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Zenda Homes for Rent
BusinessWeek "Best of the Web" Winner 3 Years Running
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This is by far the most dependable and easiest to use rental search on the internet. It has been so helpful in finding both apartments and homes for rent. - Zenda,WI
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Globe 10.0: Are Hightower and Jones super sophomores?
(Boston Globe) In this episode of Globe 10.0, Kevin Dupont and Chad Finn debate how good Chandler Jones and Dont'a Hightower will be in their 2nd season. (By Alan Miller, Globe Staff)
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subset: commoncrawl


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Lomax, T; (1998) "Making the Walsall Saddle". Walsall Metropolitan Borough, Walsall Town Centre.
Full text not available from this repository.
|"Making the Walsall Saddle"
|Additional information:
|This work formed part of the wider commission for Walsall Metropolitan Borough Council see (2001), "Nombelisk." (2001), "Walsall MBC town Centre." (2001), "Source of Ingenuity." This part of the commission was a collaboration with the artisans and managers of Jabez Cliffe the Walsall based internationally reputed manufactures of bespoke leather saddle.
|UCL classification:
|UCL > School of Arts and Social Sciences > Faculty of Arts and Humanities > Slade School of Fine Art
Archive Staff Only: edit this record


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Plan your ultimate summer escape with the new issue of Time Out
This week's magazine offers weekend adventure ideas for beach bums, hikers and foodies—plus we find out, just what the hell is up with single men who own cats?
Wed May 28 2014
CommentsAdd +
Warm temperatures seemed like a distant dream this winter, but summer has finally arrived. That means it's time to shove the boots and puffy coat under the bed, throw open the front door and start thinking about getting the hell out of the city for a few days. Weekend getaways may take a bit of planning, but this week's issue of Time Out New York has a bunch of ideas for all kinds of adventurers. The list gives off-the-beaten-path recommendations for beach-goers trying to avoid Cape Cod traffic, gay dudes searching for the next Fire Island, artsy types who are over the Beacon hype, and many more.
There's no shame in wanting to stick around the city, though—the new issue also includes a breakdown of three street fairs to choose from this Sunday alone. And if you want to stay local but get a quick taste of the tropics, we've got a list of bars around town serving boozy, refreshing summer cocktails.
Plus: Fellas with felines talk about why guys should be proud of owning and loving their cats (yes, that's plural), and a guide for staying stylish yet comfy at Governor's Ball.
Get your copy today!The new issue is on sale now, or you can go here to subscribe.
You can also download the digital edition from iTunes, Google Play and Nook.
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Get in touch
Dana Varinsky, Editor
Twitter: @dgvarins
Email: dana.varinsky at timeout.com
Will Pulos, Assistant Editor
Twitter: @willpulos
Email: will.pulos at timeout.com
Top stories
- Awesome photos of the NYC LGBT Pride March
- Marc Maron talks feral cats and returning to NYC
- The best art exhibitions to see this summer
- Patti LuPone talks theater, pepper blood and her new play at Lincoln Center
- 10 reasons why Goodfellas is the most important movie of the last 25 years
- The 20 best bagels in New York, ranked
- Game of Thrones' most shocking moments
- Amazing pictures of classic Broadway shows by Joan Marcus
- The best outdoor yoga in NYC
- Take a tour of New York's most beautiful bridges


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The Leading eBooks Store Online
for Kindle Fire, Apple, Android, Nook, Kobo, PC, Mac, BlackBerry...
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Bathroom Remodeling For DummiesWiley 2011; US$ 15.99
We're a nation of countless bathrooms that are on the brink of being torn apart, redesigned, and remodeled. Bathrooms are big! They've gone from being a convenience to a luxury. Welcome to Bathroom Remodeling For Dummies , where you'll discover how to transform your bathroom from blah to beautiful and inefficient to well-designed. Remodeling a bathroom... more...
Fix It & Flip It: How to Make Money Rehabbing Real Estate for Profit Even in a Down MarketMcGraw-Hill Education 2008; US$ 25.00
Repair, remodel, resell-and get your top investment dollar The current real estate market is ripe for rehabbing. Based on decades of successful flipping experience, this Revised and Expanded edition of the real estate classic Fix It and Flip It will help you buy the right property at the right price and use professional estimating tools to... more...
Fix It & Flip It WorkbookMcGraw-Hill Education 2008; US$ 25.95
A Fail-Safe System for Making the Right Decisions About Your Investment A real-estate classic, Fix It and Flip It is a favorite of those who purchase, improve, and sell properties for a profit. This companion workbook has all the worksheets, checklists, and spreadsheets to help you implement the strategies in the book. A bonus accompanying... more...
Coastal Cruising Under PowerMcGraw-Hill Education 2006; US$ 24.00
Coastal Cruising Under Power is your ultimate companion for exploring the coastal waters of North America with confidence in your powerboat. This beautifully illustrated guide provides countless tips on anchoring, piloting, weather, and more, along with in-depth, step-by-step instructions on numerous specific skills and techniques. more...
Home Improvement All-in-One For DummiesWiley 2011; US$ 23.99
Put on your grubbies, get out your tools, and get ready to tackle home repairs and improvements with the goof-proof instructions in this guide that combines the best of nine For Dummies home improvement books in one comprehensive volume. Whether you?re an accomplished do-it-yourselfer or a novice, the easy-to-follow instructions, complete with photos... more...
- 1
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The Ware Center Jazz-in-the-Sky Series Presents Ken Gehret's A Very Jazzy Christmas, 12/14
Settle down at one of the candle-lit cabaret tables or sink into a comfy leather sofa in the Ware Center's beautiful Grand Salon overlooking the twinkling lights of Lancaster city as you listen to Ken Gerhart perform classic holiday repertoire with a twist of jazz. Ken has been performing as a musician for more than thirty years, primarily as a guitarist/violinist, in various musical scenarios. Ken performs, teaches, composes and arranges for over 17 instruments. This cabaret is the last live performance in the Fall 2012 Artistic Season at the Center. The Winter/Spring 2013 Season does not begin again until February. The Ware Center will not be open to the public from Dec. 16 – Jan. 25 for continued renovations to the building.
Ken was born to be a musician. He says "On the day I was born, my mother spoke these words: 'My son is going to be a musician as she held my hands in hers. As time passed, my first awareness of music was hearing my "Bro-Joe" play the guitar. I knew then, music chose me and I happily accepted. My insatiable curiosity was ignited with the "flame" (of music). I think of my musical life as a kid in a candy store!"!
His musical experiences stem from primarily a country music background, performing with well-known stars (at the age of 18) including Roy Clark and Mel Tellis. He has performed live with Jim and Jesse and the Virginia Boys. His shows have been televised from the Grand Ole Opry in Nashville, Tennessee . Among his most notable achievements is he is the designer/inventor of the 10 string guitar.
Ken's philosophy is: "Some people play golf. Some play bridge. Others jump from cliffs using bungee cords. I like to play music. Music is what I do and love. I believe music is the most honest and immediate form of communication available to human beings."
Ken, who will be playing violin and providing vocals, is accompanied by Tom Witmer on guitar, Keith Mohler on bass and Dave Young on drums.
Ken's performance is sponsored by the Ware Center, Rodgers & Associates, Ann B. Barshinger, Peggy Neff, Aussie & the Fox, Fine Living Magazine and Pam & Hugh McGettigan.
Ticket Information: This event is open to the public. Tickets cost $20 for general admission, $10 for students, and $65 for a table of four. Tickets may be purchased by calling the Ticket Office at 717-872-3811 or 717-871-2038. Online, at MUTicketsOnline.com. Or, in person at The Ware Center Box Office (42 N. Prince St., Lancaster). Ticket Offices are opened Monday through Friday, 8 a.m. - 4:30 p.m. Tickets are also on sale – if still available – one hour prior to the performance. Seats are limited. Advance ticket reservations are strongly encouraged.
About Author
- Casting Roundup: Sarah Chalke, Kim Raver Headline New Pilots
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- Pilot Season Roundup: Colm Feore, Daniel Stern, Dave Annable to Star in New Shows
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- CONSTELLATIONS' Jake Gyllenhaal and Ruth Wilson Set for THEATER TALK This Weekend


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Parking tip
There is ample parking around the castle walls. I parked the Volvo in between two other cars, despite the comment of my friend that the space was too tight. NOT! Which gave us both a laugh :-)
The secret is, a wise person once said: always park backwards, however small the space is to turn your car. Because you'll need more space if you park forward and then have to turn around to drive away.
It is great fun though, to see people run into trouble by not parking correctly. The manoeuvres!
- Road Trip
This map is to give you an idea where Arnhem is located. It is in the eastern part of the country. The second red dot you see on the map is the town where I used to live: Oldenzaal
- 1
Instant Answers: Kasteel Doorwerth
Get an instant answer from local experts and frequent travelers


