text:
DonorDrive is made by fundraisers for fundraisers. Today we power the world's leading NGOs and have helped raise over a billion dollars for worthy causes.
It's flexible enough to handle Event, DIY and every type of online fundraising. DonorDrive's scalable platform easily supports your fundraising needs as you grow, no matter how large you get. Its innovative features help our clients see an average online revenue increase of 41%.
In these days when nonprofits are trying to eliminate the zero-dollar fundraiser, it's surprising how organizations are reluctant to require peer-to-peer fundraising minimums for their events. While most cycling events and marathons require fundraising minimums, some into the thousands of dollars, we're seeing more and more organizations that fundraise with DonorDrive successfully employing fundraising minimums at lesser amounts on walks as well. We spoke with Kim Kraus, in charge of Special Events at Children's Hospital at Dartmouth-Hitchcock about their CHaD HERO event that uses DonorDrive's Required Fundraising feature. The CHaD HERO is a 5K walk, 10K hike, 5K run, half marathon, family fun run and day-long community festival. The CHaD HERO has had a minimum fundraising requirement for several years. Here's how their minimums work: "Our current registration fee and fundraising minimum structure is:
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©1997-2016 Global Cloud® All Rights Reserved. DonorDrive® and Global Cloud® are registered trademarks of Global Cloud, Ltd. All other trademarks are registered marks of their respective owners.


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What's on at Tyntesfield
House Tickets - 1 July - 31 July
In 2016 the house at Tyntesfield will be open in some way, everyday (except Christmas day).
Thu 28 Jul 2016
+ 3 other dates
+ other times
Kitchen garden and glasshouse tours
Join us on a kitchen garden or glasshouse tour - Running daily throughout the Spring and Summer. For tour times, please ask a member of staff in the Ticket Office to contact a Estate Guide on the day of your visit
Thu 28 Jul 2016
+ 95 other dates
Passions & Possessions: The Material World of Antony Gibbs
Our new exhibition throws light on the Passions and Possessions of Tyntesfield's Victorian owner for the first time.
Thu 28 Jul 2016
+ 93 other dates
Feel Good Friday: Tyntesfield run
Blow the cobwebs away with a morning run round the Tyntesfield estate, enjoying spectacular scenery along the way. Open to all abilities - no one will be left behind. Led by members of the Nailsea Running Club. Price includes a cup of tea or...
Fri 29 Jul 2016
+ 10 other dates
Much ado about nothing with The Lord Chamberlain's Men
Join The Lord Chamberlain's Men this summer for Shakespeare's sparkling, battle-of-the-sexes comedy, Much Ado About Nothing.
Sat 30 Jul 2016
House Tickets: 1 August - 31 August
In 2016 the house at Tyntesfield will be open in some way, everyday (except Christmas day).
Mon 01 Aug 2016
+ 30 other dates
+ other times
The Stableyard: Antony's Legacy
Join volunteer Ken in the stables to explore Antony Gibbs' architectural and technological creation and get a glimpse into the Victorian world and our on-going care of Tyntesfield. Tours on request.
Wed 03 Aug 2016
+ 21 other dates
Summer Forest School Day Camps
Join our Forest School leader for an exciting woodland session, including cooking on an open fire, den building and wild play!
Wed 03 Aug 2016
+ 1 other date
Make a green man plaque 2016
Create your own green man or woodland spirit face, under the expert guidance of the North Somerset Bodgers.
Sat 06 Aug 2016
Food and craft market - 2016
Come along and stroll around Home Farm, enjoy the atmosphere and pick up some seasonal treats. From freshly baked bread and fine cheeses to hand made jewelry and ceramics you're sure to find something to tempt you!
Sun 07 Aug 2016
+ 4 other dates
Tyntesfield Lecture: Tyntesfield at War with David Hogg
David Hogg will introduce Michael Boyce, who wrote the book about Tyntesfield in World War II and he hopes to bring the colour film on the American Hospital for us to see.
Fri 12 Aug 2016
WW1 Commemoration Week
2015 marked the 100th anniversary of the death of Antony Gibbs' son, Eustace Lyle, who died while fighting near Ypres. Commemorate all who lost their lives in the Great War and discover life on the front line with the Tyntesfield WW1 weekend
Sat 13 Aug 2016
+ 8 other dates
TYNTEtots: Owl and the Pussy Cat
Join us for story-telling and games for under fives and their families.
Wed 17 Aug 2016
+ 1 other date
Bat walks 2016
Our Warden Darren will show you the bat haunts on the Tyntesfield estate by torchlight, don't forget yours!
Wed 17 Aug 2016
Boundary walk with our Estate Interpreters
Explore the wider Tyntesfield estate by following the perimeter wall around the 540 acre estate.
Sat 20 Aug 2016
+ 4 other dates
Live from the Lawn 2016
Back for its third year, Tyntesfield's very own mini music daytime festival will be showcasing amazing Bristol based performers and artists from further afield. Expect to find a main stage, children's workshops and intimate sessions in beautiful...
Sat 27 Aug 2016
+ 2 other dates
The Beat: Live from the Lawn at Night
One of the biggest bands of the 2-Tone movement, The Beat present a wall of sound that transcends time and ska fusion rhythms to ignite a crowd.
Sat 27 Aug 2016
Trust 10
Run 10k for free with the National Trust around the Gibbs family estate at Tyntesfield, either as part of a challenge, a training programme or just a run. Free to take part in the run. Normal garden admission applies from 10am.
Sun 28 Aug 2016
+ 4 other dates
Show of Hands: Live from the Lawn at night
Show of Hands return with Walk with Me - The Big Autumn Tour with special guest Megan Henwood.
Sun 28 Aug 2016
House Tickets: 1 September - 30 September
In 2016 the house at Tyntesfield will be open in some way, everyday (except Christmas day).
Thu 01 Sep 2016
+ 29 other dates
+ other times
House tours: Antony's Legacy
Come with us on a tour of some of the rooms on the second floor and discover what Antony and his descendants left behind. You'll go behind the scenes to see untouched rooms and the places we look after and store Antony's legacy. Tickets...
Thu 01 Sep 2016
+ 29 other dates
+ other times
Peter Pan with the Chapterhouse Theatre Company
Chapterhouse Theatre Company present the unforgettable story of Peter Pan, the boy who wouldn't grow up.
Sat 03 Sep 2016
Tyntesfield Lecture: Antony Gibbs and Sons, the family firm with David Hogg
This will tell the story of how a merchant in Exeter founded a firm of merchant bankers which went on to be one of the four largest merchant and investment houses in the City of London by 1914.
Fri 09 Sep 2016
Stool or coffee table making 2016
Cleave some ash legs, turn them on a pole lathe and then put them all together. Using traditional woodworking tools you'll learn all the basic skills you'll need to progress on to a bigger project in your own shed!
Sat 17 Sep 2016
+ 1 other date
Corde di Gioia 'Strings of Joy' concert in the House
From Bath Abbey to Petersburg, Danish trio Strings of Joy have played to the world, and will now be taking time out of their world tour to perform at Tyntesfield.
Sat 17 Sep 2016
TYNTEtots: The Elves and The Shoemaker
Join us for story-telling and games for under fives and their families.
Wed 21 Sep 2016
+ 1 other date
Tyntesfield Lecture: The Gibbs' and the Age of Guano, Part One with Lesley Kinsley
Lesley will examine how scientific enlightenment and exploration enabled Antony Gibbs and Sons to enter the guano trade and begin the period that became known as 'the Guano Age'. Moreover, she will look at company history and the networks in...
Fri 23 Sep 2016
Evening Lecture Series: 'Two great Gothic houses: Tyntesfield & Knightshayes' with Stephen Ponder
To coincide with the launch of 'Passions and Possessions' our Antony Gibbs exhibition, Curator in the National Trust's South West Region Stephen Ponder discusses 'Two great Gothic houses: Tyntesfield & Knightshayes'.
Sat 24 Sep 2016
Tyntesfield Lecture: The Gibbs' and the Age of Guano, Part Two with Lesley Kinsley
Following the introductory lecture, this talk focusses on Victorian Britain. Lesley will examine how Peruvian guano featured in the experiments of newly emerging agricultural chemists and was used by Victorian high farmers. Guano even influenced...
Fri 30 Sep 2016
Made in North Somerset festival showcase 2016
Marking the end of the Made in North Somerset festival, Tyntesfield will be hosting a large market with local producers, handmade crafts and delicious delights. Entertainment will be on hand, as well as special tours and children's activities,...
Sat 01 Oct 2016
+ 1 other date
House Tickets: 1 October - 31 October
In 2016 the house at Tyntesfield will be open in some way, everyday (except Christmas day).
Sat 01 Oct 2016
+ 30 other dates
+ other times
Make a chair with Peter Murray aka The Artful Bodger
Make your very own chair from scratch with master Craftsman Peter Murray.
Mon 03 Oct 2016
+ 5 other dates
Champion Tree Walk
Enjoy exploring a range of our Champion trees with Tyntesfield's dendrologist and tree registrar, Chris Watts.
Sun 16 Oct 2016
Introduction to green woodworking 2016
Learn the basics of green woodworking under the expert guidance of the North Somerset Bodgers. You'll cleave and turn some items to take home with you using timber from the Tyntesfield estate.
Sun 16 Oct 2016
TYNTEtots: Hansel and Gretel
Join us for story-telling and games for under fives and their families.
Wed 19 Oct 2016
+ 1 other date
Half-term Hallowe'en fun
It's Hallowe'en half term at Tyntesfield and that can only mean one thing; fearsome fun for all! There'll be a spooky Hallowe'en trail, eerie arts & crafts, a torchlight tour and pumpkin carving. Fearsome fancy dress is a must all week. Trail...
Sat 22 Oct 2016
+ 8 other dates
Pumpkin carving workshop
Come and have a go at carving your own pumpkin!
Sat 22 Oct 2016
+ 4 other dates
+ other times
Lantern making workshop
Tyntesfield House was one of the first properties in England to have electricity installed - to celebrate this, we'll be making paper lanterns on every other day of the Halloween half term at Home Farm. Suggested age range: 4-94... everyone can...
Sun 23 Oct 2016
+ 3 other dates
Tyntesfield Fungi Forays
Discover the wonderful world of fungi at Tyntesfield with our Local Fungi experts. Drop in to see finds from their annual fungus audit by booking on to one of our forays to become an expert yourself. Sorry, no dogs on the forays.
Sat 29 Oct 2016
+ other times
Hallowe'en Murder Mystery
Travel back in time with the critically acclaimed Pantaloons Theatre Co for a hauntingly hilarious experience. Meet some eerie Tyntesfield characters and put your sleuthing skills to the test to solve an immersive murder mystery. Includes an...
Sat 29 Oct 2016
House Tickets: 1 November - 30 November
In 2016 the house at Tyntesfield will be open in some way, everyday (except Christmas day).
Tue 01 Nov 2016
+ 29 other dates
+ other times
Christmas house opening
Pop over to Tyntesfield this Christmas to interact with Victorian characters, help make Christmas decorations, sing in the hall, and take in the fantastic festive atmosphere across the estate.
Sat 26 Nov 2016
+ 38 other dates

