2b).
Figure 2. Intradiscally injected MSCs remain in the nucleus pulposus at 24 weeks. (a) Disc in normal condition and one day after intradiscal injection of DiI-labeld syonovial MSCs into the normal disc. Macroscopic views of interbertebral discs and fluorescent microscopic views of nucleus pulposus are shown. (b) Fluorescent microscopic views for GFP and DiI synovial MSCs. Nucleus pulposus was aspirated in both groups, and synovial MSCs were intradiscally injected into the MSC group.
Imaging analyses for discs
The disc height index was defined as the ratio of disc height to vertebral body height by lateral radiographs of the spine (Figure 3a). The disc height index in the degeneration group decreased gradually and reached bottom at six weeks. The disc height index in the MSC group was comparable to that in the normal group up until 24 weeks. The disc height index in the MSC group was statistically higher than that in the degeneration group at two weeks and thereafter (Figure 3b).
Figure 3. Intradiscally injected MSCs maintain disc height. (a) X-ray image of normal rabbit spine for measurement of disc height index. (b) Sequential changes of disc height index after transplantation of synovial MSCs. Average percentages of the value are shown with standard deviations. **P < 0.01 between the degeneration group and the normal group or the MSC group (n = 10 at each time point) by two-factor ANOVA and Turkey-Kramer post-hoc test. (c) Disc height index at two weeks after transplantation of bone marrow or synovial MSCs. Average percentages of values with standard deviations. **P < 0.01 between the bone marrow or synovial MSC group and the degeneration group (n = 6 for each group). (d) Representative T2-weighted MR images of intervertebral discs at 2 to 24 weeks after operation.
We also used injections of bone marrow MSCs instead of synovial MSCs, and compared the disc height index in both groups at two weeks. There was no significant difference of the disc height index between the bone marrow MSC group and the synovial MSC group, though the disc height index in each MSC group was significantly higher than that in each degeneration group (Figure 3c).
T2-weighted MR images showed that the signal intensity of nucleus pulposus in the degeneration group considerably decreased at two weeks and thereafter. Contrarily, the signal intensity of nucleus pulposus in the MSC group remained high comparable with that in the normal group at two and four weeks. Though the intensity in the MSC group gradually reduced after six weeks, it remained higher than that in the degeneration group up until 24 weeks (Figure 3d).
Histological analysis
According to macroscopic views of the sagittal section of intervertebral discs at 2 weeks after operation, in the degeneration group, the nucleus pulposus tissue volume was much less than that in the normal group (Figure 4a). In the MSC group, the nucleus pulposus was clearly observed.
Figure 4. Intradiscally injected MSCs maintain microstructure of nucleus pulpous. (a) Macroscopic views of the sagittal section of intervertebral discs at two weeks after operation. (b) Sagittal sections with Hematoxylin-Eosin (HE) staining after operation. (c) Higher magnification of the framed area with type II collagen immunostaining.
In low magnified histologies, in the degeneration group, the nucleus pulposus could hardly be seen at two weeks and thereafter (Figure 4b). In the MSC group, the nucleus pulposus looked comparable to a normal one at two and eight weeks, and it was still visible at 24 weeks.
In high magnified histologies at two weeks, in the degeneration group, the nucleus pulposus was replaced with fibrous tissue (Figure 4c). In the MSC group, the nucleus pulposus consisted of sparse cells surrounded with matrix and looked similar to that of the normal group. Interestingly, type II collagen expression in the MSC group was higher than that in the normal group.
Co-culture of human synovial MSCs and rat nucleus pulposus cells
To investigate interaction between synovial MSCs and nucleus pulposus cells, human synovial MSCs and rat nucleus pulposus cells were co-cultured. Typical rat nucleus pulposus cells attached to the culture dish were bright and round (Figure 5a). Human synovial MSCs were spindle-shaped. Though a similar number of rat nucleus pulposus cells and human synovial MSCs were co-cultured, only human synovial MSCs appeared to increase in number at seven days (Figure 5a). Human microarray showed that the gene profile of human MSCs cultured alone was similar to that of human MSCs co-cultured with rat nucleus pulposus cells (Figure 5b). Contrarily, rat microarray demonstrated that the gene profile of rat nucleus pulposus cells was widely different from that of rat nucleus pulposus cells co-cultured with human MSCs (Figure 5c).
Figure 5. Synovial MSCs affect gene profile of nucleus pulposus cells in co-culture system. (a) Morphology of mono-culture of rat nucleus pulposus cells and human synovial MSCs at seven days, and co-culture of rat nucleus pulposus cells with human synovial MSCs at one and seven days. (b) Human gene profile of human synovial MSCs in mono-culture and in co-culture with rat nucleus pulposus cells. (c) Rat gene profile of rat nucleus pulposus cells in mono-culture and in co-culture with human MSCs. (d) Number of altered rat genes seven days after co-culture of rat nucleus pulposus cells with human synovial cells by duplicate of microarray analyses.
We further analyzed the gene profile of rat microarray. Among 31,099 transcripts, we first picked up 15,779 genes whose values were more than 50 and judged to be "present." Then, we selected rat genes whose expression value were two-fold higher or lower in co-culture than in mono-culture of rat nucleus pulposus cells. Two independent microarray analyses demonstrated 172 up-regulated genes and 7,922 down-regulated genes in common between the first and the second analysis (Figure 5d). Approximately 80% of the up-regulated genes and 90% of the down-regulated genes were overlapped as shown by the first and second microarray analyses, indicating reproducible results.
We next focused on the genes related to extracellular matrix, and the genes which possibly affect the extracellular matrix. We found five genes related to collagen, six genes related to proteoglycan, three genes related to tissue inhibitor of metalloproteinase (TIMP), seven genes related to matrix metalloproteinase (MMP), 10 genes related to interleukin (IL), and six genes related to tumor necrosis factor (TNF). Among these genes, up- and down- regulated genes two-fold or higher in co-culture are listed in Table 1. In collagens and proteoglycans, Col2A1, a principal component of nucleus pulposus, and Chondroitin sulfate proteoglycan 2, a member of the hyaluronan-binding proteoglycan family, were significantly up-regulated, though Aggrecan 1, another principal component of nucleus pulposus, was stable and is not listed in Table 1. Other collagen and proteoglycan related genes were mostly down-regulated. In the TIMP family, TIMP-3 was markedly up-regulated, though TIMP-1 and -2 were down-regulated. All the MMP genes listed on the microarray, especially MMP-2, -3, and -13, were down-regulated significantly. All inflammatory cytokine-related genes were also down-regulated. These data indicate that cartilage catabolic factors were suppressed and anabolic factors were enhanced, consequently contributing to the prevention of intervertebral disc degeneration.