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Sydney Opera House
Issued Monday, October 13, 2008The Famous Spiegeltent to arrive in Sydney one week late – but the shows must go on!
Sydney Opera House today regretfully announces the delayed arrival of The Famous Spiegeltent affecting its Gorgeous! program of music, comedy, cabaret and kids shows.
The Famous Spiegeltent was expected to arrive in Sydney on October 10 but the container ship transporting the tent from the United Kingdom has been delayed at sea.
The Spiegeltent will now arrive in Sydney on Friday, October 17 and will be constructed over the weekend.
Due to this unavoidable delay, performances scheduled to take place in The Famous Spiegeltent on Friday 17, Saturday 18 and Sunday 19 October will now take place in The Studio.
Access to The Studio and tickets for affected shows can be collected from the box office located in the Drama Theatre foyer on the western side of the building.
Please note that the 10am performances of Men of Steel on Saturday 18 and Sunday 19 October have been cancelled. Patrons with tickets for these performances are welcome to attend the 2pm show which will take place on both days.
From Tuesday 21 October onwards, Gorgeous! shows will take place in The Famous Spiegeltent on the Sydney Opera House Forecourt as previously advertised.
Sydney Opera House Chief Executive Richard Evans said we are committed to bringing significant international productions to our audiences.
"The nature and beauty of live performance means that even the best laid plans can go awry and Sydney Opera House apologises for any inconvenience the delay of the Spiegeltent arriving may cause," he said.
"However in the tradition of our business, the show must go on and we trust our patrons will enjoy the great acoustics and intimate surrounds of The Studio."
The Famous Spiegeltent GORGEOUS!
Dates/Times: Tuesday to Sunday from 21 October to 15 November.
Show times vary.
Venue: Sydney Opera House Forecourt
Tickets: From $18 - $49
Bookings: (02) 9250 7777 or online at sydneyoperahouse.com
Various Venues
© Copyright 2015 Sydney Opera House


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tony_sutton: @svengalie Yeah, the price is a bit on the high side. I'm sure it will drop down soon. :D
UniqGiri: If toast always lands butter-side down & cats always land on their feet, what happens if U strap toast on the back of cat & drop it? ~Wright
koehorst: Morning TWEEPS ;) The classic example of Murphy's law: If you drop a piece of toast it always falls buttered side down....SO TRUE !!!!
_kkaycee: lol im remembering when @fknMelissa asked me to drop her off at her house cuz it was down the street& it was by the other side of the city!
BigPapaPedro: Wife lost phone this AM. I went to drop off my son to school and saw 1 of the plastic covers on side of road. Walked up & down but nothing.
sca11ywag: @Chris_Boardman A. even if they weren't round they'd still land butter-side-down when you drop 'em. Law of Physics.
rainhamfestival: We are after a 12ft long (ish) Flatbed with drop down side Ifor Williams
trailer to purchase in good condition &... http://fb.me/GNKLe7qn
Amiess_: OG you can do the giant drop UP SIDE DOWN
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Bonjoviluvr17: @Mary81bjc once you get to home page, click on music at the top, from the drop down menu, choose rock..then look to the side..they are #1 ?
AlwaysEpicMB: If toast always lands butter-side down, & cats always land on their feet, what happens if you strap toast on the back of a cat and drop it?
Lisa_Bryant_16: @coreyfmurphy Go to wboc.com and fill out the tour request form. It's on the left hand side under "community" drop down menu.
Djcornbreeze: Ok, things have calmed down enough to drop the all caps. She's 33 weeks and is feeling pain on her left side, let me know, should we worry?
Covey89: @frenchgirl82 Aww thats ok :) I think you have to go onto my twitter page and select DM from the drop down menu at the side
xo__0x: @Pkittytrisha shoot first bird over and make him knock down right side of house then both yellows at left side, drop egg, done.
blinksalePD: @DamianMadray You can edit your invoice and on the right hand side, select 'No Late Fee' from the drop-down menu. Let me know if that helps.
ivebeengilmored: @ilovemakeup86 I've done that so many times, I tend to drop it down the side of my bed too.. ha!
xColeyyx: If toast always lands butter-side down and cats always land on their feet, what happens if you strap toast on the back of a cat and drop it?
chockwit: If toast always lands butter-side down, cats always land on their feet, what if you strap toast on the back of a cat and drop it?" S. Wright
1ChrisRob: @Short_N_Sweet_ ...that's grimy. I hope you drop your pie potoato side down on the floor. [if not, save me some :) ]
maryannjohanson: Devil (review): Ever drop your toast and despair to watch it land on the floor jelly side down? You know who's res... http://bit.ly/bcXC0i
samuraiacid: Always two sides to every story of course unless you drop the toast on the buttered down side and it lands on a carpet? <--- err????
bruh_itsjordan: i dont stop wen drop my side chick off.....she gotta drive out wen i slow down #BecauseImGangsta
Drop down the cord from the roof by the side of the wall for an interesting and unusual look. There are many easy ways that you can be creative in decorating your home. Use ideas you have seen on shows, but with your own twist to it
To get the offers, visit Groupon, sign-in or sign-up, then select the city that's listing the deal you want from the drop-down list of cities at the top of the Groupon site. ... All Cities (Look on the right side of the page, after the side deals, deals nearby, etc.)- Get $50 worth of maternity from Belly Dance Maternity for $25. The Groupon can be applied toward shipping. Even though this Groupon is found on each city's page, it looks like the limit is 1 total
Last up was one of the drop target optos for the right side drops. When a game was started it would reset the right side drop target bank 3 times (even though all the targets were up.) This was a sign the game wasn't reading all those ... Back in 1985 Gary Stern ran the "classic" Stern pinball company down the crapper. Apparently Gary Stern didn't learn anything from that experience. Because he's making ALL the same mistakes today with the "new" Stern Pinball company
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They lock up again and JTG with a side head lock. JTG with a drop kick followed by a hip toss and knee. JTG with a flip shoulder tackle and he gets a near fall. JTG backs Primo into the corner and he goes to the turnbuckles and punches him. ... Gabriel with a forearm but Cena with punches and a standing drop kick that connects for a near fall. Cena goes up top but Gabriel punches Cena. Gabriel with a forearm and then he hits a superplex and both men are down
Tuam kicked off the season in incredible style last week but must face a Corinthians side that cannot afford two straight defeats at this early stage. That said, the home side are in trouble with injuries and won't be much better come Sunday. ... This season, the top four sides in Division 1b will be promoted to a new second tier joining the three sides that drop down from Division 1a. That's the target for all in the league, the battle should be intense, with Buccaneers
We went to a different spot and Charity was surprised because it wasn't a typical spot for them to cross..it was a steep drop down to the water and some would make it across but then they'd get stuck on the other side due to the current
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You can now invite anyone to meet at your favorite Starbucks.
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41 year old woman | Norwood, PA, USA | Seeking men 39-49 within 25 miles
Active within 3 days
Looking for someone who sees the big picture, but appreciates the finer details.
So, here it is: the profile, the thing everyone hates to write. SWF, 40, normal. I try to surround myself with people who are honest and to the point, and that is how I want a relationship as well. I work too much and have an independent streak, ok stubborn. I am willing to try new things, consider myself a bit of a foodie, and like to find off-the-beaten-path places, like a local winery or a nature site. I have followed that sign from the interstate that says "attraction" or "historical marker." Fun facts: I have only had two cars in my life, I have two Phillies shirts and a Gagne jersey hanging in my closet, I make my grandmother's gravy for Italian night, I have too many pairs of shoes, I like the color yellow, and I am a loyal friend. I am looking for a good match to my personality. Someone who wants to do things and has ambition. Someone who wants that random text during the day that makes them smile. Someone who has a job and wants to build something together and is not just out for themselves. Is that you?
Coffee and conversation , Cooking , Dining out , Museums and art , Exploring new areas , Performing arts , Political interests , Shopping/Antiques , Volunteering , Watching sports , Wine tasting
Golf, Running, Tennis / Racquet sports, Walking / Hiking
I exercise 3-4 times per week.
Cancun, North Wildwood, DC
Louisiana State University and Agricultural and Mechanical College, Baton Rouge, LA
Cheese, Instagram, snow (less than 50 inches a year!), hugs, a balanced checkbook, finding solutions to problems
Shopping. Occasional live theatre. Watching a good classic movie like the Wizard of Oz. North Wildwood Polar Bear Plunge. Real quiet time off the computer and off social media.
Probably a magazine. I would like to read more then I actually do, but third person novels, action/adventure stuff, history stuff about the American Revolution, a fashion magazine are all go to choices.
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What she is looking for
|5'2" (157cms)
|5'5" (165cms) to 6'1" (185cms)
|Body type:
|No preference
|Blue, Brown, Hazel
|Dark brown
|Light brown, Dark brown, Blonde
|No Way
|No Way, Cigar aficionado
|Social Drinker
|Political / Govt / Civil Service / Military
|No preference
|I'll tell you later
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|Never Married
|Never Married, Widow / Widower, Divorced
|Have kids:
|No preference
|Wants kids:
|Not sure
|No preference
|White / Caucasian
|White / Caucasian
|Christian / Catholic
|No preference
|Graduate degree
|Some college, Associates degree, Bachelors degree, Graduate degree, PhD / Post Doctoral


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Julianne Hough arrives at the 64th Annual Emmy awards on September 23, 2012
Summer is upon us and that could only mean one thing: Swimsuit season is finally here.
Caitlyn Jenner is giving thanks for the freedom to be herself on this very special Independence Day.
America's founding fathers would have favorited Chelsea Handler's salute to our freedom in a video of her 'liberated' water ski ride.


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SoundCLASH Producer Showcase and Networking Event prized by iStandard
- Music
- 435 Esplanade St., New Orleans, LA, 70116
GROOVE THERAPY: A night of FUNk, R&B, Soul, Spoken Word + more
The Soundclash
Thursday, February 19, 2015 at 8:00 PM - Friday, February 20, 2015 at 12:00 AM (CST)
*FREE before 9pm with Open Mic Sign-Up
February edition featuring Mark Caesar, RQ Away, T-Ray The Violinist, and Mara KT.
Hosted by Tony Wilson and OnPoint El
Sign-Up for the Open Mic in advance online for a slot between 8pm - 9pm AND receive free admission > http://groovetherapynola.eventbrite.com/
A night of FUNk, R&B, Soul, Spoken Word + more...
In order to purchase these tickets in installments, you'll need an Eventbrite account. Log in or sign up for a free account to continue.