subset: commoncrawl


text:
Preventing a SPOF with CMAN 12c
I have already written about the use of Connection Manager 11.2 to govern access to a database. While researching this piece I have updated the original post so that it's relevant for 12c as well.
Although the idea of using Connection Manager is tempting, the obvious first question is about high availability of the process. After all, if the gatekeeper to your database environment fails, you are effectively closed for business. One option would (more...)


text:
Award-winning Texas distillery Balcones Distilling plans to open a location in the Texas Fireproof Storage building in Waco. The building dates to 1923. The new facility, expected to open in 2015, will allow the distillery to add more equipment and experiment with new products. "I believe this distillery will put Balcones on the map and will put Waco on the map," said Chip Tate, the company's president and head distiller.
Full Story:Waco Tribune-Herald (Texas)


text:
Demonstrates the features and tools of this spreadsheet application, including building great charts and creating effective formulas.
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More options
"lynda.com has made it possible and affordable to retool and get back to work after raising my kids. I can't imagine working without this resource now." —Maureen C.
more from our members »


text:
I was viewing the site that I created using wordpress on my Mac & iPhone, and views fine if not perfect, and then I decided to view from an iPad, it's totally different site. I was wondering if it was possible for me to edit the footer of the mobile theme, hide date, author, and category of the mobile theme (Jetpack mobile theme).
Please give me detailed instructions, and I'm still learning my way around wordpress.
My site URL is edreporting.imbekotours.co.za


text:
Law Office Lillo & Partnerid grew up from Law Office Ehasoo & Benevolenskaja, which was established in 1992 in Tallinn. Law Office Lillo & Partnerid was established in 2009 as a result of division of Law Office Ehasoo Benevolenskaja Lillo.
Law Office Lillo & Partnerid is modern and innovative law office, where are working acknowledged professionals. Our aim is to satisfy our clients through high-quality legal service. Our lawyers are specialized to all law branches.
Detail data of our lawyers will be found on our website.
Our clients are mostly companies, but also state and local authorities and private persons. Our lawyers can also serve foreign clients. Our working languages are Estonian, English, German, Russian and Finnish.
Law Office Lillo & Partnerid
in Tallinn: Pärnu mnt 10, Tallinn 10148, Estonia Phone: +372 6 444 900, +372 6 449 530 Fax: +372 6 313 909 E-Mail: firstname.lastname@example.com
in Tartu: Raatuse 83, Tartu 50604 Phone/Fax +372 7 444 900 E-Mail: firstname.lastname@example.com
Registry No.: 11643617, Account No. 221045633635 Swedbank, 10220103788015 SEB, www.advokaat.ee
© 2010 Law Office Lillo & Partnerid All Rights Reserved.


text:
Hotels near Bukit Bintang Station
Search hotels near Bukit Bintang Station in Kuala Lumpur
Last minute Kuala Lumpur deals
- Ceria Hotel Bukit Bintangfrom $30$18
- Capri by Fraser Kuala Lumpurfrom $92$69
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- Royce Hotelfrom $81$24
Trending now in Kuala Lumpur
- Most bookedPARKROYAL Kuala Lumpur
386 people looked at this hotel in the last hour
Most recent review
"Check in to hotel at 2am. The power point tripped. Called and they came up pretty fast to fix it. Double lock our room door and connecting door did not come with stopper. Ask them to break open the door with a fee. With great help from samatha, the fee was waive even without us asking for it."
A Traveller, Jun 2016, SG
373 people looked at this hotel in the last hour
Most recent review
"The Wolo hotel is smack in the middle of all the action at Bukit Bintang. On the ground floor is a beautiful French style bakery offering pastries that are simply sumptuous! Check in was smooth and fast. Helped with my bags as well - more efficiently than some of the big 5 star outfits. Ro..."
A Traveller, Jun 2016, AU
136 people looked at this hotel in the last hour
Most recent review
"Strategiclly located opp Sungei Wang shopping centre and opp bukit Bintang metro station. The housekeeping staff is very good and the bathroom is very big. Overall, will come and stay here again next time."
A Traveller, Jun 2016, SG
47 people looked at this hotel in the last hour
Most recent review
"Very nice hotel, I went to this hotel for 5 days with family, it was like a second house ,very comfortable with all the facilities, you don't have to bring in any thing, breakfast was very good and the staff in the kitchen was very cooperative,front desk staff was also very helpful and understand..."
A Traveller, Dec 2015, SG
39 people looked at this hotel in the last hour
Most recent review
"Very small room, not fit for big size people. Not a perfect clean room if we compare with a 5 star hotel. Room looks gloomy but for a price like this at such a convenient place, i can't expect much."
A Traveller, May 2016, AS
16 people looked at this hotel in the last hour
Most recent review
"Good location. Very friendly and helpful hotel owner."
A Traveller, Apr 2016, SG
11 people looked at this hotel in the last hour
9 people looked at this hotel in the last hour
Most recent review
"The hotel has good facilities, brilliant location, good customer service."
A Traveller, Mar 2016, AS
Most recent review
"it's nothing amazing, a good cheap hostel for backpackers, staff are friendly and helpful they are always happy to help"
A Traveller, Feb 2016, GB
- OYO Rooms Jalan Sultan Ismail ExtensionNo Guest Reviews yet
Map of hotels near Bukit Bintang Station
- Petronas Twin Towers
- Berjaya Times Square
- Suria KLCC Shopping Centre
- Sunway Lagoon Theme Park
- Aquaria KLCC
- Kuala Lumpur City Centre
- Pavilion Kuala Lumpur
- University of Malaya
- Mid Valley Mega Mall
- KLCC Park
- Sunway Pyramid Mall
- Kuala Lumpur Convention Centre
- 1 Utama
- Petaling Street
- Merdeka Stadium
- Putra University-Malaysia
- Sepang International Circuit
- Royal Thai Embassy
- Kelana Jaya Lake Park
- Kuala Lumpur Tower
Hotels in Kuala Lumpur near Bukit Bintang Station
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If you wish to see the hotels with the highest featuring discounts and deals near Bukit Bintang Station, simply filter by price/ average nightly rate. We recommend you filter by star rating and read our genuine guest reviews so you can get the best quality hotel with the best discount.
One of the new features on Hotels.com guest reviews is that also show reviews from Expedia for Bukit Bintang Station hotels and the TripAdvisor Bukit Bintang Station hotels reviews so you can make sure that you checking with a reliable source. See the review scores on our Kuala Lumpur hotel information pages.
Make the most out of your family vacation when you book your accommodation with Hotels.com – book your hotel near Bukit Bintang Station, Kuala Lumpur after reviewing the facilities and amenities listed for each hotel.
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Kuala Lumpur travel guides
Kuala Lumpur – where to stay and the areas explained
When it comes to accommodation in Kuala Lumpur, five star hotels in gleaming tall metal and glass st...
Where to shop in Kuala Lumpur – a guide to the different areas
When it comes to shopping, Kuala Lumpur isn't for the faint hearted. Malls are beyond huge with a va...
Where to eat in Kuala Lumpur – a guide of what to eat and where to go
The melting pot of Kuala Lumpur has many influences when it comes to food. The city is made up of Ma...