Table 1. Rat genes up- and down- regulated two-fold or higher in nucleus pulposus cells co-cultured with human synovial MSCs
In this study, we demonstrate that intradiscal injection of synovial MSCs prevented intervertebral disc degeneration in rabbits up until 24 weeks. Several reports have shown differentiation of bone marrow MSCs toward a nucleus pulposus-like phenotype in vitro [21-23] and the regenerative effects of bone marrow MSCs after intradiscal injection [8-10]. To the best of our knowledge, only one paper has shown in vitro differentiation of synovial MSCs into nucleus pulposus in which synovial MSCs and nucleus pulposus cells were co-cultured [24]. Ours is the first report demonstrating the effectiveness of intradiscal transplantation of synovial MSC in rabbit intervertebral disc degeneration model.
For tracking the transplanted cells, we used DiI and GFP systems. DiI is a popular dye, highly fluorescent and photostable when incorporated into lipid membrane [25,26]. It exhibits low cell toxicity [27], and retains its fluorescence for a long time in specific situations. However, there is some criticism in the use of DiI. The emission of DiI fluorescence decreases every time cells divide. If DiI leaches out of dying cells, it may be doubtful whether DiI fluorescence indicates living transplanted cells or not. In our case, if the dye had leaked from the injected MSCs, they would not have emitted significant fluorescence in the extracellular matrix of nucleus pulposus, because DiI almost never emits fluorescent in aqueous solutions. When leaked DiI transfers between intact membranes, DiI is usually negligible [28]. To verify the results of tracking cells, we also transplanted synovial MSCs derived from a GFP transgenic rabbit. We could observe GFP-positive and/or DiI labeled cells at the nucleus pulposus 24 weeks after transplantation, which demonstrates that transplanted MSCs survived for some time in the nucleus pulposus.
According to X-ray and histological analyses, the effect of MSCs could be observed at 24 weeks. Immunohistological analyses demonstrated that the amount of type II collagen in the nucleus pulposus was higher in the MSC group than even in the normal group at two weeks. Generally, type II collagen functions as a frame work in cartilage tissues [1,29]. These findings indicate that transplantation of MSCs induced a higher amount of type II collagen, which acted as a frame work in the nucleus pulposus, which resulted in the maintenance of disc height and histological features.
On the other hand, based on MR imaging, the effect of MSCs already decreased at six weeks. High signal intensity of T2 weighted MR imaging reflects the amount of water in the nucleus pulposus. Possibly, transplanted MSCs promoted synthesis of proteoglycan, in which negative charged sulfate held water. In our study, the ability of MSCs to maintain water was reduced at six weeks. During the degeneration of intervertebral disc in humans, reduction of water content in the nucleus pulposus by MR imaging precedes a decrease of disc height as shown by X-ray [30,31].
In this model, transplantation of MSCs into the intervertebral disc delayed progression of degeneration, but its effect was not maintained due to MRI imaging. This result is different from that of previous studies demonstrating regeneration of articular cartilage. After synovial MSCs were transplanted into full thickness defect of articular cartilage, cartilage matrix was filled in the defect. Then the border between the bone and cartilage was moved, and finally the thickness of the regenerated cartilage became similar to that of the adjacent native cartilage. The regenerated cartilage was maintained at least for six months [14,32,33]. In our current study, though extracellular matrix was reproduced by synovial MSC in the nucleus pulposus, the nucleus pulposus never thickened more than the normal one. These differences were thought to be caused by environmental differences. Intervertebral disc is a thicker avascular tissue than articular cartilage, and it is not surrounded by joint fluid or synovium. A severer environment around the intervertebral disc may reduce the regenerative effects of MSCs.
Species specific microarray analyses in our co-culture experiment revealed that nucleus pulposus cells dramatically changed their gene profile by interaction with synovial MSCs. Contrarily, synovial MSCs did not change their gene profile in co-culture with nucleus pulposus cells. These results demonstrate that synovial MSCs influenced nucleus pulposus cells, but nucleus pulposus cells did not affect synovial MSCs.
Co-culture of synovial MSCs increased Col2a1 expression more than 10-fold in nucleus pulposus cells. An in vivo study demonstrated that transplantation of synovial MSCs enhanced type II collagen expression in the nucleus pulposus immunohistologically. We could not detect type II collagen expression around labeled MSCs. It is well known that turnover of cartilage collagen is very slow [34]. These findings indicate that synovial MSCs promoted the remaining nucleus pulposus to synthesize type II collagen.
Among genes for proteoglycans, chondroitin sulfate proteoglycan 2 expression increased to about 3-fold in nucleus pulposus cells with co-culture of synovial MSCs. Chondroitin sulfate proteoglycan 2, known as versican, is one of the principal components of nucleus pulposus, and its expression is higher in nucleus pulposus than in articular cartilage [35,36]. A possible higher expression of chondroitin sulfate proteoglycan 2 in nucleus pulposus cells induced the holding of water, resulting in improvements of MR imaging in an in vivo study.
A number of studies have noted that degradative enzymes and inflammatory cytokines are highly produced by nucleus pulposus cells in degenerate intervertebral discs [37,38]. According to our microarray analyses, all MMP genes examined were down-regulated in nucleus pulposus cells with co-culture of synovial MSCs. In particular, MMP-2, -3, -13 were highly down-regulated, and their inhibitor TIMP-3 expression increased more than 27-fold. It is known that TIMP-3 particularly inhibits aggrecanases [38] and that MMP-13, known as collagenase-3, has a strong effect on type II collagen [39]. MMP-2 was also found in degenerated discs and had a highly significant correlation with age and histological alterations of intervertebral discs [40]. Suppression of MMPs results in an increase of type II collagen expression in vivo.
All interleukin related and tumor necrosis factor related genes listed were down-regulated. All of them have inflammatory effects. Inhibition of inflammatory cytokine-related genes could induce the suppression of MMPs [39,41] which resulted in the preventive effects of intervertebral disc degeneration.
For clinical application, two things should be considered. First, species differences will affect the results. We used rabbits in which nucleus pulposus contained more notochordal cells than found in adult humans. Notochordal cells have a higher regenerative potential than other cells in the nucleus pulposus [42,43]. Second, the choice of intervertebral disc degeneration model should be considered. In this study, disc degeneration was induced by aspiration of nucleus pulposus tissues. This model is caused traumatically and does not mimic human age-dependent intervertebral disc degeneration. However, a nucleus pulposus aspiration model is useful in that nucleus pulposus promptly decreases after aspiration, and this method could provide rapid and stable induction of disc degeneration.