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- Sea Winds
- Seaside at Anastasia
- Seaside Villas
- See Ray Shores
- Spanish Trace
- St. Augustine Beach & Tennis Club
- St. Augustine Beach & Tennis Resort
- St. Augustine Ocean & Racquet Resort
- Surfcrest Village
- Surfside Six
- Tradewinds
- Villas of Ocean Gate
- Anastasia by the Sea
- Anastasia Condos
- Beach Club
- Bryn Mawr Ocean Resort
- Captains Quarters
- Casa De Las Sirenas
This property allows you to book online with a credit card. After submitting a reservation request, the owner or manager has 24 hours to accept, at which time your card will be charged and you will receive an email confirmation
Recent St. Augustine Beach reviews
"The house was beautiful and my friends and I had a wonderful time there!"Guest: Margaret m. Date of Stay: March 2015 Review Submitted: March 29, 2015View listing
"The room was adequate..... it needs upgrades - new mattress - new sofa would be nice. The shower/tub upgrade was a plus."Guest: Anonymous Date of Stay: January 2015 Review Submitted: March 3, 2015View listing
"My family of 4, along with my parents stayed here last week. This was the first time we had ever rented a condo and it was great! The pictures provided were exactly what we got when we arrived. Great patio - large enough for us all to enjoy the view of the pool and the ocean. There's a large field behind the pool where kids would play each night. This condo is in the perfect spot for everything....from things to do in the area to the pool and the ocean. It's not in the hustle and bustle of everything, but just far enough out to be peaceful. My kids loved the pool and spent a majority of their time there. The beach is beautiful and just a short walk down the walkway. Local restaurants are all around and I don't think we had a bad meal while were there. The owner was fantastic to work with and we will definitely be back!!!"Guest: Crystal D. Date of Stay: July 2014 Review Submitted: July 29, 2014View listing
"Very nice for a short term rental. Close to A1A but overall nice layout and very clean. Would think of renting in future again."Guest: Barbara A. Date of Stay: January 2015 Review Submitted: February 23, 2015View listing
St. Augustine Beach Travel Tips
Head out to St. Augustine Beach on Anastasia Island for your next vacation. The warm waters of the Atlantic Ocean and the cool ocean breezes are sure to keep you relaxed during your stay.
St. Augustine Beach Attractions
Water activities are the No. 1 thing to do while staying in St. Augustine Beach. Whether you prefer to lay out in the sun or venture out into the Atlantic Ocean, the beach is the best place to spend your day. There are white sand beaches and the St. Johns County Pier is along the water for those who want to enjoy a little fishing. When spending time on the beach, be sure to go for a swim, learn to boogie board or build a sand castle with the family.
Anastasia State Park and Recreation Area is comprised of 1,700 acres filled with sunny Florida fun. The park is a popular spot for wildlife enthusiasts and a protected bird sanctuary. When you visit the park and recreation area, you can swim, hike nature trails and fish. There are 2,000 acres where endangered species at Guana River State Beach roam. There are two public access areas to the 4.2 miles of undeveloped beach. Visitors can participate in a number of activities at the state park including fishing, shell-hunting, swimming, sunbathing and surfing.
Golf is plentiful in St. Augustine Beach and the surrounding area. Head to St. Johns Golf Club for an 18-hole championship course that's well-maintained and perfect for players of all skill levels. The Golf Club at South Hampton features 18 holes, a medium-length layout and six sets of tee boxes, so even the little ones can work on their swing or hit a ball. Other courses in the area include King and Bear Golf Course, Marsh Creek Country Club, The Palencia Club, Royal St. Augustine Golf and Country Club, St. Augustine Shores Golf Course and Slammer and Squire Golf Course. Since the weather is almost perfect year-round in St. Augustine Beach, you can hit the green every time you visit.
There are several types of tours offered in the St. Augustine area including Eco Tours, Flagler's Legacy Tours and Old City Water Excursions. The Flagler's Legacy Tour is one you don't want to miss as you make your way through the former Hotel Ponce de Leon. It was developed by millionaire Henry M. Flagler in 1887 and its Spanish Renaissance architecture has been captivating visitors for decades. Along the tour you'll get to stop in the dining hall where there are 79 Louis Comfort Tiffany stained glass windows. The hotel's original grand parlor is now the Flagler Room and visitors will find Tiffany Austrian crystal chandeliers, an onyx Thomas Edison clock and a photo gallery of the Flagler family.
Things to Do
Shopping: In the heart of downtown St. Augustine there are specialty shops, boutiques and art galleries. The brick streets are populated with Spanish Colonial homes and the storefronts sell apparel, unique gifts, art, blown glass, pottery and custom crafted jewelry. Head over to A1A Highway where shoppers will find specialty stores as well as boutiques, art galleries and stores with the equipment and accessories you'll need for a day at the beach. The St. Augustine Premium Outlet Mall and the Prime Outlets are home to many designer stores that offer massive discounts.
Dining: Whatever you're craving during your stay in St. Augustine Beach, you're likely going to find it. From seafood to Italian, eateries in this getaway location are serving up delectable dishes that are sure to satisfy your taste buds. You can try restaurants including A1A Ale Works, Acapulco Mexican Restaurant, Amici Italian Restaurant, Buck's Pizza, Cafe Eleven, Flavors Eatery, Papagallos Bistro and Woody's Barbecue.
Dow Museum of Historic Houses: This museum is a combination of interactive experiences, objects and exhibitions depicting Florida's historical and cultural development. There are nine homes that make up the Dow Museum of Historic Houses, which range in date from 1790 to 1910. You'll get to learn about more than 400 years of St. Augustine history. This block-long district is also the site where the Emancipation Proclamation was read in 1863 to free all of Florida's slaves. The Prince Murat House is one of the oldest surviving Colonial structures in the city. Prince Murat was the Crown Prince of Naples and after living in the home he moved to Tennessee where he married George Washington's great-grandniece Catherine Gray, who became the first American-born princess. Other homes in the Dow Museum of Historic Houses include the Spear House, the Howells House, the Rose House, the Carpenter's House, the Canova House, the Dow House and the Worcester House.
Churches: There are several historic places of worship in St. Augustine including The Cathedral Basilica, Mission of Nombre De Dios, Grace United Methodist Church, Memorial Presbyterian Church, Ancient City Baptist Church and Saint Augustine Seventh-day Adventist. The Cathedral was constructed in 1793 but later destroyed by fire in 1887. It was rebuilt and has become an important part of the city's history. Mission of Nombre De Dios is located on the site where St. Augustine was founded in 1565. Memorial Presbyterian Church was built in the late 1800s and is the only structure to incorporate Venetian Renaissance architecture in the U.S. Henry Flagler commissioned the church to be built in memory of his daughter, Jennie Louise.


text:
Michael DeNicola
Maybe it's because I still have a rod up my ass from Saturday's loss. Maybe it's because this was one of the worst ideas ever. I'm going to go with a mixture of the two.
Today around noon, Flyers fans came together on the deck of the Moshulu to toss a few bags of tea overboard into the Delaware River. Sort of a reenactment of the Boston Tea Party that took place in 1773, Boston, MA.
Because these simpletons think it's funny, and/or for some reason believe it's a thumb in Boston's eye.
"TAKE THAT, BRUINS FANS!", as one Philadelphian hucks a crate of Lipton over her head.
I can just imagine Boston's reaction to this.
So what's going to happen next? Will Bruins fans all over New England start burning cheesesteak meat? Take a bell and smash cracks into it? Kick the living shit out of any old man resembling Benjamin Franklin? Piss on a painting of Betsy Ross? Wipe their asses with a copy of the Declaration of Independence?
I dunno. What's this got to do with hockey? Absolutely f*cking nothing.


text:
Dennis Pitta
Aaron Wilson Joins Scott & Jeremy For Full Training Camp Update
Aaron Wilson of the Baltimore Sun joins Scott & Jeremy for a full update from the castle in Owings Mills.
Glenn Younes: Ravens – No Surprises
I can already tell you what the #Ravens slogan is… or at least should be. NO SURPRISES. Think about it. It's the theme of the year.
Vinny Cerrato: Ravens Camp – Impression Of Offense
Vinny breaks down the Ravens offense after checking out their minicamp at the Castle in Owings Mills.
Scott Garceau: Ravens Tighten Up
Owen Daniels welcome to Baltimore! The 2-time Pro Bowl pick has agreed to a 1-year deal and should be a nice 1-2 tight end punch with Dennis Pitta.
Last Call: Are The Ravens Heading In The Right Direction?
Clifton Brown joined Zinno on Last Call to talk about the Ravens off season activity.
Scott Garceau: Ravens Slow Roll
Really, should we be surprised that the Ravens didn't dive head first into free agency? Not if you've been paying attention for 18 years.
Ravens TE Pitta Calls 5-Year Contract 'A Blessing'
It wasn't long ago when Baltimore Ravens tight end Dennis Pitta wondered if a serious injury would end his NFL career.
Dennis Pitta Joins The Scott Garceau Show
Baltimore Ravens Tight End Dennis Pitta talks with Scott and Jeremy about a number of things…
John Eisenberg On Chances Of Eugene Monroe Returning To Ravens
John joins Ed and Steve to discuss the Ravens free agency and the Dennis Pitta extension.
Joe Platania On The Ravens And Dennis Pitta Together For 5 More Years
Joe joined Ed & Terry to talk about the Ravens re-signing Dennis Pitta to a new 5 year contract.
Jeremy Conn: Five More Years Of Pitta..
So the Ravens have signed Dennis Pitta to a 5 year deal with a base value of $32 million over five years, which works out to an average of $6.4 million per year. It's a good […]
Ravens, Dennis Pitta Agree To 5-Year Deal
The Baltimore Ravens and tight end Dennis Pitta reached agreement on a five-year contract Friday.