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We spoke to her in order to know more and she said, "Yes, I have injured my wrist. I cannot even move my hand as it is swollen. I am lucky enough that I have an off today so I can take rest."
Krishma Solanki.
Monday, March 18, 2013
2 Comments | By Krishma Solanki
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I want to put a specific border like below around my element. How can I do this with css.
I use this css for showing a border around an element:
.ui-selected {
-moz-box-shadow: 0 0 1px 1px black;
-webkit-box-shadow: 0 0 1px 1px black;
box-shadow: 0 0 1px 1px black;
But I want to show border like in the image. Is this possible?
I want to put those eight square around an element.
I use $('#element').addClass('ui-selected') to add and $('#element').removeClass('ui-selected') to remove.
I want css classes, is it possible


text:
Top Apps
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Cantera is a suite of object-oriented software tools for problems involving chemical kinetics, thermodynamics, and/or transport processes. It can be used from MATLAB, Python, C++, or Fortran.259 weekly downloads
Blit - Block Iterative Sparse Solvers
Blit contains a group of highly efficient iterative sparse solvers that can handle multiple right-hand-sides (i.e. block solvers). We will implement BLQMR, BLGMRES and other block algorithms in MATLAB, FORTRAN 90, C/C++, CUDA and OpenCL.
Elvis solver
1-dimensional Eulerian Vlasov-Maxwell solver, used primarily for studies of kinetic effects in plasma waves and for stimulated Raman scattering.


text: It Has Been a Bad 24 Hours For StrippersJordan Sargent1/03/13 9:05pmFiled to: StrippersFloridaOhioAtlantaLauren blockArlene menaCorey john capels132EditInvite manuallyPromoteDismissUndismissHideShare to KinjaToggle Conversation toolsGo to permalink It's almost never a good thing for a stripper to make the news. The last 24 hours — which has seen strippers hospitalized, arrested and fired — has proven at least that much.In Cleveland, a 22-year-old stripper named Lauren Block — pictured above — is currently in critical condition after falling from a balcony at Christie's Cabaret. Block was dancing for a client when she attempted what police are calling a "jump-dance" move, sending her headfirst over a railing. Block reportedly suffered "massive head trauma." Hopefully she can make a full recovery.A South Florida (#Florida) stripper named Arlene Mena was likely pretty drunk, high or some combination thereof yesterday when she decided to do some "artwork." Mena was arrested after tossing a traffic cone and spitting at a motorist driving along A1A in Hallandale Beach. According to a witness, Mena said she was part of a television show (she was not), then that she's a federal agent (she is not) and then that she is friends with Hallandale Beach mayor Joy Cooper (she is not). Instead, she is out on $1,000 bond.In Atlanta, a male stripper named Corey John Capels was fired from Bliss Atlanta for hating gays and announcing so on his Facebook page. Imagine that. Capels called police to report damage to his Maserati that he suspected was done by one of the club's bartenders. After an incident with the club's manager as the two reviewed surveillance, Capels took to his Facebook to write "I hate all faggots burn in Hell" — clearly he's not a stripper trying to pay for college. In any event, Capels was fired. So, if you ever decide to become a stripper — and if it means owning a Maserati, I'd at least consider it — make sure you at least manage to stay out of the news. [photo via ABC News via Facebook]Reply132 repliesLeave a reply


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Enterprise content management software platforms
ECM software platforms
Enterprise content management software platforms News
June 08, 2016
Open APIs key to successful collaboration platforms
APIs could be the future of collaboration platforms, enabling enterprise content management data to be available.
May 04, 2016
Slack kicks up dust in collaboration software tool market
Slack is taking aim at traditional communication tools like instant messenger and email. But its integration with other services bespeaks a broader strategy.
February 10, 2016
ECM software integration still puts companies through paces
Companies are bringing efficiency and productivity to their processes with ECM software. But some are waiting on integration with other systems in order to gain all the benefits.
December 16, 2015
Box-Salesforce partnership brings ECM features to CRM
A new partnership will bring the cloud file-sharing capability of Box to Salesforce.
Enterprise content management software platforms Get Started
Bring yourself up to speed with our introductory content
SharePoint sprawl
SharePoint sprawl occurs when end users create a new site to categorize content that could have been stored in an existing site, or categorize content poorly so it's difficult to find later on. Continue Reading
information rights management (IRM)
Information rights management (IRM) technologies help control, secure and manage content from unwanted access. Continue Reading
Cloud and mobile drove content management trends in 2015
Cloud and mobile fill expanding roles for Enterprise Content Management. Continue Reading
Office 365 cloud services encounter turbulence
Office 365 cloud services can hasten Microsoft's flight to the cloud, but effective migration depends on willing users and worthy features.
By submitting your email address, you agree to receive emails regarding relevant topic offers from TechTarget and its partners. You can withdraw your consent at any time. Contact TechTarget at 275 Grove Street, Newton, MA.
You also agree that your personal information may be transferred and processed in the United States, and that you have read and agree to the Terms of Use and the Privacy Policy.
Evaluate Enterprise content management software platforms Vendors & Products
Weigh the pros and cons of technologies, products and projects you are considering.
Microsoft's Office 365 strategy blurs lines between desktop, mobile
Office 365 strategy is decidedly cloud-first and mobile-first, but how well has Microsoft fared in making this happen? Continue Reading
Comparing traditional collaboration platforms with new alternatives
Conventional collaboration platforms can be a hurdle in terms of usability. New apps like Basecamp provide an alternative, but are they comprehensive enough? Continue Reading
SharePoint Online Public Website drops off, pricing doesn't
Microsoft is killing off support for SharePoint public websites, and companies will now have to go to third parties for support. For some shops, that could increase costs. Continue Reading
Manage Enterprise content management software platforms
Learn to apply best practices and optimize your operations.
Identifying must-have features in file-sharing programs
Choosing a file-sharing program takes some planning and understanding of your business requirements. Continue Reading
How to restore SharePoint business intelligence features
Microsoft removed business intelligence features from SharePoint 2016, and only an upgrade to two other platforms allows IT pros to get them back. Here's how. Continue Reading
SharePoint 2016 RTM highlights Microsoft's cloud path
SharePoint 2016 RTM is out and highlights the path Microsoft is paving for shops to the cloud. Continue Reading
Problem Solve Enterprise content management software platforms Issues
We've gathered up expert advice and tips from professionals like you so that the answers you need are always available.
OpenText Content Suite targets governance and compliance
This overview examines OpenText Content Suite, a set of ECM tools for managing content from capture through archiving and disposition. Continue Reading
Cloud file sharing pushes ECM content for mobile apps
Cloud file sharing has been a boon for remote collaboration. Now file-sharing apps are poised to help users manage content for mobile experiences. Continue Reading
Google shift underscores importance of mobile Web design
Google is adding mobile performance to its SEO equation, and that has companies reevaluating Web strategy to avoid losses in search rankings. Continue Reading


text:
Success in taking a protein alive in an artificial capsule-A protein was encapsulated within a self-assembled capsular molecule- : Professor Makoto Fujita & Lecturer Sota Sato, Department of Applied Chemistry
October 3, 2012
The research group of Professor Makoto Fujita in Department of Applied Chemistry, School of Engineering, The University of Tokyo succeeded in encapsulation of a whole protein within a synthesized capsular molecule with the diameter of seven nanometers.
In nature, biomolecules like proteins or DNA are encapsulated within huge capsular materials like virus cages, where the structures or activities of the biomolecules are controlled or the biomolecules are stored until they are used. In an artificial chemical phenomena, small organic molecules are encapsulated within hollow capsular molecules, called "host molecules", where the structures or activities of the encapsulated guest molecules are also controlled. However, huge biomolecules like proteins have never been encapsulated because the sizes of the artificial capsular molecules with precise structures are limited.
Here, the research group synthesized huge metal-organic complexes (artificial capsules) utilizing self-assembly process, where metal ions and organic compounds are just mixed in a flask and the coordination bonds between the starting materials automatically construct the ordered precise structures. A whole protein was successfully encapsulated in the capsular complex.
A wide variety of applications in the fields of industry, drug discovery, and so on are expected, because the encapsulation of proteins could be useful for structural analysis or functional improvement of the trapped proteins.
The structure of the self-assembled, huge capsular molecule encapsulating a whole protein from building units of organic molecules and palladium ions. The product has a single, precise structure, where sugar chains make an internal hydrophilic interface to hold the protein stably.