The preventive effect of synovial MSCs was similar to that of bone marrow MSCs in our study. Though it may be thought that the availability of synovium in human is lower than that of bone marrow, it really is not. We are currently conducting a clinical study of transplantation of autologous synovial MSCs into cartilage defect of the knee. Synovial tissue can be harvested from the knee joint under local anesthesia without arthroscopy in clinical practice. Synovial MSCs can be expanded faster and greater with autologous human serum than bone marrow MSCs [15], which is an advantage of synovial MSCs. Synovial MSCs are a potential cell source for intervertebral disc regeneration therapy as well as bone marrow MSCs.
We summarize a possible mechanism of prevention for intervertebral disc degeneration by intradiscal transplantation of synovial MSCs shown in Figure 6. After aspiration of the nucleus pulposus, intervertebral disc space rapidly decreases. Synovial MSCs injected into the nucleus pulposus space promoted the remaining nucleus pulposus cells to synthesize type II collagen. Also, synovial MSCs affected the remaining nucleus pulposus cells by inhibiting their expressions of degradative enzymes and inflammatory cytokines, resulting in maintaining the structure of the intervertebral disc.
Figure 6. Possible mechanism of prevention for intervertebral disc degeneration by intradiscal transplantation of synovial MSCs. After aspiration of the nucleus pulposus, intervertebral disc space rapidly decreases. Synovial MSCs injected into the nucleus pulposus space promote synthesis of type II collagen for the remaining nucleus pulposus cells. Also, synovial MSCs affected the remaining nucleus pulposus cells by inhibiting expressions of degradative enzymes and inflammatory cytokines, resulting in maintaining the structure of the vertebral disc.
Intradiscal transplantation of synovial MSCs prevented intervertebral disc degeneration in vivo. Co-culture assay in vitro revealed that nucleus pulposus cells dramatically changed their gene profile by interaction with synovial MSCs to inhibit expressions of the genes for degradative enzymes and inflammatory cytokines.
αMEM: α-minimal essential medium; DHI: disc height index; EDTA: ethylenediaminetetraacetate; FBS: fetal bovine serum; FOV: field of view; GFP: green fluorescent protein; IL: interleukin; IVT: in vitro transcription; MMP: matrix metalloproteinase; MRI: magnetic resonance imaging; MSC: mesenchymal stem cell; PBS: phosphate-buffered saline; TE: echo time; TIMP: tissue inhibitor of metalloproteinase; TNF: tumor necrosis factor; TR: repetition time.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
TMi participated in the design of the study, carried out the animal experiments, analyzed the results, and drafted the manuscript. TMu participated in the design of the study and provided the administrative and financial support. TT carried out the MR imaging. KM and HS carried out the microarray and participated in the evaluation of the results. KT participated in the design of the study and provided the financial support. IS participated in the design of the study, provided the financial support, and completed the final manuscript. All authors read and approved the final manuscript.
We thank Kenichi Shinomiya, MD, PhD, Atsushi Okawa, MD, PhD, and Tsuyoshi Kato, MD, PhD, for their continuous support; and Miyoko Ojima for her expert help with histology. This work was supported by a grant from the Japanese Ministry of Education Global Center of Excellence (GCOE) Program, the International Research Center for Molecular Science in Tooth and Bone Diseases, Tokyo Medical and Dental University, Tokyo, Japan. GFP transgenic rabbits were provided by Kitayama Labes Co., Ltd. Bone morphogenetic protein 7 was provided by Stryker Biotech.
Humzah MD, Soames RW: Human intervertebral disc: structure and function.
Anat Rec 1988, 220:337-356. PubMed Abstract | Publisher Full Text
West J Med 1985, 143:259-265. PubMed Abstract | PubMed Central Full Text
Deyo R, Weinstein J: Low back pain.
N Engl J Med 2001, 344:363-370. PubMed Abstract | Publisher Full Text
Walsh A, Bradford D, Lotz J: In vivo growth factor treatment of degenerated intervertebral discs.
Spine (Phila Pa 1976) 2004, 29:156-163. PubMed Abstract | Publisher Full Text
Masuda K, Imai Y, Okuma M, Muehleman C, Nakagawa K, Akeda K, Thonar E, Andersson G, An HS: Osteogenic protein-1 injection into a degenerated disc induces the restoration of disc height and structural changes in the rabbit anular puncture model.
Spine 2006, 31:742-754. PubMed Abstract | Publisher Full Text
Cui M, Wan Y, Anderson D, Shen F, Leo B, Laurencin C, Balian G, Li X: Mouse growth and differentiation factor-5 protein and DNA therapy potentiates intervertebral disc cell aggregation and chondrogenic gene expression.
Spine J 2008, 8:287-295. PubMed Abstract | Publisher Full Text
Nomura T, Mochida J, Okuma M, Nishimura K, Sakabe K: Nucleus pulposus allograft retards intervertebral disc degeneration.
Clin Orthop Relat Res 2001, 94-101. PubMed Abstract | Publisher Full Text
Crevensten G, Walsh A, Ananthakrishnan D, Page P, Wahba G, Lotz J, Berven S: Intervertebral disc cell therapy for regeneration: mesenchymal stem cell implantation in rat intervertebral discs.
Ann Biomed Eng 2004, 32:430-434. PubMed Abstract | Publisher Full Text
Zhang YG, Guo X, Xu P, Kang LL, Li J: Bone mesenchymal stem cells transplanted into rabbit intervertebral discs can increase proteoglycans.
Clin Orthop Relat Res 2005, 219-226. PubMed Abstract | Publisher Full Text
Sakai D, Mochida J, Iwashina T, Hiyama A, Omi H, Imai M, Nakai T, Ando K, Hotta T: Regenerative effects of transplanting mesenchymal stem cells embedded in atelocollagen to the degenerated intervertebral disc.
Biomaterials 2006, 27:335-345. PubMed Abstract | Publisher Full Text
Sakaguchi Y, Sekiya I, Yagishita K, Muneta T: Comparison of human stem cells derived from various mesenchymal tissues: superiority of synovium as a cell source.
Arthritis Rheum 2005, 52:2521-2529. PubMed Abstract | Publisher Full Text
Mochizuki T, Muneta T, Sakaguchi Y, Nimura A, Yokoyama A, Koga H, Sekiya I: Higher chondrogenic potential of fibrous synovium- and adipose synovium-derived cells compared with subcutaneous fat-derived cells: distinguishing properties of mesenchymal stem cells in humans.
Arthritis Rheum 2006, 54:843-853. PubMed Abstract | Publisher Full Text
Yoshimura H, Muneta T, Nimura A, Yokoyama A, Koga H, Sekiya I: Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle.