text:
On Tue, Jun 1, 2010 at 5:03 PM, Shelley Powers <firstname.lastname@example.com> wrote: > Something for people to keep in mind, now -- the chairs don't judge based on > the proposals or counter-proposals, only the objections raised in the > surveys for both. I don't believe this was clearly stated in the decision > process. What are you basing this conclusion on? ~TJReceived on Wednesday, 2 June 2010 00:48:39 UTC
This archive was generated by hypermail 2.3.1 : Wednesday, 7 January 2015 16:25:48 UTC


text:
Former Sparta mayor accused of using state car, gas card & police databases for personal purposes
Brian Brady of Sparta, a suspended police captain with the state Human Services police and a former township mayor and councilman, was indicted Friday on charges that he submitted fraudulent time sheets that indicated he was working when, in fact, he was traveling out of state for personal pleasure or business, or gambling in Atlantic City.
Brady, 50, is also charged with filing fraudulent firearms qualification certificates, misusing police databases for personal purposes, and using a state vehicle, state gas card and state-issued E-Z Pass on the personal trips.
State Attorney General Jeffrey S. Chiesa said Brady is charged in a nine-count state grand jury indictment with three counts of official misconduct, one count of pattern of official misconduct, one count of theft by deception, two counts of tampering with public records or information, and two counts of computer theft. If convicted, he would face a sentence of five to 10 years in prison on each official misconduct charge, including five years without possibility of parole, and a consecutive sentence on the pattern of official misconduct charge.
The charges stem from an investigation by the state Division of Criminal Justice Corruption Bureau and state Department of Human Services.
Brady is the third highest ranking officer in the Human Services police, reporting to the chief and the director. The Human Services police provide services at state developmental centers and psychiatric hospitals. They are also assigned to protect offices and case workers for the state Division of Youth and Family Services. DHS immediately suspended Brady when he was charged by complaint in the case in May.
"It is deeply troubling that a police supervisor, who has a sworn duty to uphold the law, is instead charged in this indictment with violating the public's trust through multiple criminal acts of dishonesty and theft or abuse of police resources," Chiesa said. "By aggressively prosecuting official misconduct, we will deliver a loud and clear message that nobody is above the law."
"This captain allegedly falsified timesheets in order to collect pay as if on duty for days when he was away on vacation or traveling for personal business," Criminal Justice Director Stephen J. Taylor said. "On top of that, he allegedly used a police vehicle and state-funded gas for those personal trips. We will not tolerate that type of abuse of public office."
In connection with the first count of official misconduct, it is alleged between March 2007 and October 2010, Brady took blocks of personal time without submitting documentation for the appropriate leave time. Instead, Brady, whose annual salary is $101,000, allegedly submitted false time sheets indicating he had worked on days when he was away on personal trips, including ski trips to Vermont, trips to New York for his personal consulting business, and visits to Atlantic City and Delaware to gamble.
On some of these personal trips, including travel outside of the state, Brady allegedly used a state vehicle and a state-issued E-Z Pass, and purchased gas using a state gas card.


text: what a day today, looked beautiful this morning so we rented bikes to ride across the Golden Gate Bridge. As we near the bridge (I'm towing Amelia in a sidecar pod thingy, and I am pretty out of shape), the fog rolls in heavy and the temp drops about 15degrees, then gail force winds kick in. We didn't let it get in the way though, we pushed on and made it to (forgive my spelling) Salusito, a nice little town on the other side. The sky cleared a bit and we took the ferry back after 5. I really like San Fran a lot, several parts remind me of Wellington, NZ. I know we will not get to do near enough so we are considering this a scouting trip. Next time we come to the US San Fran will be our gateway (since they have NZ direct flights) and we'll be able to hit the ground running. My legs are going to be in some serious pain tomorrow, anyone have a Segway I can borrow?


text:
Dark Prints
Started Dec 23, 2012 | Discussions thread
Curious to know how does the original picture print when using the HP software ?
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text:
Portugal boosted their precarious hopes of qualifying for the 2010 FIFA World Cup South Africa™ finals with a hard-fought victory over Hungary in Budapest. Pepe's goal proved the difference between the sides this evening as Carlos Queiroz's team moved on to 13 points in Group 2 and leapfrogged their hosts into third spot.
The result leaves the Portuguese five points behind pool leaders Denmark and two adrift of second-placed Sweden with two rounds of matches remaining.
Portugal have endured a disappointing qualifying campaign and run the risk of missing out on a place at South Africa 2010, with their destiny not in their own hands in next month's final qualifiers.
Despite their worries, Portugal started brightly tonight. Real Madrid's Pepe, occupying a defensive midfield role, converted the visitors' first noteworthy chance of the match in the tenth minute.
A free-kick from Deco on the left side found Pepe, who had timed his run perfectly to nod home what proved to be the winner. Even after conceding an early goal, Hungary kept things tight and looked to hit their opponents on counter-attacks which rarely materialised.
Portugal, too, were wary of compromising their early lead, which made for a cagey first half. The game continued along the same lines after the break, with the match only opening up at all when the hosts upped the tempo in the last 15 minutes.
Cristiano Ronaldo had another quiet night in national team colours and hit his best opportunity of the match only into the body of Hungary goalkeeper Gabor Babos.
Ronaldo's former Manchester United team-mate Nani had a sight of goal two minutes from time, but failed to test Babos.


text:
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text:
I Like to Be Little (Paperback)
With her finely tuned ear for the concerns and cadences of childhood, Zolotow records a little girl describing all the things she likes that grown-ups usually do not. This tale, adapted from Zolotow's I Want to Be Little and newly illustrated with appealing watercolors, will strike a pleasurable chord with adults and children.
About the Author
Charlotte Zolotow author, editor, publisher, and educator has one of the most distinguished reputations in the field of children's literature. She has written more than seventy books, many of which are picture-book classics, such as Mr. Rabbit and the Lovely Present and William's Doll. She lives in Hastings-on-Hudson, New York.
Erik Blegvad was born in Denmark and studied at the School of Applied Arts in Copenhagen. Mr. Blegvad has illustrated more than one hundred children's books, including "Twelve Tales" by Hans Christian Andersen, "Riddle Road" by Elizabeth Spires, "Hurry, Hurry, Mary Dear!" by N. M. Bodecker, and "Sea Clocks: The Story of Longitude" by Louise Borden. The Blegvads divide their time between England, France, and Wardsboro, Vermont.


text:
Time: 2 Hours Tools: Sockets and screwdrivers Talent: (3) Applicable Years: 1984 to 1996 Tools: ¼-inch drive socket set and metric combination wrenches Tab: $100 Tip: You don't have to replace the entire spoiler. Remember it comes in three pieces. The way these spoilers get abused you might want to consider doing one section each… Read more »
Items Posted January 2007
Clutch Hydraulics on the 1984 to 1996 Corvette
Time: 3 Hours Tools: 3/8" drive Metric sockets, box end and flare nut wrenches, GM hydraulic clutch fluid and vinyl hose. Talent: (3) Applicable Years: 1984 to 1996 Tab: $150 Tip: Replace both the master and slave units at the same time When the 1984 Corvette was introduced it used a totally different system for… Read more »
Automatic Transmission Service for the 1984 to 1996 Corvette
Time: 1 hours Tools: 3/8" drive metric sockets Talent: (4) Applicable Years: 1984 to 1996 Tab: $50.00 Tip: Make sure you ask a lot questions before you let a shop tear into your transmission. The expense is great and a lot of things can happen to your car while it's in the shop. Make sure… Read more »
Servicing the Overdrive on the 4+3 Transmission
Time: 1 hour Tools: Drain pain, sockets and 3/8" torque wrench Talent: (3) Applicable Years: 1984 to 1988 Tab: $45.00 Tip: Treat this job the same way you would treat changing the fluid and filter with an automatic transmission. You'll probably end up making just as big a mess on your garage floor. Complementary Work:… Read more »
The LT1 Opti-Spark Distributor
Time: 2 hours Tools: A Visa card with a large credit line. Talent: (4) Applicable Years: 1992 to 1996 Tab: $500 to $1,000 Tip: Don't ever let any water get in the area of the Opti-Spark if you detail your engine compartment. Complementary Work: This would be a good time to install new spark plug… Read more »


text:
Potential students who are interested in pursuing programs of study leading to information technology degrees may enjoy the idea of being involved in creating and/or maintaining technologies used in many businesses and by individuals. Earning an information technology degree may offer many career paths, since computers and telecommunications are used in many facets of our daily lives.
Programs of study leading to information technology degrees can span a broad spectrum of fields. Students looking for a career in information technology may find opportunities in software, mobile communications, networks and information systems. Program courses may address these areas, as well as problem solving, critical thinking, communication and teamwork.
The ITT Technical Institutes are a leading provider of technology education in the United States. Some ITT Technical Institutes offer several different programs of study involving information technology in the School of Information Technology.
Meet a Graduate
Jered Kuhfahl
Information Technology - Computer Network Systems
Associate of Applied Science
Graduated 2011
Omaha, NE
Meet Jered