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Coachella cool! Kelly Osbourne and famous friend Dita Von Teese cut a stylish pair at music festival
With their bright red lipsticks, sunglasses and stylish outfits, Kelly Osbourne and Dita Von Teese certainly made for a striking pair at the Coachella festival yesterday.
The friends sat together on the grass for a chat while checking out some of the musical acts at the California event
The 27-year-old looked festival ready with lilac coloured hair, vest top and several necklaces as she sat next to Dita, 39.
Girl time: Kelly Osbourne and Dita Von Teese hung out together at Coachella Festival on Sunday
Dita covered up in a black and white heart motif maxi dress and the classically styled pin-up's eyes were fixed on the stage.
The ladies regularly take on Hollywood night life together with the burlesque dancer's new beau, Theo Hutchcraft from the Hurts.
But while the rocker was glued to her lips yesterday, today he was nowhere to be seen.
As the women enjoyed some girly time in front of a jam-packed line up which includes Swedish House Mafia, Snoop Dogg and Dr Dre, Kelly's mind seemed to be on other things.
It's a tense weekend for the lavender-haired television presenter as she spent much of it eagerly posting on Twitter, urging Britons to vote for her singer friend Tyler James.
The gentleman is a contestant on the UK's The Voice show and Osbourne was not shy to admit how annoyed she was to be missing his performance.
Somewhere to be: The friends clearly had contrasting things on their mind with Dita intrigued by the bands
Reunited: Osbourne was glued to her phone after retrieving it from a friend's car
Although she had asked her beloved followers to keep her updated on his progress in the competition she forgot her mobile phone in a friend's car so was forced to wonder whether her long-time associate would blow judges Tom Jones, Jessie J, Will.i.am and Danny O'Donoghue away.
In some heartfelt messages, once she had retrieved her belongings, she told the performer: 'You got through im so proud of you I LOVE you so much! you are kicking ass on @bbcthevoiceuk EVERY TIME I SEE YOU SING I CRY!
'Tears of joy that is you got this im here 4 you when ever you need me i cant wait to sit on the audience of @bbcthevoiceuk
'You will never be able to understand how proud i am of you! for god sake look where you were 6 months ago.'
Good luck: Kelly has been talking about her friend form the UK's The Voice show all weekend
I'm coming! Kelly Osbourne has been supporting her associate on Twitter, letting him know she would be visiting him in the UK
The night before she only had eyes for her man.
He has romanced various beautiful ladies including English television presenter Alexa Chung and German model Shermine Shahrivar.
But while Hutchcraft has opted to keep his past relationships relatively under cover, the musician had no qualms about revealing his latest girlfriend Von Teese.
It's no surprise, the singer wanted the world to know he was dating the world's most famous burlesque dancer.
Loved up: Gorgeous burlesque model Dita Von Teese kisses Hurts frontman Theo Hutchcraft during the Swedish House Mafia's set at the Coachella Music Festival in Indio
The British rocker is clearly besotted with the star and was openly affectionate with her this weekend.
The 25-year-old was eager to let fellow festival goers know that the American sex symbol was his lady as they danced in Indio, California.
Wearing his signature black shades in the form of a black shirt, trousers and shoes, one half of the music duo act only had eyes for the black-haired older lady.
Sex symbol: The burlesque dancer looked stunning in a cherry print jumpsuit, red bracelets, lipstick and nail polish
Fashion icon: The 39-year-old added a black sash and carried a black Chanel bag
Changed her tune: Von Teese has opted for a younger man after being dating to Marilyn Manson some years ago
He leaned in to passionately kiss her, placing his hands on her tiny waist.
Dita stood out from the crowds in a multi-coloured cherry print jumpsuit which she paired with a red bracelet, nails and lipstick.
Von Teese – who in the past was engaged to Marilyn Manson - added to her ensemble with a black waist sash, Chanel shoulder bag and patent wedges.
On fire: Swedish House Mafia light up California with their performance
She recently opened up her wardrobe for the world to see her footwear collection.
She told Yahoo!: 'A lot of people like to go into my shoe collection because I have a really close friend named Christian Louboutin and so I get a lot of custom shoes made and so people like to see what he does for me.'
The couple were seen earlier this weekend leaving Chateau Marmont in West Hollywood with their friend Osbourne but opted to keep their feelings under wraps.
Other celebrities enjoying the second weekend of music performances include Joe Jonas, Paris Hilton, Busy Phillips, Melanie Griffiths, Alexander Skarsgard, Rosie Huntington Whiteley and Emma Roberts.
Disney roots: Pop star Joe Jonas was joined by a posse of men at the popular
Style for the heat: Cougar Town's Busy Phillips wore a mint dress
Music lovers: Kristen Stewart kept a low-profile and Melanie Griffith enjoyed her second day of fu
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[Watch] Here's What Happens When Your Side Chick Crashes Your Wedding
It's a scene out of a Tyler Perry movie.
Steve Harvey's book Act Like a Lady, Think Like a Man was a media sensation, topping the New York Times bestseller list and landing Harvey on Oprah and The View. The book gives blunt and humorous advice for women looking to be in relationships or who are already committed.
Two years ago there was word a film would be released. Now, the producer, Will Packer (Stomp the Yard, Takers) revealed via Twitter that the movie starts filming this summer. He tweeted, "MY NEXT MOVIE BASED ON @IAmSteveHarvey's bestseller "Act Like A Lady, Think Like A Man" shoots this summer… All my real up and coming actors stay tuned for casting info… All my 15MIN OF FAME ppl stay tuned to learn how you can win a walk on role. I'll be on @IAmSteveHarvey's show next Tues to give details."
Let's hope this movie is nothing like another relationship advice book-turned-film, He's Just Not That Into You -- in other words, a dry and dull romantic comedy. Here's to Harvey bringing a little more flavor!
(Photo: Dimitrios Kambouris/Getty Images)
It's a scene out of a Tyler Perry movie.
A few fellow students have taken to Instagram displaying an appalling lack of remorse


text:
Skydiving.com features a network skydiving venue serving Columbus, along with various other cities within Burlington County and the whole state of New Jersey. Exactly what are you waiting on? Contact our team right away at 1-800-617-7948 for more details. Don't forget to ask our team about current skydiving specials in the Columbus area!
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text: Amanda Doyle Twenty-Four Things To Do In St. Louis This Summer By Erica Smith • Jul 17, 2014 Romondo Davis Summer in the city. There's nothing like it, and no shortage of things to see, do and experience in St. Louis. From parks to concerts and festivals, frozen custard to marionettes, farmers markets to museums, there's an event (or 20) for everyone. Author Amanda Doyle has written a second St. Louis guidebook. She said being an outsider affects her view of St. Louis. "You can't be born in a place and appreciate everything about it," she said. From Candy Cane Lane To Cahokia Mounds: Winter Around St. Louis By Camille Phillips • Dec 17, 2013 Mark S. Abeln Whether it's sledding on Art Hill, ice skating at Steinberg Skating Rink, drinking whiskey or hibernating with cats (all responses we received on our Facebook page), St. Louisans have a variety of ways to keep warm and have fun in the winter. What's your favorite wintertime activity in the St. Louis area? Submit your answer on our Facebook page.


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Excellent Hotel if you are travelling with your beloved dog. The Hyatt Regency Hotel in Düsseldorf caters for every wish your animal may have. They supply you with a luxurious doggy bed in camel leather, food and water bowls with Evian water and a lovely doggy pate especially made for the
Hyatt Regency Hotel. They even offer a doggy babysitter who will look after your pet while you are at the theatre or sightseeing. They take him or her for their regular walks. What was so wonderful was the personal letter they write to your dog on your arrival.
Thumbs up to this great hotel and service!
- Official Description (provided by the hotel):
- Experience Hyatt Regency Dusseldorf, an elegant hotel for business and leisure travellers located directly at the Media Harbour in Dusseldorf. The hotel was opened December 2010 and is located in a 19-storey building at the tip of a peninsula directly on the Rhine with a stunning view of the Media Harbour, Old Town, the Rhine Tower and the skyline of Dusseldorf. The hotel offers 303 rooms and suites with a contemporary, elegant design, complemented by a comfortable setting. Dining facilities include DOX restaurant with classic regional cuisine, the self-service restaurant Cafe D as well as DOX Bar and Lounge with a wide range of beverages, classical snacks and local dishes. The Pebble's Terrace catches the eye with a stunning view of the Media Harbour. Flexible meeting spaces with floor-to-ceiling-windows and a total area of 850 sq m are ideal for business meetings, seminars and conferences. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Expedia, Booking.com, TripOnline SA, Hotels.com, Hotwire, Hyatt Hotels Corporation, Cancelon, Priceline, Travelocity, Agoda and Odigeo so you can book your Hyatt Regency Dusseldorf 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:
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text:
First visit to Madeira and by dint of good fortune we found ourselves staying in this excellent hotel. The staff were friendly and efficient. They were kind enough to present us with surprise champagne on ice to celebrate our joint birthday ...still dont know how they knew about this! Also they were very helpful when I contracted the dreaded winter vomiting illness (I hasten to add not their fault in any way). The hotel is situated high in the town giving a superb view, however this does mean transport is needed however the free hotel minibus is readily available during the daytime (except Sunday). Also the Official Sightseeing Tour Yellow Bus stops on the road immediately outside hotel.
- Official Description (provided by the hotel):
- Situated in the west hillside of Funchal, with superb views of the sea, the bay and magnificent mountains. With a relaxed and friendly atmosphere, this is the ideal place for an unforgettable holidays. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Expedia, TripOnline SA, Hotwire, Orbitz, HotelTravel.com, Hotels.com, Priceline, Booking.com, Travelocity and Otel so you can book your Madeira Panoramico Hotel 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:
- Hotel Madeira Panoramico
- Madeira Panoramico Funchal
- Madeira Panoramico Hotel Funchal


text:
The "Koch-sperts" must have had a hand in the creation of the RNC Platform, because the list of ALEC-approved policies in the platform is stunning.
The Republican platform eviscerates the "hard working American," denying workers of their right to unionize while targeting vulnerable communities.
Matt Renner is Director of Development and Communications at Truthout.