Cell Tissue Res 2007, 327:449-462. PubMed Abstract | Publisher Full Text
Koga H, Muneta T, Nagase T, Nimura A, Ju Y, Mochizuki T, Sekiya I: Comparison of mesenchymal tissues-derived stem cells for in vivo chondrogenesis: suitable conditions for cell therapy of cartilage defects in rabbit.
Cell Tissue Res 2008, 333:207-215. PubMed Abstract | Publisher Full Text
Nimura A, Muneta T, Koga H, Mochizuki T, Suzuki K, Makino H, Umezawa A, Sekiya I: Increased proliferation of human synovial mesenchymal stem cells with autologous human serum: comparisons with bone marrow mesenchymal stem cells and with fetal bovine serum.
Arthritis Rheum 2008, 58:501-510. PubMed Abstract | Publisher Full Text
Takahashi R, Kuramochi T, Aoyagi K, Hashimoto S, Miyoshi I, Kasai N, Hakamata Y, Kobayashi E, Ueda M: Establishment and characterization of CAG/EGFP transgenic rabbit line.
Transgenic Res 2007, 16:115-120. PubMed Abstract | Publisher Full Text
Lü D, Shono Y, Oda I, Abumi K, Kaneda K: Effects of chondroitinase ABC and chymopapain on spinal motion segment biomechanics. An in vivo biomechanical, radiologic, and histologic canine study.
Spine (Phila Pa 1976) 1997, 22:1828-1834.
discussion 1834-1825PubMed Abstract | Publisher Full Text
Kato A, Homma T, Batchelor J, Hashimoto N, Imai S, Wakiguchi H, Saito H, Matsumoto K: Interferon-alpha/beta receptor-mediated selective induction of a gene cluster by CpG oligodeoxynucleotide 2006.
BMC Immunol 2003, 4:8. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Gene Expression Omnibus [http://www.ncbi.nlm.nih.gov/projects/geo/] webcite
Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E: Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.
Cytotherapy 2006, 8:315-317. PubMed Abstract | Publisher Full Text
Risbud MV, Albert TJ, Guttapalli A, Vresilovic EJ, Hillibrand AS, Vaccaro AR, Shapiro IM: Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro: implications for cell-based transplantation therapy.
Spine 2004, 29:2627-2632. PubMed Abstract | Publisher Full Text
Steck E, Bertram H, Abel R, Chen B, Winter A, Richter W: Induction of intervertebral disc-like cells from adult mesenchymal stem cells.
Stem Cells 2005, 23:403-411. PubMed Abstract | Publisher Full Text
Richardson S, Hughes N, Hunt J, Freemont A, Hoyland J: Human mesenchymal stem cell differentiation to NP-like cells in chitosan-glycerophosphate hydrogels.
Biomaterials 2008, 29:85-93. PubMed Abstract | Publisher Full Text
Chen S, Emery S, Pei M: Coculture of synovium-derived stem cells and nucleus pulposus cells in serum-free defined medium with supplementation of transforming growth factor-beta1: a potential application of tissue-specific stem cells in disc regeneration.
Spine (Phila Pa 1976) 2009, 34:1272-1280. PubMed Abstract | Publisher Full Text
McCauley D, Bronner-Fraser M: Neural crest contributions to the lamprey head.
Development 2003, 130:2317-2327. PubMed Abstract | Publisher Full Text
Mothe A, Tator C: Proliferation, migration, and differentiation of endogenous ependymal region stem/progenitor cells following minimal spinal cord injury in the adult rat.
Neuroscience 2005, 131:177-187. PubMed Abstract | Publisher Full Text
Crawford J, Braunwald N: Toxicity in vital fluorescence microscopy: effect of dimethylsulfoxide, rhodamine-123, and DiI-low density lipoprotein on fibroblast growth in vitro.
In Vitro Cell Dev Biol 1991, 27A:633-638. PubMed Abstract | Publisher Full Text
Andrade W, Seabrook T, Johnston M, Hay J: The use of the lipophilic fluorochrome CM-DiI for tracking the migration of lymphocytes.
J Immunol Methods 1996, 194:181-189. PubMed Abstract | Publisher Full Text
Nerlich A, Boos N, Wiest I, Aebi M: Immunolocalization of major interstitial collagen types in human lumbar intervertebral discs of various ages.
Virchows Arch 1998, 432:67-76. PubMed Abstract | Publisher Full Text
Berlemann U, Gries N, Moore R: The relationship between height, shape and histological changes in early degeneration of the lower lumbar discs.
Eur Spine J 1998, 7:212-217. PubMed Abstract | Publisher Full Text
Kim K, Yoon S, Li J, Park J, Hutton W: Disc degeneration in the rabbit: a biochemical and radiological comparison between four disc injury models.
Spine (Phila Pa 1976) 2005, 30:33-37. PubMed Abstract | Publisher Full Text
Koga H, Muneta T, Ju YJ, Nagase T, Nimura A, Mochizuki T, Ichinose S, von der Mark K, Sekiya I: Synovial stem cells are regionally specified according to local microenvironments after implantation for cartilage regeneration.
Stem Cells 2007, 25:689-696. PubMed Abstract | Publisher Full Text
Koga H, Shimaya M, Muneta T, Nimura A, Morito T, Hayashi M, Suzuki S, Ju Y, Mochizuki T, Sekiya I: Local adherent technique for transplanting mesenchymal stem cells as a potential treatment of cartilage defect.
Arthritis Res Ther 2008, 10:R84. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Verzijl N, DeGroot J, Thorpe S, Bank R, Shaw J, Lyons T, Bijlsma J, Lafeber F, Baynes J, TeKoppele J: Effect of collagen turnover on the accumulation of advanced glycation end products.
J Biol Chem 2000, 275:39027-39031. PubMed Abstract | Publisher Full Text
Sztrolovics R, Grover J, Cs-Szabo G, Shi S, Zhang Y, Mort J, Roughley P: The characterization of versican and its message in human articular cartilage and intervertebral disc.
J Orthop Res 2002, 20:257-266. PubMed Abstract | Publisher Full Text
Roughley P: Biology of intervertebral disc aging and degeneration: involvement of the extracellular matrix.
Spine (Phila Pa 1976) 2004, 29:2691-2699. PubMed Abstract | Publisher Full Text
Roberts S, Caterson B, Menage J, Evans E, Jaffray D, Eisenstein S: Matrix metalloproteinases and aggrecanase: their role in disorders of the human intervertebral disc.