text:
ASUS 1015E-DS01 10.1" Notebook Computer, Intel Celeron 847, 2GB RAM, 320GB HDD, Windows 8 - 90NB00J4-M00750
|The 1015E-DS01 10.1" Notebook Computer from ASUS features a compact form factor and a stylish look, complete with a matte black finish. The 10.1" HD widescreen LED-backlit display on this ASUS notebook has a 16:9 aspect ratio and a 1366 x 768 native resolution, supported by integrated Intel HD graphics.
The system is powered by a dual-core 1.1GHz Intel Celeron 847 processor. It also has 2GB of DDR3 RAM, a 320GB 5400rpm hard drive, a 4-in-1 media card reader, 10/100Mbps Fast Ethernet, 802.11b/g/n Wi-Fi, Bluetooth 4.0, an integrated webcam and microphone. There are also built-in stereo speakers with SonicMaster technology.
Windows 8 (64-bit) is the installed operating system. Windows 8 replaces the traditional Start menu with a redesigned Start screen. The Start screen features tiles, which provide notifications in real-time. Windows 8 also focuses more on cloud storage as well as apps, which can be downloaded from the Windows Store. Windows 8 provides a unique user experience that differs from previous versions of Windows.
Customer Reviews of ASUS 1015E-DS01 10.1" Notebook Computer, Intel Celeron 847, 2GB RAM, 320GB HDD, Windows 8
great laptop7/10/2014
i was expecting this pc to come a couple days later then it did but when i got it i was satisfied with what i bought there are some problems with the headphone jack but that is a minor problem
good notebook for the price1/13/2014
It is a nice little net book it's fast but it does heat up fast so I do worry about it overheating and the graphics are great want it even better go to bios and change the graphics memory from 64m to 512m It's now even faster I love it And I see how everybody hates windows8. But I love it Oh shipping was fast Thank you again I will refer a friend
This once again is not what I expected The keyboard is defective. once I start to type I get through one or two words and the cursor jumps to another area and then I have to reclick on the area where I was typing to start again. Once again the product description was inconclusive because It does not have a dvd/cd component to install programs. and it is not touch screen either. I would like to return this product for a refund, but no one seems interested in answering any of my emails , so here I