text:
Employers & Recruiters
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All jobs and internships are posted on the College's online job board. These listings can be accessed by students/schools in the NPEC consortium including: King's College, Marywood University, Misericordia University, the University of Scranton, and Wilkes University.
To post a job or internship opening online visit College Central Network and register as an employer. You will gain site access that allows you to post positions and search resumes of registered Keystone College students and alumni.
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Individuals wishing to post full or part-time jobs that include yard work, childcare, tutoring, or odd jobs are welcome to advertise through our college. All such opportunities will be advertised on our bulletin boards only. Students take positions at own risk.


text:
The Genizah at the House of Shepher
The Genizah at the House of Shepher
Toby Press, Hardcover, 9781592640850, 400pp.
Publication Date: April 15, 2005
List Price: $19.95*
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A scholar, returning to her family home in Jerusalem becomes embroiled in a family dispute over a discovered Codex, brought home originally her great-great grandfather. Set against the backdrop pf a hundred and thirty years of change, this is a novel of exile and belonging, displacement and the quest for both love and a true promised land.
About the Author
Tamar Yellin was born in the north of England. Her father was a third generation Jerusalemite and her mother the daughter of a Polish immigrant. She began writing fiction at an early age, and the creative tension between her Jewish Heritage and her Yorkshire roots has informed much of her work. She received the Pusey and Ellerton Prize for Biblical Hebrew from Oxford University, and has worked as a teacher and lecturer in Judiasim. Her first novel The Genizah at the house of Sheper was awarded the Sami Rohr Prize, the Ribalow Prize and was shortlisted for the Wingate Prize.


text:
11921 Eastwick Circle
Carmel, Indiana, 46033
Commute to Downtown Carmel
Almost all errands require a car.
About your score
11921 Eastwick Circle has a Walk Score of 5 out of 100. This location is a Car-Dependent neighborhood so almost all errands require a car.
This location is in Carmel. Nearby parks include Founders Park, River Heritage Park and Flowing Well Park.
Explore how far you can travel by car, bus, bike and foot from 11921 Eastwick Circle.
This location is in the city of Carmel, IN. Carmel has an average Walk Score of 17 and has 79,191 residents.


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Unibond Grey Floor Tile Grout (W)3.75kg
Unibond Grey Floor Tile Grout (W)3.75kg
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This 3.75kg floor tile grout is on for ceramic & porcelain floor tiles. Simply apply and leave for 24 hours to dry. Each pack covers 8 m² on average.
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text:
Wasted (Paperback)Mark Johnson
Paperback Published: 03/04/2008
- Can be ordered from our supplier
Click & Collect From your local shop
Mark Johnson's father had 'LOVE' tattooed across his left hand, but that didn't stop the beatings. The Johnson children would turn up to school with broken fingers and chipped teeth, but no one ever thought of investigating their home life. Mark just slipped through the cracks, and kept on falling. For years. Constantly in trouble at school, Mark began stealing at the age of seven, was drinking by the age of eight, and took his first hit of heroin aged eleven. A sensitive, intelligent boy, he could never stay on the right path, and though Art College beckoned, he ended up in Portland prison instead. With searing honesty, WASTED documents Mark's descent into the depths of addiction and criminality. Homeless, hooked on heroin and crack, no one - least of all Mark - believed he would survive. And yet - astonishingly - he somehow pulled himself through, and now runs his own thriving tree surgery business, employing and helping other recovering addicts. His story is at once shocking and inspiring - a compelling account of his struggle to save himself, and help save others in the process.
Publisher: Little, Brown Book Group
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This book really tells all the nitty gritty about drugs and how they can take over your life. But it also shows there is hope for all additcs if they want to get clean. Once I started to read it I couldnt put it down... More
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text:
"...a must read in economic literacy." (USA Today, November 29, 2010)
"Finally, an economics book that is neither dull nor inscrutable and that won't put you to sleep. Greg Ip gives us a lucid and entertaining understanding of 'the dismal science' and reveals how economic concepts and institutions affect our daily lives. This little gem can turn all of us into sophisticated and educated citizens."
—Burton G. Malkiel, Professor of Economics, Princeton University; author of A Random Walk Down Wall Street and The Elements of Investing
"Greg Ip is one of the world's best economic journalists. The Little Book of Economics will teach you much more than a little about the forces that shape all of our lives."
—N. Gregory Mankiw, Professor of Economics, Harvard University; author of Principles of Economics
"Here's the perfect way to understand the economy without breaking a sweat. Clearly written and easy to understand, The Little Book of Economics guides you through what you need to know. Ip's bright light illuminates places previously darkened by insider jargon and arcane formulae."
—Robert B. Reich, Professor of Public Policy, University of California, Berkeley; former U.S. Secretary of Labor
"The book is an excellent introduction to basic economic concepts and ideas explained in clear and thoughtful ways. A must read in economic literacy." "
—Nouriel Roubini, Professor of Economics, New York University; Co-founder and Chairman of Roubini Global Economics
"Greg Ip has the rare talent of making even the toughest topics easy to understand. In The Little of Book of Economics, he tells you what you need to know with superb clarity and memorable examples. I recommend this book to anyone who wants a clear explanation of how the forces of economics shape the world."
—Michael J. Mauboussin, Chief Investment Strategist, Legg Mason Capital Management; Author of Think Twice
From the Inside Flap
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One side effect of the deepest economic downturn since the Great Depression is that it has ignited a fresh desire among citizens and investors to better understand the economy. The Little Book of Economics is an accessible, engaging, and entertaining guide to all of the wonderful and wicked ways in which the economy functions and what it all means to you.


text:
This is the last photo taken of the children of Earl and Delcie (Crisler) Ash before the parents' deaths. After, the children were split apart. The picture was probably taken in Flint, Michigan.
In this photo:
Margaret Ash, Elton B Ash (born 1917), Clayton R Ash (1922 - 2005), Francis Ash (born 1924), and Winona M Ash (born 1920)


text:
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Gov. Scott Walker said he would consider a special session this summer to pass the new voter ID legislation.
Who's to blame over a painkiller scandal at the Tomah Veteran's hospital?
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Getting Kerry-ed away
From Kerry's convention speech: "You don't value families if you force them to take up a collection to buy body armor for a son or daughter in the service…"
Truth: The body armor bake sale myth is debunked by Eliana Johnson here.
Posted in: John Kerry
As a show of unprecedented openness, the State Dept. can release all Hillary-related emails by 2091
June 7, 2016 09:11 AM by Doug Powers
Transparency, Iran deal-style! Mysterious video editing gremlin roaming halls at the State Dept.
June 1, 2016 09:04 PM by Doug Powers
John Kerry should tell the White House about his 'borderless world' prediction
May 8, 2016 11:08 PM by Doug Powers
Perfect: John Kerry opposes offshore tax havens the same way he leads by example on climate change
April 26, 2016 09:21 AM by Doug Powers
John Kerry will LOVE Bono's idea: What needs to be deployed against ISIS is … comedy
April 13, 2016 07:59 AM by Doug Powers
Categories: Al Qaeda, Barack Obama, Gitmo, Hollyweird, John Kerry, Terrorist attacks, War


text:
Real life is more important than comics, Em. Skip a week. We will cope
or maybe no update today either... tbh this might have to be a week i skip
YAY! Cherry Book
ps update will be tomoz as i've been busy today.
new cherry books are here and available to buy! link
But finding a hand to write with?
Why not? It's not like they'd have trouble finding a pen...
Silly Nilam, ducks can't be penpals.
@dralou she's the most powerful (and trolly) sorceress in the faerie realm... her clothes do whatever she wants them to
Truly impressive is when Neb starts being Keti's pen pal from inside faery prison.