Spine (Phila Pa 1976) 2000, 25:3005-3013. PubMed Abstract | Publisher Full Text
Le Maitre C, Freemont A, Hoyland J: Localization of degradative enzymes and their inhibitors in the degenerate human intervertebral disc.
J Pathol 2004, 204:47-54. PubMed Abstract | Publisher Full Text
Le Maitre C, Freemont A, Hoyland J: The role of interleukin-1 in the pathogenesis of human intervertebral disc degeneration.
Arthritis Res Ther 2005, 7:R732-745. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Weiler C, Nerlich A, Zipperer J, Bachmeier B, Boos N: 2002 SSE Award Competition in Basic Science: expression of major matrix metalloproteinases is associated with intervertebral disc degradation and resorption.
Eur Spine J 2002, 11:308-320. PubMed Abstract | Publisher Full Text
Séguin C, Pilliar R, Madri J, Kandel R: TNF-alpha induces MMP2 gelatinase activity and MT1-MMP expression in an in vitro model of nucleus pulposus tissue degeneration.
Spine (Phila Pa 1976) 2008, 33:356-365. PubMed Abstract | Publisher Full Text
Hunter C, Matyas J, Duncan N: The notochordal cell in the nucleus pulposus: a review in the context of tissue engineering.
Tissue Eng 2003, 9:667-677. PubMed Abstract | Publisher Full Text
Miyazaki T, Kobayashi S, Takeno K, Meir A, Urban J, Baba H: A phenotypic comparison of proteoglycan production of intervertebral disc cells isolated from rats, rabbits, and bovine tails; which animal model is most suitable to study tissue engineering and biological repair of human disc disorders?
Tissue Eng Part A 2009, 15:3835-3846. PubMed Abstract | Publisher Full Text


text:
With the launch of the SEC Network on Thursday, Aug. 14, at 6 p.m. ET, Gator fans will gain 24/7 coverage of the Southeastern Conference and various Florida Gators' athletic events.
Click here for the original story.
Welcome to SECTalk.com
|Welcome to SECTalk.com -- The Home of 6 Straight National Titles!
You are currently accessing our site as a guest which means you can't access all of our features such as social groups, sports betting, and many more. By joining our free community you will have access to all of these great features as well as to participating in our forums, contacting other members, and much more. Registration only takes a minute and SECTalk.com is absolutely free, so please join today!
If you have any problems registering or signing in, please contact us.
Latest Topics
Latest News
Top Bettors
Top High Fived
Fall Gator Sports Broadcast Schedule on the SEC Network
Started by gatoraus, Aug 13 2014 03:22 PM
Similar Topics
|Started By
|Last Post Info
Bulldogs Fall to #10 Gonzaga 88-76
Started by TheRealBrave, Today, 12:42 AM In: The East → Georgia Sports → Georgia News
- 0 replies
- 88 views
Tigers Fall To No. 2 Wildcats 72-53 In Maui
Started by SECTalk.com, 24 Nov 2014 In: The East → Missouri Sports → Missouri News
- 0 replies
- 39 views
Vols Fall To #19/20 Mizzou, 29-21
Started by SECTalk.com, 22 Nov 2014 In: The East → Tennessee Sports → Tennessee News
- 0 replies
- 50 views
Gamecocks Fall To Charlotte In Charleston Classic Semifinals
Started by GAMECOCK_FAN, 21 Nov 2014 In: The East → South Carolina Sports → South Carolina News
- 0 replies
- 153 views
Gator Baseball Announces Weekly Practice Schedule To Complete Fall Ball
Started by gatoraus, 19 Nov 2014 In: The East → Florida Sports → Florida News
- 0 replies
- 45 views
0 user(s) are reading this topic
0 members, 0 guests, 0 anonymous users


text:
View Entire Collection
By Clinical Topic
By Journal
By Specialty
By Category
By State Requirement
Faith Community Nursing
Future of Nursing Initiative
Welcome NursingDrugGuide.com and NDHnow.com Users
You are being redirected to NursingCenter where you may earn your CE certificates instantly. You must be logged in to take a test on NursingCenter. Membership is FREE and entitles you to save your test to your personal account for future access. Upon clicking on a "Take Test" link, you will be prompted to either join NursingCenter or log in if you are already a member.
Listed below are CEs you can take at discounted prices. Each CE has a coupon code associated with it. Use these coupon codes during check-out to receive your discount! Click here to learn more on how to use the coupon codes.
Take the following CEs at a 50% discount:
For more information about taking CE on NursingCenter, view our Help and Accreditation Information.
To earn CE at discounted prices, use the coupon code listed under the CE title you have taken. During the purchase process, enter the appropriate coupon code in the following section:
For life-long learning and continuing professional development, come to Lippincott's NursingCenter.
Caring for...Patients of different religions
Nursing Made Incredibly Easy!, November/December 2014
Expires: 12/31/2016 CE:2 $21.95
Autoimmune disease: Cost-effective care
Nursing Management, November 2014
Expires: 11/30/2016 CE:1.5 $17.95
CE: Original Research: Staff Nurses' Perceptions Regarding Palliative Care for Hospitalized Older Adults
AJN, American Journal of Nursing, November 2014
Expires: 11/30/2016 CE:2.5 $24.95
More CE Articles
Subscribe to Recommended CE
Dogs as Pets, Visitors, Therapists and Assistants
Home Healthcare Nurse, November/December 2014
Free access will expire on January 5, 2015.
Tracheostomy Care
Nursing2014 Critical Care, November 2014
Free access will expire on December 22, 2014.
Effective management of ARDS
The Nurse Practitioner, 13December 2014
Free access will expire on December 22, 2014.
More Recommended Articles
Subscribe to Recommended Articles
Back to Top


text:
I lead Delta 14 on gmod.
I love modding. I like Gmod. I do a lot of fun stuff.
We're sorry, but no images, videos or audio files have been added to this gallery.
If you would like to share media with the community, sign up and you can.
No one is friends with lookovertheir awww. You should invite your mates to the site, or make new ones.


text:
State Seeks To Prevent Falls By Older Ohioans
A state initiative to prevent falls by older Ohioans is aiming to warn shoppers of all ages what to look for and how to avoid falling hazards amid holiday shopping.
A judge has granted a request from three AIDS patients to block changes to Ohio rules that they say would drastically reduce eligibility for a free or reduced-medication program.
More court hearing are scheduled in two weeks, but if upheld, the directive from the Franklin County Common Pleas court blocks the state from implementing new rules.
The group argues the new rules were not properly adopted and will "arbitrarily deny funding for potentially life-saving medications" to Ohioans with HIV or AIDS-related symptoms. They say the change will drastically reduce eligibility for Ohio's AIDS Drug Assistance Program for the low-income, uninsured and under-insured.