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Page 1
The past, present and future of cell-free
protein synthesis
Federico Katzen1, Geoffrey Chang2and Wieslaw Kudlicki1
1Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008, USA
2Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, CB-105, La Jolla, CA 92037, USA
Recent technical advances have revitalized cell-free
expression systems to meet the increasing demands
for protein synthesis. Cell-free systems offer several
advantages over traditional cell-based expression
methods, including the easy modification of reaction
conditions to favor protein folding, decreased sensitivity
to product toxicity and suitability for high-throughput
strategies because of reduced reaction volumes and
process time. Moreover, improvements in translation
efficiency have resulted in yields that exceed a milligram
of protein per milliliter of reaction mix. We review the
advances on this expanding technology and highlight
the growing list of associated applications.
Since the pioneering studies conducted by Nirenberg and
Matthaei more than four decades ago [1] cell-free protein
synthesis has been a valuable tool for understanding how
mRNAs are translated into functional polypeptides. In
addition, it has been used for antibiotic drug discovery and
has been a means for small-scale expression of toxic
products. With the advent of the proteomics era, the field
has experienced a technical renaissance expanding into a
myriad of applications covering both functional and
structural proteomics. Significant improvements made to
the configuration, energetics and robustness of reactions
have led to productivities that far surpass the milligram
levels of product per milliliter of reaction. Also, important
progress has been made on key reaction components so
that it is now possible to obtain a polypeptide with its
corresponding post-translational modifications or, in the
case of a membrane protein, properly imbedded into a lipid
bilayer. Finally, the ability to easily manipulate the
reaction components and conditions makesin vitro protein
synthesis particularly amenable to automation and min-
iaturization, enabling application to the fields of protein
arrays, in vitro evolution and multiplexed real-time
labeling, among others. In this article we review the key
aspects of this field, emphasizing the most recently
developed applications for both basic and applied
research. A review of the entire field is beyond the scope
of this article and further details in this regard can be
found in previously published literature [2–6].
Configurations and history
In vitro translation systems are based on the early
demonstration that cell integrity is not required for
protein synthesis to occur. In its simplest form, translation
can be accomplished using a crude lysate from any given
organism (that provides the translational machinery,
accessory enzymes, tRNA and factors) in combination
with exogenously added RNA template, amino acids and
an energy supply. This classical in vitro translation
scheme is called 'uncoupled' as opposed to the 'coupled'
or 'combined' transcription and translation configuration
in which the mRNA is transcribed in situ from a DNA
template added to the reaction (see [2] for details). Usually
coupled systems exhibit higher protein yields and are
easier and faster to operate than systems that are not
coupled although they require supplementing the reaction
with additional NTPs and a highly processive RNA
polymerase, such as those encoded by T7, T3 or SP6
bacteriophages. The use of plasmid or PCR templates
rather than purified mRNAs resulted in the emergence of
a variety of new applications.
Sources of lysates
Although any organism could potentially be used as a
source for the preparation of a cell-free protein expression
system, the most popular are those based on Escherichia
coli, wheat germ and rabbit reticulocytes (for recent
reviews see [2,6,7]). The choice of the system should be
determined by the origin and biochemical nature of the
protein and the specifics of the downstream application. In
general, E. coli-based systems provide higher yields and
more-homogeneous samples suitable for structural
studies. The protein yields of E. coli-based systems
range from a few micrograms up to several milligrams
per milliliter of reaction, depending on the protein and the
reaction format [8]. Eukaryotic-based systems, although
less productive, provide a better platform for functional
studies particularly for post-translationally modified
proteins. Reported protein yields for the rabbit reticulo-
cyte lysate are in the microgram or in the fraction of a
microgram per milliliter of reaction range whereas protein
yields of the wheat germ extract are typically two orders of
magnitude higher [2,6,9].
Crude cell lysates are not the only source of the
enzymatic machinery. Despite being one of the most
complicated basic cellular processes, the whole transla-
tional mechanism from E. coli was recently reconstituted
Corresponding author: Kudlicki, W. (firstname.lastname@example.com).
Available online 26 January 2005
TRENDS in BiotechnologyVol.23 No.3 March 2005
www.sciencedirect.com0167-7799/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2005.01.003
Page 2
in vitro starting with O100 individually purified com-
ponents [10]. The system exhibits high translational
efficiency with the added advantage of simpler manipu-
lation of the reaction conditions and easy purification of
untagged protein products.
Productivity and automation
The principal limitation of the first generation batch-
formatted reactions (Figure 1a) is their short lifetimes
(!1 hr) and consequently their low yield. This is primarily
because of the rapid depletion of the high-energy phos-
phate pool, which occurs even in the absence of protein
synthesis [11]. In turn, this leads to the accumulation of
free phosphates, which can apparently complex with
magnesium, and further inhibit protein synthesis. This
problem was first overcome by Spirin and coworkers with
the introduction of the continuous-flow cell-free (CFCF)
translation system, which relies on the continuous supply
of energy and substrates and the continuous removal of
the reaction by-products (Figure 1b) [12]. The reaction
time can then be extended for 20 h and increases the
product yield by two orders of magnitude. Despite the
improvement in yield, the operational complexities make
this system extremely impractical. The technology was
later simplified by the development of a semi-continuous
or continuous-exchange cell-free (CECF) method, in which
a passive rather than active exchange of substrates and
by-products extended the reaction lifetime [13,14]
(Figure 1c). However, semi continuous systems are not
easily applicable to high-throughput processes, which
require miniaturization and automation.
To this end several laboratories have focused either
on developing high-throughput-friendly systems or on
maximizing the energetics of the batch reaction. Endo
and coworkers have devised a highly efficient bilayer
diffusion system devoid of membranes that is compa-
tible with high-throughput formats [9,15] (Figure 1d).
Swartz's group has consistently sought more efficient
and economical alternatives to the traditional ATP
and/or GTP regeneration systems with promising
results. For example, they have recently demonstrated
that is possible to produce close to a milligram of
protein per milliliter of reaction using a batch format
(see [16] and references therein). The same group has
recently shown that a higher efficiency cell-free system
can be obtained simply by mutating genes involved in
the catabolism of certain amino acids [17].
Folding and post-translational modifications
A key goal for cell-free translation systems is to
synthesize biologically active proteins. Currently, the
primary issues are protein folding and post-transla-
tional modifications. A clear advantage that these
systems have over in vivo protein synthesis is that
the environmental conditions can be adjusted easily.
Strategies to improve protein folding and post-transla-
tional processing include the addition of a variety of
reagents and folding catalysts to the reaction.
Feeding buffer
Batch Continuous flow
Continuous exchange Bilayer
Feeding buffer
(a) (b)
feeding buffer
TRENDS in Biotechnology
Figure 1. Current formats of cell-free protein expression systems. Formats are classified according to how the reaction is fed: (a) batch (b) continuous-flow cell-free
(c) continuous exchange cell-free and (d) bilayer. Reaction components include ribosome, translation factors, tRNA, aminoacyl tRNA synthetases, template (RNA or DNA),
and RNA polymerase (when necessary). The feeding buffer includes amino acids, energy components, NTPs (when necessary), co-factors and other accessory reagents.
Yellow arrows indicate the flow of buffer components and red arrows represent the flow of protein product.
TRENDS in BiotechnologyVol.23 No.3 March 2005151
Page 3
Reports on the exogenous supply of chaperones to cell-free
protein synthesis reactions suggest that the effect that
these catalysts have is protein-dependant. For example
addition of purified DnaK, DnaJ, GroEL and GroES has
been reported to be beneficial for the synthesis of single
chain and fragment antigen binding (Fab) antibodies
[18,19] but it appears to have no effect on the folding or
activity of luciferase [20].
Membrane proteins
One of the greatest and potentially most far-reaching
impacts of in vitro translation could be in the area of
membrane protein production. Membrane proteins
account for nearly a third of the genes encoded by most
fully sequenced genomes. However, only a handful of
integral membrane protein structures (!80) have been
solved to high resolution. The paucity of membrane
structures reflects the hurdles that must be overcome
when expressing and preparing these proteins. This
difficulty is particularly daunting when over-producing
large quantities of eukaryotic or mammalian integral
membrane proteins. Over-expression of membrane pro-
teins in vivo frequently results in cell toxicity largely
owing to their hydrophobicity, protein aggregation, mis-
folding and low yield. In some cases, the over-expression of
integral membrane proteins, such as ion channel proteins,
transporters and receptors can disrupt the integrity of the
cell membrane and lead to cell lysis. What is exciting is
that nearly all of these obstacles can be overcome by cell-
free expression. In vitro translation offers a unique
opportunity to use the highly efficient bacterial transcrip-
tion and translational machinery while introducing
natural mammalian or other synthetic lipids. In addition,
in vitro expression can circumvent the problem of post-
translational modification that is essential in vivo and can
impair crystallization. For example, milligram amounts of
active transmembrane multi-drug transporters have
recently been synthesized in vitro [21,22]. The presence
of mild detergents or lipid mixtures during the reaction
notably eased aggregation and insolubility issues and
apparently did not interfere with the translation activity.
Moreover, membrane proteins that require their cognate
partner protein in the proper molar ratios for folding and
solubility can easily be co-expressed by adjusting the
relative amounts of template encoding the two different
subunits [21].
Disulfide bond formation
Disulfide-bonded proteins are ordinarily formed in extra-
cytoplasmic compartments, such as the periplasm of
prokaryotes and the lumen of the endoplasmic reticulum
(ER) of eukaryotes in which conditions are more oxidizing.
Although cell-free protein systems have two intrinsic
features that might prevent the formation of disulfide
bonds (reducing agents that stabilize the protein synthesis
machinery and the lack of compartments with oxidizing
redox potential) these hurdles can easily be overcome. One
method eliminates dithiothreitol from the cell-free extract
before the translation reaction, which results in the high
production of single-chain antibodies with dual disulfide
bonds [23]. Also, the combination of alkylation of the
extract with iodoacetamide, a suitable glutathione redox
buffer and a disulfide bond isomerase added to the in vitro
reaction can have a profound positive effect on the
production of active proteins with multiple disulfide
bonds [18,24].
Glycosylation and other post-translational modifications
Glycosylation is the most widespread and complex form of
post-translational modification in eukaryotes (for a review
see [25]). A major problem for the production of glyco-
proteins is that they are ordinarily produced as a mixture
of glycoforms. Only the glycosidic core remains relatively
uniform while the protein is in transit within the ER. In
cell-free systems, core glycosylation can be achieved by
supplementing extracts with microsomal fractions [26].
Proteins are translocated to the lumen of the vesicles
where their leader peptide is cleaved and they acquire the
oligosaccharide chain. Given that intracellular transport
in cell-free protein synthesis systems is disrupted, further
processing of the oligosaccharides is being prevented.
However, some variation in the glycosylation pattern can
still be observed in cell-free protein synthesis systems
owing to inhomogeneous folding that apparently restricts
the access of the glycosylating enzymes [27]. Recently, the
generation of a Spodoptera frugiperda 21 cell-based lysate
has been reported [28]. The system provides core protein
glycosylation enzymes without the need for supplement-
ing the reaction with membrane vesicles. The newest
approach for the in vivo synthesis of homogeneous
samples of glycoproteins exploits the use of a non-natural
amino acid linked to a monosaccharide moiety [29]. This
strategy could easily be adapted to cover in vitro protein
synthesis. When incorporated into a protein, the mono-
glycosylated amino acid can be further modified by
glycosyltransferases added to the in vitro reaction result-
ing in more-complex glycoforms.
Other post-translational modifications, such as phos-
phorylation, myristylation, farnsylation, isoprenylation
and adenylation have been observed in lysates from
higher eukaryotes (for further details see [2,6,30] and
references therein). With all these modifications, the
dynamic complexity of post-translational modifications
makes it difficult to produce homogeneous protein
samples. Methods for creating artificial post-translational
modification mimics (artificial modifications that imitate
the structure of the natural ones) have been proposed as a
solution for this problem (for a review see [31]). Cell-free
systems seem to be the most favorable platform for this
novel strategy.
Until several years ago, the use of cell-free protein
systems was restricted to low-throughput applications
with low protein concentration requirements. Today,
thanks to significant progress made to the miniaturiza-
tion, automation and optimization of fed-batch and
semicontinous reactions, in vitro synthesis of proteins
has found a large variety of low- and high-throughput
applications suitable for functional and structural
TRENDS in BiotechnologyVol.23 No.3 March 2005 152
Page 4
Structural proteomics
Proteins that pose obstacles for expression and analysis
are under-represented in the protein structure databases.
Toxic proteins, membrane proteins, complex assemblies
and proteins that exhibit insolubility issues are examples
of under-characterized proteins. The flexibility that cell-
free protein synthesis offers for manipulating reaction
conditions and directing all metabolic resources to protein
production has already proven to be effective in filling this
void. First, the productivity of in vitro systems based on
Esherichia. coli and wheat germ extracts has surpassed
the milligram levels per milliliter of reaction and the
products can be directly used for structural studies [8,32].
Second, several cytotoxic proteins, which are difficult or
impossible to overexpress in cellular environments can be
readily produced in vitro (for an example see [33]).
Selenomethionine labeling, which is often used by protein
crystallographers to facilitate protein phasing by multi-
wavelength dispersion, frequently leads to poorly sub-
stituted proteins in vivo owing to its cytotoxicity, but it is
efficiently incorporated in vitro [34]. The cell-free tech-
nique can also overcome the problem of large-scale cell
growth using expensive media and materials. And finally,
integral membrane proteins that are notoriously difficult
to express in vivo can be produced in vitro at more than a
milligram per milliliter of reaction and are suitable for
biophysical analysis by nuclear magenetic resonance
Today, NMR spectroscopy techniques enable research-
ers to gather information from relatively large and
supramolecular structures [35]. However, the application
of this technology on proteins expressed from cell-based
systems presents two major shortcomings. First, it
requires extensive isotope labeling, which involves costly
expression procedures in vivo. Second, the dual amino
selective labeling technique [36], which helps to identify
the signals, can only be used with certain amino acids
owing to limited uptake and amino acid scrambling. With
the absence of bilayer barriers, reduced amino acid
metabolism and small reaction volumes, an in vitro
protein expression system is a more convenient and
economically feasible option for heavy isotope labeling.
Several successful examples of amino-selective or uniform
stable-isotope labeling using cell-free systems have been
reported (for a recent example see [37] and for a review see
[38]). More importantly, heteronuclear single quantum
coherence spectra can be recorded directly from the
reaction without further purification of the products
facilitating the simultaneous handling of multiple
samples [39].
High throughput and functional proteomics
With the vast and growing amount of genetic information
available today, it is becoming increasingly difficult to use
the traditional cell-based expression systems to rapidly
screen protein activities or interactions using a multi-
plexed approach. By contrast, batch-fed cell-free protein
synthesis offers tremendous flexibility for fast and parallel
analysis of the products. One of the earliest strategies was
the so-called 'in vitro expression cloning', in which small
plasmid pools are used to express desired protein products
and screen for the presence of a desired biochemical
activity. The subsequent positive pools are then progress-
ively subdivided until a single plasmid coding for the
active protein is isolated [40]. This strategy can be
further improved by the use of the Gateway technology
(http://www.invitrogen.com/gateway) which has recently
proven to be convenient for the rapid screening of large
libraries using protein cell-free synthesis [41]. Using this
technology a large collection of genes can be tagged in
tandem and screened for expression and solubility in a few
hours (Figure 2).
With the finding that PCR products can be used as
templates for the reaction, the procedure could be
expanded to the construction and screening of random
mutagenesis libraries (for a recent example see [42]).
Other strategies are based on tandem PCR reactions that
add specific 50and 30elements to the amplified product
[43]. However, in this case the use of extremely long
primers increases the chances of introduction of unwanted
mutations. In our laboratory we have improved this
approach by combining the power of PCR with the
robustness of directional topoisomerase I-mediated join-
ing to create DNA fragments optimized for in vitro
expression (http://www.invitrogen.com/Content/Focus/
Protein evolution
Directed evolution is a powerful tool for accelerating the
improvement of enzymes and interacting proteins. The
key principle behind this strategy is the creation of a
physical link between the nucleic acid and the protein that
permits multiple rounds of amplification and selection.
Cell-free translation-based display technologies offer
faster procedures, lower costs and wider dynamic range
when compared with traditional cell-based strategies. The
most established methods are ribosome display, in vitro
virus (also known as RNA-peptide fusion or mRNA
display) and in vitro compartmentalization (for a review
see [44]). Ribosome display uses cell-free extracts to
translate mRNAs devoid of a stop codon, resulting in the
RNA–ribosome–protein ternary complex remaining
intact. For in vitro virus, a puromycin-tagged DNA is
ligated to the 30-end of the template messenger. The
translating ribosome stalls at the RNA–DNA junction and
the attached puromycin serves as a covalent acceptor of
the polypeptide chain. In the third method a DNA library
of enzyme mutants together with the transcription/
translation mix are partitioned in water-in-oil-emulsion
at an average of one DNA molecule per droplet. The
synthesized product either modifies or attaches to the
DNA from which it was produced (e.g. by the use of
biotinylated DNA and a streptavidin tag). Then the
emulsion is collapsed and the corresponding selection
and amplification steps are performed.
Unnatural amino acids and in vitro protein labeling
The incorporation of non-natural amino acids, particu-
larly those with chemically or physically reactive side
chains, has the potential to be one of the most useful tools
for functional and structural proteomics. A variety of
labels, including fluorescent dyes for functional studies,
TRENDS in BiotechnologyVol.23 No.3 March 2005 153
Page 5
biotinylated moieties to facilitate purification and many
others, including those for structural studies and for post-
translational modifications, can be sequence-specifically
incorporated into proteins. An efficient way to incorporate
artificial amino acids into polypeptides is to supplement
the cell-free extracts with chemically aminoacylated
suppressor tRNAs that recognize a particular stop codon
[45]. Genes containing the desired nonsense mutation at
the appropriate site are used as templates. For example
the incorporation of a single label at the N-terminal
position, highly desirable for the preparation of protein
micro arrays, can be readily accomplished by using an
amber initiator suppressor tRNA and a DNA template
with an amber codon instead of the normal initiation
codon [46].
In vitro co-translational labeling is not limited to the
use of unnatural amino acids. For example, puromycin
derivatives can be used in cell-free expression systems to
specifically label proteins at the C-terminus [47]. Recently,
a novel tetracysteine motif was shown to specifically bind
biarsenical ligands that become fluorescent only after
binding [48]. Using a fluorometer, these compounds have
been directly added to cell-free transcription–translation
systems to monitor real-time protein synthesis in high-
throughput expression format (Figure 3). This approach is
particularly useful for high-throughput screening of
pharmacological agents with translation-inhibiting
activity. Although some of these labeling techniques can
be applied to cell-based systems, problems such as
cytotoxicity of the compounds, reduced protein yields,
low label incorporation, or transport across membranes
are issues largely reduced or eliminated when using a cell-
free expression systems.
Finally, protein interaction validation studies should be
greatly facilitated in cell-free expression systems, for
instance by combining dual labeling with fluorescence
cross-correlation spectroscopy [49]. This method allows
monitoring whether two molecules labeled with different
fluorophores bind to each other or not by the identification
of fluctuations that occur simultaneously in two channels
of a fluorescence correlation microscope.
Miniaturization and protein arrays
A clear application of cell-free protein expression reactions
is in the area of miniaturization and protein arrays. For
example, protein 'macro' arrays can be generated by small
25 ml reactions to synthesize tagged products that are
in situ immobilized in separate wells coated with tag-
binding beads [50].
Reactions can be downscaled to levels (nanoliter scale)
that are unimaginable for cell-based approaches [51] and
yet still synthesize enough products to perform individual
enzymatic assays in 96-well glass microplates. Finally,
cell-free protein synthesis offers tremendous advantages
to the construction of protein micro arrays. Ramachan-
dran and coworkers developed a self-assembling protein
chip starting with DNA gene microarrays, which are
transcribed and translated by a cell-free system. The
resulting proteins, fused to glutathione-S-transferase
(GST), are immediately captured in situ because of an
antibody, anti-GST, printed simultaneously with the
expression plasmid [52]. This technique saves consider-
able labor, time and costs by eliminating the need to
express, purify and print proteins separately.
Diagnostics and therapeutics
In the field of genetic diagnostics, cell-free expression has
attracted considerable interest with the protein trunca-
tion test as a method for detection of nonsense or
frame-shift mutations in marker genes. Briefly, gene
specific RT–PCR or PCR amplified products are added to
transcription and translation reactions and truncated
TRENDS in Biotechnology
Figure 2. Rapid high-throughput protein expression screening method. A collection of human open reading frames were recombined in tandem into a vector that encodes a
tetracysteine motif N-terminal fusion tag (pEXP3, Invitrogen, http://www.invitrogen.com). Proteins were synthesized in parallel using a commercial E. coli cell-free lysate
(Expressway HTP Cell-Free E. coli Expression Kit, Invitrogen) and loaded in a 96-well SDS–PAGE (E-PAGE 96 Protein Electrophoresis System, Invitrogen) in the presence of a
biarsenical fluorescein derivative [48] (Lumio Green Detection Kit, Invitrogen). Products were visualized in-gel with no further staining under a laser scanner (Typhoon 8600
Variable Mode Imager, Amersham Biosciences, http://www.amershambiosciences.com). Abbreviations: M, Molecular weight marker.
TRENDS in Biotechnology Vol.23 No.3 March 2005154