text:
Translation of highway robbery in Spanish:
- 1 (History)asalto (masculine) (en un camino)
salteamiento (masculine)
Definition of highway robbery in:
What do you find interesting about this word or phrase?
Comments that don't adhere to our Community Guidelines may be moderated or removed.
Grammar and usage help for Spanish:
= trending


text:
About this Business
From Our Editors
Just because you're rushing doesn't mean you have to miss out on a delicious meal — Green Bay's The Pancake Place will fill you up in a hurry. Keep your diet in check at The Pancake Place, a local restaurant with gluten-free and low-fat menu items. If you're in need of a booster seat, this restaurant's got you covered. This is a great spot for the whole family.
Feeling a little shy? Carryout is available.
There is parking close to the restaurant. The Pancake Place offers parking for all diners, including those who travel by bike.
The breakfast dishes at the restaurant really bring the crowds in, though lunch and dinner are also served. For a deliciously quick bite to eat, be sure to stop by The Pancake Place.


text:
Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://wileyonlinelibrary.com/onlineopen#OnlineOpen_Terms
Version of Record online: 14 MAY 2012
DOI: 10.1111/j.1558-5646.2012.01676.x
No Claim to original U.S. government works. Evolution© 2012 The Society for the Study of Evolution.
Volume 66, Issue 10, pages 2993–2999, October 2012
Additional Information
How to Cite
Uriz, M. J., Agell, G., Blanquer, A., Turon, X. and Casamayor, E. O. (2012), ENDOSYMBIOTIC CALCIFYING BACTERIA: A NEW CUE TO THE ORIGIN OF CALCIFICATION IN METAZOA?. Evolution, 66: 2993–2999. doi: 10.1111/j.1558-5646.2012.01676.x
Publication History
- Issue online: 1 OCT 2012
- Version of Record online: 14 MAY 2012
- Accepted manuscript online: 21 APR 2012 12:31PM EST
- Received August 9, 2011 , Accepted April 6, 2012 , Data Archived: Dryad doi:10.5061/dryad.ff586c8k
- Abstract
- Article
- References
- Cited By
- Biomineralization;
- calcibacteria;
- marine sponges;
- symbiosis
Sponges show the highest diversity of associated bacteria among marine invertebrates. Immunological evidence traces the origin of the sponge bacterial symbioses to the Precambrian era. Hence, sponges appear to be ideally suited for studying the evolutionary origins of prokaryote–metazoan associations. Sponges produce either calcareous or siliceous skeletons, which only coexist in a relict group of demosponges, the sclerosponges. We report here, for the first time, intensive calcification in nonsclerosponge siliceous demosponges. Calcification is mediated by endosymbiotic bacteria (calcibacteria) located in archeocyte-like sponge cells. These calcibacteria are devoid of bacterial walls and divide within sponge cells until they became surrounded by a calcitic sheet, being subsequently extruded to the sponge subectosomal (subepithelial) zone. Thousands of bacteria-produced calcitic spherules cover the surface of the host sponges, forming a cortex-like structure that mimics a rudimentary peripheral skeleton. Calcibacteria are vertically transferred to the sponge larvae during embryogenesis. Calcium detoxification may have generated this symbiotic association, with some additional benefits for the sponges, such as skeletal formation and deterrence from predation. This unique symbiosis holds implications for sponge biology and may advance discussions on the role of bacteria in early biocalcification processes in metazoans.
Sponges (Porifera) are the earliest branching metazoans (Li et al. 1998; Philippe et al. 2009), and they show the highest diversity of associated bacteria among marine invertebrates (Schmitt et al. 2011). Immunological evidence has tracked the origin of sponge bacterial symbioses to before the Cambrian period (Wilkinson 1984). Therefore, sponges appear to be appropriate organisms for studying the evolutionary origins of prokaryote–metazoan symbioses.
Extant sponges have either siliceous or calcareous skeletons (Uriz 2006), which rarely coexist in the same species. So far, the only exception to this clear-cut dichotomy in the mineral nature of sponge skeletons is the so-called coralline demosponges or sclerosponges that produce a massive calcium carbonate skeleton and free siliceous spicules simultaneously (Vacelet 1985). Extant sclerosponges are considered relict, but representatives of this group proliferated and built reefs in the late Permian (Paleozoic) and late Triassic (Mesozoic) periods (Reitner 1989). The adaptive potential of a double mineralization is illustrated by examples of extant sclerosponges (e.g., genus Merlia). This genus usually shows both types of mineral skeletons, but produces mainly a calcareous skeleton in silica-depleted habitats (i.e., M. lipoclavidiscaVacelet and Uriz 1991) or mainly a siliceous skeleton in silica-rich habitats (i.e., M. deficiensVacelet 1980). The calcareous skeletons of sclerosponges are made of intracellular spherical structures (spherulites) that are excreted to the sponge mesohyl, where they grow epitaxially, by successive superposition of aragonite-made layers, to build walls and chambers (Willenz and Hartman 1989; Wood 1991; Wörheide 1998). The possible role of symbiotic bacteria in the formation of the sclerosponge spherulites has been considered. A bacterial signature was found in the spherulites of the sclerosponge Astrosclera willeyana (Jackson et al. 2010). Later, Jackson et al. (2011) reported the presence of a protein (spheruline) in the spherulites of this species, which, according to several lines of evidence (Jackson et al. 2011), seems likely to be encoded by a sponge gene horizontally acquired from a bacterium. Thus, there is growing indication of the participation of prokaryote genes in sponge biocalcification, but the direct involvement of symbiotic bacteria has never been proved.
Here, we present the first evidence of endosymbiotic calcifying bacteria (hereafter calcibacteria) within nonsclerosponge sponges of the genus Hemimycale (Demospongiae: Poecilosclerida, Van Soest 2002). These calcifying bacteria may have implications for sponge biology and may support the hypothesis of an early endosymbiotic origin of calcification in metazoans.
Fresh sponges of the genus Hemimycale were collected from several seas (H. columella: NE Spain, Mediterranean Sea; H. arabica: Red Sea, Hemimycale sp.: E Africa, Indian Ocean) and immediately processed for light, epifluorescence Transmission Electron (TEM) and Scanning Electron (SEM) microscopy studies, and Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM). The presence of calcibacteria was microscopically verified less than 1 h after collection.
To determine the amount of calcium carbonate (as a proxy for calcibacteria abundance), specimens (N= 5 per species) were dried at 80 °C for 48 h in a stove and then weighed (A) using an analytical balance. The dried samples were placed in an oven at 600 °C to remove the organic matter. The remaining minerals, mainly consisting of siliceous spicules and calcibacterial coats, were weighed (B) and put in a Pyrex fingerbowl, where the calcareous material was removed through boiling in nitric acid. This process left a residue mostly consisting of siliceous spicules that were rinsed with distilled water and dehydrated with absolute ethanol before being dried and weighed (C). The weight of the calcibacteria coats was calculated as B–C and was referred to as the sponge dry weight in percentage by the formula (B–C) × 100/A.
Small pieces, approximately 3 mm in diameter, were obtained from living sponges (H. columella, H. arabica), fixed for TEM (Uriz et al. 2008), and embedded in Spurr. Ultrathin sections were lead citrate stained and observed through a JEOL JEM-1010 TEM (Scientific-Technical Services of the University of Barcelona). Isolated calcibacteria were dehydrated, gold palladium metalized, and observed through a Hitachi S-3520N SEM (Microscopy Service ICM-CSIC, Barcelona). Calcibacteria cryofracture was performed in liquid nitrogen to observe the inner part of the calcibacterial coat. For light microscope observation, fresh samples were dissociated in seawater by stirring them with a pipette and a drop of cell suspension was observed.
EDX analysis was performed to determine the chemical nature of the calcibacterial coat (RÖNTEC Microanalyzer ICM-CSIC, Barcelona) according to standard practice (Turon et al. 2000).
DNA staining and in situ hybridization with bacterial and archaeal probes was performed on sponge sections. A fragment of a living individual of H. columella was fixed in 4% buffered paraformaldehyde (PANREAC, Spain). This fragment was dehydrated and embedded in paraffin, cut into 5-μm-thick sections, mounted on slides and stored at −20 °C. Sponge sections were DAPI stained and used for catalyzed-reported deposition whole cell fluorescence in situ hybridization (CARD-FISH) with bacteria-specific (EUB338) and archaea-specific (ARC915) probes (Pernthaler and Amann 2004; Auguet and Casamayor 2008). Other bacterial probes were also assayed for Betaproteobacteria (BET42a), Bacteroidetes (CF319a), and Actinobacteria (HGC69a), but they gave negative results. All probes were synthetized by Molecular Probes (Invitrogen, USA). The hybridized DNA was observed with a Zeiss epifluorescence microscope using the appropriate filter sets for DAPI (Zeiss filter set 01, BP365/12 FT396 LP397) and Alexa-Fluor 488 (Zeiss filter set 09, BP450–490 FT510 LP515).
Sponge calcibacteria were extraordinarily abundant (their calcareous coats represented 30–60% of the sponge dry weight) in all three currently known species of the genus Hemimycale (Demospongiae: Poecilosclerida) inhabiting different seas, that is, H. columella (Bowerbank 1874), Atlanto-Mediterranean; H. arabica (Ilan et al. 2004), Red Sea; and Hemimycale sp. (taxonomic status currently being determined by the authors), Indian Ocean, E. Africa (Fig. 1 A–C).