The rules were set to take effect today.
Click here for hear Mandie Trimble's reporting on how many Ohioans have struggled to get HIV and AIDS medications.


text:
Most Popular in:
New in Treatments (page 131 of 152)
AAD Issues Statment Urging Public to Be Sun-conscious
With warmer weather quickly approaching, the American Academy of Dermatology has released a statement encouraging people to be smart about their sun exposure in regard to skin health.
Sunscreen Smarts
By Laura J. Goodman
New research and government rulings are making it more important than ever to stay up to date on sun care.
Spa, MD: Client Safety First
By Elizabeth Roche, MD, with sidebar by Louis Silb…
With the rise in laser treatments being offered in medical spas, it's important to know how to provide security for your clients and your business.
Viewpoint: Anti-inflammation and Visible Skin Aging
By Carl Thornfeldt, MD
The fountain of youth may be discovered through reducing inflammation--both internally and externally.
The Oat Approach
By Cathy Christensen
Ingrain oats into your spa meny for delicious results.
The Convenience of Technology Tools
By David Suzuki
Recent advancements in the range of technological skin care offerings can give spas a leg up on the competition.
International Trends: Loving Lemongrass
By Sarah Kajonborrirak
Sweetly scented and full of healing properties, this plant is a spa's dream.
Music Becoming Popular in Medical Treatments
Music is coming to play a larger role in the treatment of a range of illnesses, indicating a growing interest in alternative wellness therapies.
Facelift Patients Face Possible Risk of MRSA Infections
A recently released study has found the antibiotic-resistant staph infection MRSA in patients who have undergone facelifts.
Creams May Have Longer-Term Moisturizing Benefits Than Serums
Researchers in Belgium have found creams may have more long-term moisturizing benefits, while serums can be more effective in the short term.


text:
Video: The link between abuse and suicide Add to ...
Featuring video from CTVNews.ca
Last updated
Scientists in Montreal are finding that children who were physically or mentally abused in childhood may have higher odds of committing suicide later in life
Watch More Video:
When I was 7 years old, my uncle sexually assaulted me: Actress Teri Hatcher
Shoppers be warned: 'Tis the season for vehicle break-ins
How to turn your leftovers into a sumptuous feast
Turkey Day in America now includes the gym for most people
This Fort McMurray cocktail proves that beer can be a dessert
Tour this bright St. Lawrence Market condo that went just under asking
Muhammad Ali's daughters offer insight on boxer in new documentary
Keep your back happy and your hamstrings healthy with these yoga moves
Are Black Friday cross-border deals a big con?
Calgary's last video rental store still open for business
World's biggest Christmas tree lights up in Germany
Ohio turkeys selected for Presidential pardon
How to maintain your heart health this holiday season
Amazing video of cheeky Saskatoon parrot goes viral
Taylor Swift honoured at American Music Awards
Audience shows love for Cosby in Florida
Love pandas? Plan a trip to the San Diego Zoo
Chef Susur Lee shares the secrets of his signature dish, the Singaporean Slaw
How to prune your garden like a professional
Got to get rid of an obnoxiously big plant or tree? Tip No. 1: Don't cry about it


text:
Click here to learn more.
Call it CSI — for animals. The
New Jersey Society for the Prevention of Cruelty to Animals' newest hire is
forensic veterinarian Dr. Ernie Rogers, DVM, Ph.D., an expert in investigating animal abuse and solving crimes against dogs,
cats and the occasional alligator.
He's also the first employee with that title on the agency's payroll.
At a time when few industries are gaining jobs, animal welfare officials say that veterinary forensics and animal crime scene investigators are bright spots in the field. The SPCA credits the upswing to shows like Animal Planet's Animal Cops, as well as heightened media coverage of abuse cases.
As he joins the agency's law enforcement division, we chatted with Dr. Rogers about what it means to be a forensic veterinarian — and the crimes against animal-kind he helps solve on a day-to-day basis.
A: Dr. Ernie Rogers: "I went to medical school and got my degree in veterinary medicine, and then I got a Ph.D. in pharmacology and toxicology. I'd always wanted to work with the police force, so I started by working with police dogs.
Part of what you need is a scientist who can view the crimes from the point of view of an animal: Is it poisoned? What was it poisoned with? How long has it been dead? How long has it been decomposed? That's normally what a medical examiner does, but that's what I do."
A: "People are becoming more and more aware of animal crime and how it affects their lives. A lot of animals that have been stolen out of yards have been used as baiting animals in dog fighting, for example. There's also 'trunking,' which is when they put two dogs in the trunk of a car and drive around. Then they open the trunk and the last dog living is the 'winner.' "
A: "I have a full-time veterinary practice, but when there is an incident, the police department will get in touch with me and say, 'Doc, I have this case. Do you think this is abuse?'
For example, there was a horse left out in a snowy field without any water, and the owner said, 'It doesn't matter; he can eat snow.' But when I did the calculations, I realized that he would have to eat 250 feet of snow every day because snow is a lot less nutritious than water."
A: "We had a guy who stabbed his girlfriend's cat to get back at her. People who do these things aren't far behind doing other bad things. If you're willing to take life from an animal, what else are you willing to do?
I had a case many years ago where an animal was shot in the head and it died instantly. I got the bullet, and when the officer took it to his lab, the forensic evidence tied the gun and bullets to two other human murders. I also have a case involving alligators and dogs. It's an ongoing investigation, so I can't say more, but that's the most unusual animal I've had the opportunity to deal with."
A: Well, I think there's been a thing put out there that's a bit of a misnomer. Most vets put themselves in places where they want to help people, whether that's law enforcement or healing a sick animal. I just happen to have a badge that helps me get into crime scenes."
A: "It's good in some ways, and it's bad in some ways. I know my partner will get called into a situation because one neighbor doesn't like the other, so they'll call the animal cops to get him. In the other sense, some people are more willing to go out on a limb now and say, 'I saw my neighbor kicking his dog. He can't walk, and he's so emaciated.' "
A: "The NJ SPCA doesn't receive tax dollars, so we appreciate anyone who can help us by raising funds. There is a 2012 calendar — and I'm actually in it. I have my clothes on though."
Like this article? Have a point of view to share? Let us know!
Thank you for subscribing to Petwire. Look for the latest newsletter each Wednesday.
Ebola survivor Nina Pham celebrated
Bentley the Cavalier King Charles
Spaniel's second birthday Thursday.