text:
Sharon added, "After acquiring the retail travel agency, it made sense to form a partnership with the two products and offer our Holiday letting Shop customers an even greater service as part of the RubyBlue Group".
Holiday Letting Shop Customers will also benefit with discounts and special offers from the travel agency when they deposit or rent their accommodation through Holiday Letting Shop.
Sharon also added "I have decided to take the business in a new direction and expand our whole travel services package; I believe that both products enhance each other perfectly and this is a natural progression for the group".
"We have a team of designers working on our new website at the moment as part of the whole development plan, and I am very excited about the re-launch of the new and improved Holiday Letting Shop".
# # #
Holiday Letting Shop has been developed to bridge the gap between individual timeshare week owners and mature or HOA-run resorts with stock to rent and companies and holidaymakers looking for resort-based accommodation.


text: FORT MYERS, Fla. – After throwing four scoreless innings in his final spring training outing, Jon Lester relayed his excitement over being named Opening Day starter for a third straight season. "All the words – honor, privilege – all of that, especially for this organization, to be named that," he said. "I take it with great pride, and I'll go out thee and give it a good start and hopefully get this team off to a good start this season."


text:
Brasil Things to Do
visit the zoo
A garotada que quiser comemorar o Dia das Crianças no Zoológico de São Paulo vai se divertir muito com as novidades que o Parque oferece.As surpresas vão desde o lançamento de um Programa de Educação Ambiental com animais vivos até a oportunidade de conhecerem os principais biomas brasileiros: Mata Atlântica, Cerrado, Pantanal e Amazônia.more
visit the nature and research
Instituto desenvolve estudos e pesquisa básica na área de Biologia e de Biomedicina, relacionadas, direta ou indiretamente, com a saúde pública. Produz vacinas e soros para uso profilático e curativo. Realiza missões científicas no país e no exterior através da Organização Mundial e Panamericana da Saúdemore
embu das artes
The Sunday is the best I gave to visit it, since some as many apples, the step to the traffic is cut to install a craft fair, its streets are of cobblestones, and on them the positions with their crafts are installed. The place is surrounded by mountains, has an incredible view. I was with friends of the place, that knew the road since the center...more
Brasil Hotels
Transamerica La Residence Pau
ALAMEDA JAU 1606, Brasil, SP 01420 002
Brasil Nightlife
walk in thew night in the city
walk in the night in sao paolo city is the huge experience, in the new year time, yo ucan see the light and the and the big building casualmore
walk in the beach
walk in the new year time in the beach of santos with all lighting, people and the ocean is the great experience, casul clothsmore
Brasil Hotels1 Hotels in Brasil
Brasil Local Customs
Feijolada is a traditional Brazilian dish. It is basically black beans soup that accompanies deep-fried fish, onions, tomato. So yiammy!!! We had it in Isla Grande by the beach while enjoing the sunshine!! Gr8 feeling..
Brasil Warnings and Dangers
Put your money where you cant see...
This tip is useful for most underdeveloped or poorer places (Bolivia not included). Especially RIO.
0) Prepare well before you leave home.
1) Dont bring anything you value too much and dont walk around flashing jewlery and watches. I mean, look discrete in all ways.
2) You can make smaller hidden money/value pockets in your clothes, bye elastic band bags to keep underneath your clothes - around the legs, whatever.
3) Keep the money in smaller portions - but dont forget where!
As well as the credit cards - bring several, in case you should "loose" one or two, and keep them seperate.
4) Give someone you trust back home authority to send/wire you money from your account in case you should loose everything.
5) Have a wire with a padlock attached to secure the backpack. You might want to have a cape (fx a waterproof one) to keep hands out of the backpack while youre wearing it. If travelling on bus, try to keep it with you inside the bus and not in the luggage compartment if you can get away with it.
If not, keep an eye out whenever the bus stops - someone might run away with your stuff.
5) Be alert, it happens when you least expect it!
6) For RIO - dont bring anything you cant live without to the beach and know which areas to go to and which not!
7) To me the most precious part of my luggage are my photos and maybe things that I buy along the way - so send them home before you loose them - but keep the negatives or vice versa. I dont even trust the mailman.
These are simple tips - but things you need to be aware of.
I might sound paranoid - but a smaller bad experience can end up ruining a beautiful journey.Related to:
- Budget Travel
Brasil Tourist Traps
Telephone Cards
Not exactly a tourist trap but I GOT TRAPPED:)) HEHe
I paied 9 reals for a Calling Card to make a call home (to Greece) and I only managed to talk for 2 minutes!! Very expensive I would say.. I prefered postcards after this..:)
Fun Alternatives: EMAILS EMAILS EMAILS!!
Brasil What to Pack
Pack as little as you can!
Luggage and bags: Best choise is an everyday one hand luggage and a small backpack!
Clothing/Shoes/Weather Gear: We went on August: Flip-flops, hikking shoes (if u want to get adventurous), 1 BIKINI (better to by some over there) and sharongs. That all u need!! PACK AS LITTLE AS U CAN!
Toiletries and Medical Supplies: Get ur meds from home as you will find it hard to describe ur problem to the pharmacist! I had some issues there that were very funny but didn't manage to find what I needed!:)
Photo Equipment: We had no prob with Cameras getting nicked. However, we tryed to be discreet with all our expensive belongings.Related to:
- Backpacking
Brasil Favorites
Don't be afraid/relax
Favorite thing: Mareietatia
Don't be afraid of Brazil, its a crazy wonderful place, you just need to have the same common sense you use when travelling in any big city- not wearing expensive jewelry, or showing too much wealth when walking the street. When I travel back to Brazil each year I pay mainly with Credit/debit cards taking enough cash for taxis, and inscidental purchases. I've been married to a wonderful Paulistana for 12 years, lived in Sao Paulo for 3 of those, and now travel, back with my family at least once a year. Brazil is a giant country with many facids aside form Rio/Sao Paulo, by all means visit them, but visit the real country in the Northeast, with its great beaches and people.
Fondest memory: I have too many, but I think it was my wife & I in Maceio, on varios excursions we took on a small tour bus, with a group of people we just met.
Instant Answers: Brasil
Get an instant answer from local experts and frequent travelers