The calcibacteria were coccoid in shape and 200–800 nm in diameter (about 500 nm on average) and were found within vacuoles of amoeboid, archeocyte-like sponge cells (hereafter calcibacteriocytes) of about 15 μm in diameter (Fig. 2A, B). Degenerated calcibacteria were also observed in the sponge mesohyl as a result of calcibacteriocyte lysis. The bacterial nature of the calcibacteria was confirmed by electron (TEM) and epifluorescence microscope observations (DAPI staining and CARD-FISH techniques with specific prokaryote probes). Calcibacteria showed positive hybridization with the universal eubacterial probe (Fig. 2C, F) and only rare signals with the universal archaeal probe (Fig. 2D).
Viewed through electron microscopy, the calcibacteria showed loose inner material and appeared to be devoid of a typical cell wall; they were instead surrounded by a 100-nm-thick calcareous coat. Their organic content was arranged in a peripheral ring bounded by the bacterial membrane and an inner nucleoid (Fig. 3A, B). The electron microscopy observations indicated that recently replicated calcibacteria were not calcified and were able to replicate within the calcibacteriocyte vacuoles (Fig. 3C); subsequently, they secreted a calcareous coat consisting of 20–30 nm nanospherules (Fig. 3D, E). Finally, the cytoplasmic content was progressively reduced, and only the empty spherules remained (Fig. 3E). EDX microanalysis showed that the coat did not contain P, but Mg and Ca in a ratio corresponding to low-Mg calcite crystallization (Fig. 4A). Interestingly, the calcification process can be initiated even before bacterial division is complete (Fig. 4B). Calcibacteriocytes completely filled with calcibacteria were concentrated at the sponge periphery, conferring a whitish tinge to the sponge surface (Fig. 1D), where they lysed and released the calcibacteria to the mesohyl near the sponge surface.
We observed the vertical transmission of calcibacteria during the reproductive cycle of H. columella. The maturation of sponge embryos involves the incorporation of maternal cells (nurse cells). Maternal calcibacteriocytes were observed surrounding an embryo (Fig. 1E) and were phagocytized by the growing embryo, as it is reported for typical nurse cells. Once engulfed, the calcibacteriocytes disintegrated and released the calcibacteria into the embryo's mesohyl (Fig. 3F). The noncalcified calcibacteria replicated and were phagocytized by ameboid (archeocyte-like) embryonic cells, after which calcification proceeded. Thus, the sponge larvae contained abundant calcibacteria upon release, and recently settled sponges (rhagons) already exhibited an accumulation of calcibacteriocytes in their surface tissues (Fig. 1F).
To the best of our knowledge, this is the first report of intracellular calcifying bacteria living in symbiosis with marine invertebrates. Vacelet et al. (1987) first reported the presence of irregular calcareous bodies in the sponge Hemimycale columella but did not ascertain their nature. Irregular calcareous bodies similar to those reported in H. columella have also been described from the tropical Spirophorida Cinachyrella alloclada (Rützler and Smith 1992), which suggests the presence of calcibacteria in the later species as well.
The genus Hemimycale belongs in the order Poecilosclerida that mainly harbors "low bacterial abundance" sponges (Hentschel et al. 2003). However, our findings show that a sole morphotype of calcibacteria contributed up to 60% of the consortium weight in Hemimycale spp., whereas the diverse bacterial assemblages that characterize "high microbial abundance" sponges only accounted for up to 38% of the sponge volume (Vacelet 1975). This result supports the recently manifested need to reevaluate earlier sponge categorizations according to the abundance of their associated bacteria (Thacker and Freeman 2012).
Ankyrins, adhesion-related proteins (ARP) and tetratricopeptide repeat domain-encoding proteins (TPR), which may allow sponge cells to discriminate between food and bacterial symbionts, have been detected in the genome of several sponges harboring bacterial symbionts (Thomas et al. 2010; Liu et al. 2011), which denotes the capacity of sponge cells to identify potential symbionts. Whether these proteins are expressed in all sponge cells or are specific to archeocytes remains unknown. Hemimycale calcibacteria were contained in archeocyte-like cells, but they were absent from the more specialized sponge cells: choanocytes (water pumping), collencytes (collagen secretion), sclerocytes (spicule formation), and pinacocytes (pseudoepithelia formation). Archeocytes are pluripotent ameboid cells that move across the sponge mesohyl, phagocytizing cell debris and bacteria (Boury-Esnault and Rützler 1997) and are involved in immunologic processes (Sabella et al. 2007). Due to these abilities, archeocytes may detect "foreign" material in the sponge tissues, including microbes, and are thus particularly suitable cells for developing bacterial symbiosis. Once phagocytized, the microbes are recognized as a food and undergo digestion in archeocyte lysosomes or are identified as "potential partners" and kept alive and allowed to divide within the vacuole.
The phylogenetic identification of the Hemimycale calcibacterium is underway. A 16S rRNA gene tag-pyrosequencing of H. columella tissue has revealed that the most frequently recovered sequence (65%) was a representative of the Alphaproteobacteria class (authors' current research), within which mitochondria and many parasitic and symbiotic bacteria of invertebrates fit. These abundant bacteria may correspond to the Hemimycale calcibacteria, which is in agreement with the fact that the Bacteroidetes, Betaproteobacteria, and Actinobacteria probes did not gave positive signal in the CARD FISH assays on the sponge tissue. However, most in-depth studies, including in situ hybridization with other specific molecular probes, need to be conducted to confirm their taxonomic identity.
In contrast to other endosymbiotic bacteria previously reported in sponges, the calcibacteria of Hemimycale lack bacterial walls, show scant cytoplasmic material, and are contained in cytoplasmic vacuoles, as has been reported for some obligate endosymbionts of other invertebrates (e.g., Baumann et al. 2000; Shigenobu et al. 2000; Pérez-Brocal et al. 2006). These characteristics suggest evolutionary adaptations for living within eukaryotic cells. Overall, the evidence presented in this work suggests a consistent symbiotic sponge–calcibacteria relationship for the following reasons: (i) calcibacteria are present and abundant in all representatives of the host taxon (genus Hemimycale) reported from several oceans under distinct ecological conditions, (ii) there is a lack of obvious adverse effects in either symbiont or host, and (iii) calcibacteria are vertically transmitted to sponge progeny. This symbiosis likely has profound implications for the sponge biology, with positive, predictable effects. The improvement of sponge fitness through a series of beneficial functions, such as Ca2+ detoxification and supplementary skeletal formation, is the most obvious purported benefits.
Additional advantageous functions derived from calcibacteria can be proposed. Hemimycale are not edible by fish, sea urchins, or other benthic predators (Becerro et al. 1997), although the siliceous skeleton, which has been reported to deter fishes from consuming some sponges (Burns and Illan 2003), is poorly developed. Hemimycale columella also seems to be weakly defended chemically, as its secondary metabolites only display antimitotic activity (Becerro et al. 1997). The enormous density of calcibacteria at the sponge surface is likely to contribute to the reported deterrence against potential predators. However, the putative antipredatory role of calcibacteria and the environmental conditions preventing or enhancing their proliferation deserve future ecological investigations.
On the other hand, the swift proliferation of the bacteria and their consequent accumulation in the host tissues suggest potential interference with the sponge's biological functions. Curiously, calcibacteria accumulation does not seem to harm the sponge in any appreciable way. In fact, Hemimycale sponges grow faster (according to the authors' current research) than other demosponges in the same habitat (e.g., Turon et al. 1998; Blanquer et al. 2008; De Caralt et al. 2008), perhaps assisted by the supplementary biomass of replicating calcibacteria. Calcification within the calcibacteriocyte vacuoles could be a mechanism that maintains bacterial replication below a threshold that could damage the sponge. Furthermore, calcibacteriocytes accumulate at the sponge periphery and release calcibacteria to the sponge periphery in a similar way to that reported for spherulous or excretory sponge cells (Uriz et al. 1996). Together, these mechanisms may prevent calcibacteria from harming the sponge and, as a by-product, allow for the formation of a rudimentary skeletal cortex on the sponge surface.
The extent of the sponge–calcibacteria association remains unknown, but this symbiosis may be more common than presently thought. The customary use of nitric acid to remove the organic matter for spicule cleaning dissolves calcium carbonate and may have prevented their detection in typical taxonomic studies. Calcibacteria have also recently been found in the poecilosclerid Crella cyatophora from the Red Sea (authors' current research) and, as stated above, may be present in the Spirophorida Cinachyrella alloclada (Rützler and Smith 1992). If calcibacteria occur in at least two distant Orders of demosponges (Poecilosclerida and Spirophorida), and might have been overlooked in others, then a phylogenetic study of these bacteria may give insights on the evolutionary origin of this association and possibly trace it back to a common sponge ancestor. This scenario might support the potential role of endosymbiotic bacteria on the origin of calcification processes in basal metazoans, backing implications of previous studies suggesting horizontal transfer of bacterial calcification genes to sclerosponges (Jackson et al. 2011). As in sclerosponges, the siliceous skeleton of Hemimycale has been reduced considerably compared with taxonomically close species, perhaps to accommodate the extensive calcareous sclerites. However, we have shown here that unlike in sclerosponges, in which calcification is ultimately produced by the sponge (Jackson et al. 2007, 2011), the calcium carbonate skeleton in Hemimycale is a direct result of the associated bacteria.