"Polar vortex" weather conditions might
but be taking over the country, but these
winter-loving breeds…
The animal lovers in your life will surely
appreciate calendars, jewelry, chocolate
and more that benefit animal…
A tiny hamster and his tiny dinner guests
enjoy a tiny Thanksgiving dinner while
wearing tiny pilgrim hats.
In the days between Thanksgiving and
New Year's, your dog or cat will probably
sneak his fair share of leftovers.
From deliciously rich holiday fare to
enticingly shiny decorations, the season
brings plenty of risky temptations…
The playful Shih Tzu will love to be your lap dog and prized canine companion, even if you're not a Chinese emperor.
If the video doesn't start playing momentarily,
please install the latest version of Flash.
Thank you for subscribing.


text:
Gary Blair
Sunday June 30
Community Music Center
3350 SE Francis St.
3350 SE Francis St.
Where: Community Music Center
Phone: 823-3177
Address: 3350 SE Francis St.
Website: communitymusiccenter.org
Good luck getting a ticket—both shows have been sold out for ages—but maybe you can sit at home and smoke a bowl while your friend Skypes you in. Abbi Jacobson and Ilana Glazer's Comedy Centr ...
[JAZZ FLUTE] While male saxophonists and trumpeters rule the bandstands and CDs, flutists are much rarer in jazz, and so are women. New York's Jamie Baum is both, ...


text:
Adult faith formation series on prayer, given at Corpus Christi Parish. 11/18
The last petitions of the Lord's prayer -- from "Give us this day our daily bread" to the end.
Saturday, November 22, 2014
Adult Formation Series on Prayer, 11/18 - The Last Petitions of the Lord's Prayer
Daily Sermons, November 18-22
Daily Mass Homilies at Corpus Christi Parish, Great Falls, MT
The Dedication of the Basilicas of Sts Peter and Paul, St Elizabeth of Hungary, the Presentation of Mary, St Cecilia
The Dedication of the Basilicas of Sts Peter and Paul, St Elizabeth of Hungary, the Presentation of Mary, St Cecilia
Thursday, November 20, 2014
Did you know that Mary was raised in the Temple and made a vow of virginity?
November 21st, The Presentation of the Blessed Virgin Mary in the Temple
"Congratulate me, all ye that love the Lord, because when I was a little one I pleased the Most High." (from the Common Office of our Lady)
Though it is related nowhere in the sacred books, ancient tradition tells us of the presentation of the Blessed Virgin Mary in the Temple when she had been weaned from her mother at the age of three. This presentation is quite diverse from that of our Lord in his fortieth day – for Blessed Mary was given over to the Temple for her rearing and education, as Joachim and Ann returned to Nazareth, she remained in Jerusalem until her fourteenth or fifteenth year.
It was during these twelve years of dedicated service in the Temple that our Lady made her vow of virginity, which God then protected through the ministry of St. Joseph.
Let us consider the outline of the history of the presentation of Mary in the Temple, and then further the vow of Blessed Mary to remain a virgin.
Posted by Father Ryan Erlenbush 18 comments
Labels: Mary, Sacred Scripture
Tuesday, November 18, 2014
Sunday Sermon, 11/16 - Damned if you do not pray, saved if you do pray
Listen online [here]!
Sunday Sermon, November 9 -- Why Catholics venerate relics, and How to become a saint
Listen online [here]!
Advent mini-retreat, November 15 -- From meditation to contemplation
These sermons were given as part of a mini-retreat for the perpetual adoration society of Corpus Christi Parish and Great Falls, MT.
Friday, November 14, 2014
The one talent, prayer, gains all the rest
33rd Sunday in Ordinary Time
Matthew 25:14-30
To one servant he gave five talents; to another, two; to a third, one.
In the parable of the talents, our Savior teaches that of each an accounting will be required for whatever gifts God has bestowed. From the one who has received much, much is expected; from him who has received more, still more is expected.
But what shall we say of the servant who has received but one talent? We seek to discern what this talent symbolizes, so that we may know how to make it fruitful in our own lives.
Following that great Doctor of the Church, St. Alphonsus, we will consider how the parable of the talents instructs us of the necessity of prayer as the great means of our salvation.
Posted by Father Ryan Erlenbush 2 comments
Subscribe to: Posts (Atom)


text:
The opposite of Badong also used as the oppsite to the act of killing as killing is bad and wrong, its so bad there should be a new word for it like badong. killing is badong! and gnodab is the oppsite of killing.
To give life - Gnodab
To give life - Gnodab
From now on i will stand for the opposite of Badong...... Gnodab!
Garrick tərəfindən 24 May 2004
the opposite of badong or bad-wrong. Hence why gnodab is just badong spelled backwards.
that led zepplica concert was awesome! Some would say it was Gnodab! Douche Canoe's would say it was Badong.
Daniel the tiger of Ra tərəfindən 07 Yanvar 2008
The opposite of Badong.
killing is badong i will now stand for the opposite of badong gnodab
DA SHIZNIT tərəfindən 18 May 2004
something that is the opposite of "bad" and "wrong", or Badong.
"I will stand for the opposite of Badong... gno... Gnodab!"
Chainsaw Dudley tərəfindən 31 Mart 2003
the opposite of bad; good
The opposite of killing is GNODAB!
Wuddup gnodab buddy?
Wuddup gnodab buddy?
MIMI and KIKI tərəfindən 05 Sentyabr 2003


text:
Dow Jones Global Indexes | Global Stock Markets
If European equities are so cheap, why is it so difficult to find stocks to buy?
It's a conundrum that has been puzzling investors. At the risk of sounding like a broken record, Europe looks relatively appealing on a variety of measures: The Stoxx Europe 600 trades at about 12.4 times forecast 2013 earnings, compared with 13.7 times for the S&P 500, and corporate earnings are expected to grow 11% in Europe this year versus 10% in the U.S.
Logic would suggest that there should be plenty of buying opportunities in European stocks, but that doesn't seem to be the case. Far from it.
There are plenty of European companies with single-digit price-to-earnings ratios, generous dividend yields, and low levels of debt. But picky investors are looking for something extra. "The difficulty is finding a combination of qualities, including good growth prospects, that makes them attractive," says John Chisholm, chief investment officer at Acadian Asset Management in Boston.
BLEMISHES ARE INDEED abundant. Researchers at Société Générale regularly screen stocks against popular investment models, and they have found that the number of shares that meet the various criteria has dropped sharply in the past 12 months. For example, the number of stocks worldwide that pass the Graham and Rea value screen– whose criteria include earnings growth, dividends, and debt targets–has fallen from 145 in May last year to 58. So, there are fewer easy pickings.