text:
President Obama's National Day of Making!
I was raised to listen to authority. I took it seriously (most of the time) and today, I think we should all listen to our president's declaration of the National Day of Making! While I do realize that Mr. President is trying to encourage innovation and entrepreneurship in technology and the like, I don't think we have to discount our need (and love) for the decorative arts! So I say…make robots, make jewelry, make cupcakes!
Here's a fun, quick, and easy little project that you can do with your kids on this first National Day of Making!
What you'll need:
- Raw wood beads from Oriental Trading
- Paint pens
- Chinese knotting cord (1.4 mm or smaller) from Tangles n Knots
- Scissors
- Small bead
- Decorate beads with paint pens and let dry. While they are drying, wrap cord around 2 fingers about 8-10 times. Cut bundle from the spool.
- Cut a 20″ piece of cord and thread it through the loops of the bundle.
- Tie a knot to keep the bundle together in the center of the 20″ cord.
- Cut the loops at the bottom and thread the long strings through the 2 beads to make a necklace. (not shown)
Today is the first ever White House Maker Faire (oh how I'd love to be there!)—visit the website to learn, watch, and make! Post what you are making on social media with the hashtag, #nationofmakers .Add a Comment
Tags: national day of making | Categories: Crafts, DIY, Gifts, Inspired


text:
Senior Member
Last Activity: Today 11:11 PM
About Me
- About ZhenMing
- Service provider
- Malaysia - Maxis
- City
- Penang
- Home country
- Malaysia
- Current Phone:
- Nexus 5 rooted running OmniROM + Linaro r34t2
Standby Phone:
- Samsung Galaxy Tab 7.7 3G version (P6800) running OmniROM.
Fully retired Phone:
- Google Nexus One loaded with CM7.2 RC2
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Hi guys, Just try it. It is awesome. I installed this GAPP on my PA2.5 (CM10) and it work like charm. http://forum.xda-developers.com/showthread.php?t=1964726 If you are interested, you might try yourself. But please to make a nandroid first b...
I'm very excited with this news. I can't wait to try it out. How about you? http://www.android.gs/android-5-0-l-factory-images-for-nexus-5-and-nexus-7-available-tomorrow/ Sent from my Nexus 5 using XDA Free mobile app
Believe or not, I had soft brick for the 3rd time and just get it resolved a moment ago. It was due to the data folder corruption. The last 2 times I tried this way and manage to resolve it and revived my precious in no time. But I lost all my dat...
- make sure you did nandroid before proceed. - put the services.jar in the /system/framework. - Set the permission to -rw-r--r-- or you use terminal to run chmod 644 service.jar. - restart your system. CAUTION: If you don't follow all the steps ab...
So far only managed to get 50% supercharger only... Was on holiday now in my hometown which only have EDGE internet connection and away from my PC. Will dig Supercharger thread how to get 100% supercharged Sent from my GT-P6800 using Tapatalk 2 ...


text:
Report: Steelers worth $1.118 billion
Steelers/NFL Videos
The Steelers' estimated value went up, but their rank among the world's 50 most valuable franchises took a tumble.
According to Forbes' annual survey, the Steelers are worth an estimated $1.118 billion, but they fell four spots to 27th.
Forbes released the survey Wednesday.
The Steelers were estimated to be worth $996 million in 2012 and $1.1 billion in 2013.
The Steelers trail AFC North rival Baltimore ($1.227 billion) but are ahead of Cleveland ($1.005 billion) and Cincinnati ($924 million).
The NFL dominated Forbes' list, with 30 of the league's 32 franchises represented. Dallas led the way at $2.3 billion (No. 5) followed by New England ($1.8 billion) at No. 8, Washington ($1.7 billion) at No. 9 and the New York Giants ($1.55 billion) at No. 10.
The only two NFL teams not represented were Jacksonville and Oakland.
Soccer giants Real Madrid ($3.44 billion), Barcelona ($3.2 billion) and Manchester United ($2.81 billion) top the list.
The Pirates and Penguins did not make the list.
Just six MLB teams are represented, and Toronto ($1.15 billion, No. 26) is the only team from the NHL.
Mark Kaboly is a staff writer for Trib Total Media. Reach him at firstname.lastname@example.com or via Twitter @MarkKaboly_Trib.
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text:
The Ministry of Natural Resources, Energy and Environment is coordinating the UN CC:Learn Project in Malawi in cooperation with the Ministry of Finance, Economic Planning and Development.
UN Support to Address Climate Change in Malawi
The UN in Malawi is coordinating its support for climate change activities through a Climate Change Joint Programme Team. A One UN Fund has been established to simplify funding and reporting.
Agencies active in the area of climate change in Malawi include inter alia:
- The UN Development Programme (UNDP) assists the government through the National Climate Change Programme to respond to emerging development challenges posed by climate change. This Programme aims at mainstreaming and addressing climate change issues in the national development agenda. It consists of two complementary projects: the National Programme for Managing Climate Change in Malawi – formulation phase (CCP) and the Africa Adaptation Programme - Building Capacity for Integrated and Comprehensive Approaches to Climate Change Adaptation in Malawi (AAP-Malawi). The UN CC:Learn Project is complementing the Programme by reinforcing the human resources and skills development dimension and engaging national learning institutions.
- The UN Food and Agriculture Organization (FAO) is active in the area of climate-smart agriculture.
- The UN Children's Fund (UNICEF) is working on addressing the impacts of climate change on children.
- The World Health Organization (WHO) is working on the interface of climate change and health, including support for a vulnerability and adaptation assessment.
- The World Bank is supporting projects related to climate finance and low-emission development.


text: We all know that video cards drop in price over time. However, this price drop on the GTX280/260 so soon after launch is very unusual. I guess we have ATI to thank for this, and it's just one more example of no matter which cards you prefer, competition is best for everyone. Still, I bought my GTX 280 from Newegg just over 2 weeks ago, and the price has dropped $110!!! I'm tempted to return my card under their return policy and order another at the new price, but I was wondering if Newegg will just issue a credit to my account instead of me having to return this card. I've heard that Newegg won't do this, but it doesn't make a lot of sense for me to return a card I want to order another at a lower price.


text:
There will be quite a buzz when you complete your honey bee or honey witch costume with these cute black and yellow striped girls tights!
Sting and stun like a bee in the Yellow and Black Striped Tights. These black and yellow striped tights will complete any bee costume.
Create a buzz this Halloween in this Bumble Bee Adult costume. This unisex Halloween costume features a yellow and black striped top with matching wings and antennae. Pair it with black pants or tights and watch them make a bee-line in your direction!
Honey, you'll be one busy bee in this sexy Daisy Bee adult womens costume. Create a buzz when you arrive in the black and yellow striped dress which features a daisy applique, black tutu skirt and comes complete with matching leg warmers, antennae headpiece and wings.
Sting them all on Halloween wearing this Buzzin Around Girls Costume. No one will fear this bubbly bumble bee when you wear black and yellow striped tutu dress, decorated wings, antennae, glovelettes, and leggings. Just try not to buzz everyone's ears off.


text:
UCLA's Adam Plutko is one of the top starting pitchers in the Pac-12.
The 6-foot-3, 192-pound right hander was selected in the sixth round by the Houston Astros in the 2010 MLB draft. However, Plutko turned down the Astros' offers and enrolled at UCLA. Continue reading CBD Scouting Spotlight: Adam Plutko (UCLA)