The elimination of excess cellular Ca2+, which is incompatible with life, has been proposed as one of the evolutionary forces triggering a variety of mineral exoskeletons in marine invertebrates (Simkiss 1977; Brennan et al. 2004). Relatively early in animal life, the Ca2+ ion became a messenger of basic cell functions, which forced cells to maintain intracellular calcium ions at concentrations several orders of magnitude lower than those in the ambient seawater (William 2000). Thus, once cells were threatened due to calcium overshoot, natural selection would have acted to fix mechanisms for either actively reducing intracellular Ca2+ concentrations or tolerating the intracellular precipitation of calcium salts (Lowenstam and Margulis 1980). It has been hypothesized that calcium extrusion first evolved in prokaryotes to protect magnesium enzymes and to prevent phosphates from precipitating (Hoehler et al. 2001). According to the widely accepted "seriated endosymbiosis theory" (Margulis 1981), it is conceivable that bacteria also played a role in calcium regulation in early cell consortia. However, calcifying endosymbiotic bacteria have not been reported in metazoans until now.
The Hemimycale species certainly have the potential to serve as exceptional living models for the experimental study of bacteria-mediated calcification in metazoans and the biological interactions between calcibacteria and eukaryotic cells.
Associate Editor: J. Wernegreen
The authors thank the Scientific-Technical Services of the University of Barcelona, in particular N. Cortadellas and A. García for processing the TEM samples; J. M. Fortuño from the Instituto de Ciencias del Mar (ICM), Consejo Superior de Investigaciones Cientficas (CSIC) for processing the SEM samples; C. Gutiérrez and J. C. Auguet for laboratory help with the FISH techniques; P. Galand for help with the pyrosequencing blasts; A. R. Pérez-Porro for providing one sample of a ripe sponge for the TEM study; E. Macpherson for helpful discussions; and P. Erwin for improving the English style. We especially want to pay homage to the late Prof. Lynn Margulis, who provided insightful advice and useful discussions on our results. The study has been funded by projects CTM2010–22218, MICINN and SGR2009–655, Generalitat de Catalunya to MJU, and CGL2009–13318, MICINN to EOC.
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Well, I'm just waiting to go to lunch so I can see if a dog in my mother's neighborhood is loose or in her own yard still Yesterday she was out and sitting in the street in the rain. Poor little Chi. This time she came right to me and I picked her up. No one was home, but the front door was unlocked, so I put her inside and left. This morning she was out in her yard again. They leave her out all day, it seems everyday and she goes under the fence and around the 'hood. If I catch her out again, I'm taking her home where she'll be secure until they come home and I can talk with them. Wish me luck - wish her luck.
Sounds like a plan MaggieNae. When we were in Florida and lived on a farm my dogs stayed outside all day but we had good fencing. Our neighbors had a yorkie that would get out constently and come and play with my shepherds. After a while I stopped bringing her back. My neighbors finally got better at making sure she was inside or watched her when she was out.
My day started out horribly - I woke to horrendous pain. A storm moved in during the night and it has hit my poor body like a ton of bricks. I got up and fed the brood and was bent over just aching all over - got to my chair and remembered that I had invited a member of our ward to lunch today - her daughter is having a terrible time back east with her health and I wanted to give the friend a lunch out and some loving kindness... I thought Oh i should message her and cancel as it is not a good day -but I didn't got myself in the shower and left for lunch... Yep it was a great lunch - the Lord got me through it and it was wonderful for her - she thanked me so much - she is home alone as her youngest daughter is off at girl's camp and DH is out of town this week... she needed this badly and i was so glad that I got myself up to do it.
It was hard but the rewards are wonderful. I am back home now - in my chair as I don't think anything else is going to get done today... Not a dang thing - maybe later as this eases up...
I did have a good thing also to tell you - whomever buys our home is going to have an awesome house... we put in a water softener a few years back and the man who put it in didn't clean up after him self so my kitchen sink got rust spots in it - it was one of the white sinks - beautiful when it was cleaned up but I just couldn't get this stain out.. so told DH we needed a new sink and I had seen that new one hole water faucet at my son's house while up there and liked it - the end pulls out for your sprayer - very nice... so I had gone looking on Amazon for a sink and faucet and found one for $700 on sale for $115... bought it and the faucet and a single hole plate to cover the two holes... well it came and DH put it in yesterday and Oh My it looks so good - my kitchen is looking awesome - Bathroom all modernized - kitchen all modern looking and now DH says I like our house now why are we selling it ... then he laughs as he knows we are going north to be with our grand kids... it is rough though as I really do love our home...
Anyway, I am asking for a few little prayers said my way to help me through the rest of this day... My BP is high as the stress I have been under lately has been rough - some nasty emails came my way and made me cry - a good friend lifted me up and I lifted another friend up but today is painful and I need a few prayers - I know they work and I know the wonderful ladies on here when they pray for me do wonders for me - so thank you in advance.
I am feeling better already although I doubt I will be moving out of my chair for a few hours...
Hi Thea. I'm so sorry you're having such a bad day. That's no fun. I will pray that you feel better quickly and hope the nasty emails stop. Just sit back and relax. :)
Thea, praying for you!! May you have a goodnight of rest tonight and a better tomorrow ! I can't fully understand all your pain , but my little DD can , she has been on the couch all day , even with meds she can't control her arm to feed herself!
was sweet of you to take the dear lady to lunch! :)
🌺 You know you are a quilter when the only scraps under your table are fabric.🐾
Thea I am praying for you. Glad your lunch went well. I always feel better when I am serving someone else. Hang in their. Maybe you should get a blessing. That always boosts my spirits and helps me deal with the pain.
Mamagshouse Donna Gilliam:
Zoetc that looks like fun. Was it hard to use them.
Zoetc that looks like fun. Was it hard to use them.
No, not at all. I was nervous at first and always as we went through intersections. I felt surely I will fall or a car will jolt out, but none of that happened.
Cool Zoetec I'll have to try one one day.
This is what I completed today. My DS Guitar Crochet Quilt.
Good afternoon!
I have one thing on my mind today: donuts. It's national donut day! I even posted something about it on QCA's Facebook. (check it out-it's a donut quilt :) ) If there's one thing I have a problem with, it's my sweet tooth. I haven't had a donut today, but a delicious bakery is on my way home. It would be terrible if I did not celebrate such an important holiday!
In other news, I have wonderful plans to rise bright and early and go to our downtown farmer's market. Des Moines has an amazing market, and it's usually packed before it even opens! And then it is off to outdoor yoga at Grey's Lake. It's beautiful scenery, relaxing, and free!
What are all of your plans for the weekend? I would love to hear them!
I love donut's. Yummy. Not to many plans this weekend. Having a BBQ tomorrow night at the Club House with the Elders Quorum at church. Waiting for WeeZee to post her Clue #4 then I will post mine.
Mamags, the quatiar fabric is so cool, and I love blue!!
Qcarachael, I have a cup of coffee and a donut! One of the church lady's made some for me. I had sewn a dress for her DD. That was my pay!! Love homemade donuts!
Quite weekend here. Blessings to all this weekend!
Thanks Deb.
Mamagshouse Donna Gilliam:
Scanned al the photos I am going to use for my DM's memorial quilt. I have to get printable fabric sheets. Has anyone used the ones below or can you recommend any?
Scanned al the photos I am going to use for my DM's memorial quilt. I have to get printable fabric sheets. Has anyone used the ones below or can you recommend any?
Yes have used those and just about everyone that is on the market. Do you use a lot of printer sheets? Have you thought of making your own? It's very easy.
No sewing so far today...only yard work. Spent 3 hours pulling some notoriously hard to pull Nevada weeds...these, if not pulled soon, will turn into tumble weeds. Pulled 4 large garbage sacks (the kind used for construction so they are 50 gallon bags) and haven't really made a dent...but the area around the front fence is pretty clean now. Also spent about an hour picking cherries, but now it is too hot and the sun is too intense to be out there. Will try to get the rest done this evening, if I still have some energy left.
I've got to give the dogs a bath later today...they are so covered in dirt. They have dug a big tunnel trying to go after the voles...they can both disappear in the tunnel at the base of the dead apple tree. I'm sure the voles killed the tree by eating the roots. It didn't even have good apples on it anyway, always worm infested, even if we sprayed them. Sometime this fall we are going to have to take it out, should be fairly easy as all of the roots are exposed now.
Time to think of something healthy and not fattening for lunch...it won't be easy. My diet is so restricted right now because of Type 2 diabetes and exceptionally high cholesterol and triglycerides.
But I have lost 5 pounds this week, so that is keeping me motivated right now.