One big problem: Equity prices over the past few months have outpaced fundamentals, argues Andrew Lapthorne, head of quantitative equity research at Société Générale in London, adding that investors now are "getting less bang for their buck."
Among the different models that Société Générale tests, the most successful at the moment is Greenblatt's Magic Formula, which has returned more than 20% in each of the past few years on global, European, and euro-zone bases. It ranks stocks from cheap to expensive and, separately, from high- to low-quality. The two rankings are then added together and reranked. The strategy recommends buying the 30 top-ranked equities.
European names screened include gaming operator OPAP (ticker: OPAP.Greece), luxury and sporting goods company PPR (PP.France) and Telecom Italia TIT.MI -1.1963023382272975% Telecom Italia S.p.A. Italy: Milan EUR0.9085 -0.011 -1.1963023382272975% /Date(1416845298000-0600)/ Volume (Delayed 15m) : 18827261 P/E Ratio 15.141666666666667 Market Cap 16674128621.7317 Dividend Yield 2.201430930104568% Rev. per Employee 289380 More quote details and news » (TI). They all have their imperfections.
OPAP is a reasonable bet, but Greek equities aren't for the timid, given the country's problems. OPAP is hamstrung by the proposed sale of a 33% stake by the government and the implementation of a new 30% gaming tax that will be mitigated only marginally by a new video-lottery business. Earnings this year are expected to drop to 48 European cents per share from €1.58 ($2.05) in 2012. The monopoly generates gobs of cash and can offer steady returns, but its dividend of about 9% on 2012 earnings won't be repeated. OPAP shares closed Friday at €6.76 and trade at about 14 times forecast 2013 earnings.
At Gucci brand owner PPR, a bright outlook for luxury goods is offset by disappointing guidance from sport-lifestyle unit Puma pum.xe 0.2145347278090641% Puma SE Germany: Xetra EUR186.85 0.4 0.2145347278090641% /Date(1416845299000-0600)/ Volume (Delayed 15m) : 758 P/E Ratio N/A Market Cap 2812125342.00908 Dividend Yield 0.2675943270002676% Rev. per Employee 265853 More quote details and news » (PUM.Germany). PPR's shares, which closed Friday at €177.55, trade at 14.3 times forecast 2014 earnings of €12.34. They've climbed almost 40% in the past 12 months and will struggle to go higher without top-line growth at Puma.
Telecom Italia's stock also is on hold, due to concerns that capital expenditures will hurt earnings. The stock closed Friday at $7.70, or five times expected 2014 net of $1.55 a share.
Lapthorne calls Greenblatt's Magic Formula "a mix of cheapness and possibilities," and prefers a quality and income model, whose characteristics include above-average balance-sheet strength and dividend yields. A quality-and-income screen throws up some unexciting European names like British American Tobacco BTI 0.16358157554885924% British American Tobacco PLC ADS U.S.: NYSE MKT USD116.34 0.19 0.16358157554885924% /Date(1416607324041-0600)/ Volume (Delayed 15m) : 334167 AFTER HOURS USD116.25 -0.0900000000000034 -0.07735946364105209% Volume (Delayed 15m) : 1493 P/E Ratio 18.555238839545563 Market Cap 108247334052.023 Dividend Yield 2.6479800584493725% Rev. per Employee 263636 More quote details and news » (BTI), energy supplier Centrica CNA.LN -0.9897260273972602% Centrica PLC U.K.: London GBP289.11 -2.89 -0.9897260273972602% /Date(1416841686000-0600)/ Volume (Delayed 15m) : 1777765 P/E Ratio 0.2198857924214128 Market Cap 14513104099.1993 Dividend Yield 3.528068901110304% Rev. per Employee 775523 More quote details and news » (CPYYY) and fashion retailer Hennes & Mauritz HM-B.SK 0.2245749117741418% H&M Hennes & Mauritz AB Series B Sweden: Stockholm SEK312.4 0.7 0.2245749117741418% /Date(1416845293000-0600)/ Volume (Delayed 15m) : 296524 P/E Ratio 26.700854700854702 Market Cap 515885962603.516 Dividend Yield 3.0409731113956466% Rev. per Employee 1791270 More quote details and news » (HM-B.Sweden).
THE AMERICAN DEPOSITORY RECEIPTS of BAT and Centrica have turned in steady—if unspectacular—performances over the past 12 months, gaining 6.8% and 8.6%, respectively. BAT, which closed Friday at $106.77, trades at 13.9 times forecast 2014 earnings and yields 3.9%. Centrica, which ended the week at $21.13, fetches 11.5 times forecast 2014 earnings and yields 4.5%.
H&M is less appealing because it faces higher costs to fund expansion in the face of tough competition. Its shares have been flat over the past 12 months, although they offer a 4% dividend yield. The stock closed Friday at 237.40 Swedish kronor ($37.08), or a pricey 19.3 times expected 2014 profit.
Bouncing Back
Spanish stocks jumped 5.4% as all Europe's major exchanges gained.
E-mail: email@example.com


text:
I've got the WE Diablo Rojo kit and they say it's ready to drink at 4 weeks. I'm entering the final stages. I'm adding the last of the packets it came with soon and here's my question. With these cheaper kits will I add a noticeable improvement by racking the wine and letting it bulk age in the carboy? How long should I let it age and should I cover the carboy to protect it from light?
Answers are a strong YES. If you can bulk age definitely do so. Even bottle aging has an impact. You will note changes even a month from now. But in a year that kit wine should be much improved. Try to hold out for 90 days, six months.
Motto: quel che sara sara
Yeah you can bulk age all of the kit wines. They're great right away but they definitely improve. P.S. Saramc, sorry I'm not following you around, I just happen to be going to a lot of these same posts lol.
My experience with those 4 week wine kits is that they don't improve with bulk aging. They are not at all complex, and are drinkable shortly after bottling. Aging didn't hurt it, but it definitely didn't improve it.
It's also a little of a common misconception, or so it seems. It's not really ready to drink at 4 weeks, as much as it could be ready to bottle at 4 weeks. Bottle it at 4 weeks or so, and let it bottle age for a couple of months.


text:
Denis Guibard will take up the reins at Télécom Ecole de Management (TEM) as of December 1, 2014.
Northern Illinois University, new international partner as part of exchange program.
This Recruitment Forum is intended for management and engineering students who want to do an internship or find a job
S. Slaughter, a well-known Professor presented a Research Seminar
This story is about Pierre one of the students of Telecom EM following an internship at WappZapp an Amsterdam based startup.
Ranked 25th spot worldwide Télécom Ecole de Management gains 9 places in the international Financial Times survey released today