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"This invitation turned out even better than I thought it could. I asked for a special request because I didn't like the 50 in the circle where it fel on the uploaded picture of my parents. The designer was able to accomodate my change request (AND very quickly). When I first started designing the card, I didn't even realize there was the whole back side I could also add pictures and captions. It turned out really nice - I'M SO PLEASED!!!! I had to call customer service to ask about the special request and everyone I spoke with was very professional and helpful. I will definately be back next time I'm in need of a card!"
"Friends and family always rave over my cards. This is my 5th year in a row to use Tiny Prints and they never fail to impress me. The paper quality is so nice and thick. The images always come out crisp, exactly as seen on the computer. And the design selection can't be beat."
"I couldn't wait to see these cards! My parents 50th anniversary is such a special event that I wanted everything to be perfect, especially the invites. After placing the order, I panicked. Seeing reviews about bad envelopes sent my nerves reeling. The most perfectly packaged box arrived with the invites. Inside was exactly what I had hoped for! Beautifully printed, excellent quality, fantastic design. After taking a breath, I opened the envelopes. PERFECT! Not one bad envelope in the bunch! Hint: I used my computer's 'paint' program to increase photo resolution on my parent's 50 year old picture. I believe this is key to getting great photo quality. I highly recommend tiny prints and can't wait for the event!"
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"I ordered this card for my parents' 60th anniversary and my mother was happy with the result. Excellent quality in all aspects."


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Learn how to ensure the data submitted to your web forms is complete, accurate, and nonmalicious with HTML5, JavaScript, jQuery, and PHP form processing and validation.
Add dynamic data to a PHP-enabled web site in Dreamweaver CS5 and CS6.
"The site has given me an amazing renewal of long-lost inspiration. I feel I have my creativity back for the first time in many, many years." —Louise L.
more from our members »

subset: commoncrawl


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No entertainment complex would be complete without great food and drink varieties. Listed below are the live racing dining options available at Canterbury Park. For a list of food options during the non-live racing season, click here
Open Live Racing Thursdays & Fridays: Buffet Style Dinner from 6PM - 8:30PM.
Saturdays: Brunch Buffet from Noon - 3PM. Menu
Night Out Package
Buy 2 Thursday or Friday night buffets, get a
FREE Clubhouse Table. Good thru June.
Must be reserved in advance.
Reservations can be made by calling 952-445-7224
For reservations, contact our Guest Services Department at 952.445.7224. Menu
We have a new Kid's Menu! Menu
This 3rd floor location serves up a variety of delicious daily specials, as well as regular staples such as burgers, chicken sandwiches, pizza and more.
Cure your corn dog craving and more at the Pronto Pup Stand
Stop by for some BBQ ribs or a grill-cooked hamburger.
Specialty pub food.
Tacos, Burritos, Nachos Grande
Ice Cream, Root Beer Floats, Malt Cups
Hot Dogs, Chips, Cold Sandwiches, Fresh Salads
Formerly known as "UBetchas", located trackside
Located in the Card Casino • Open 24 Hours • Offers a wide array of sandwiches, soups, grilled items, tacos, pizza and daily specials.
Located in the Card Casino • Full Service Bar • Menu Available


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UCL recognises outstanding achievement during Eighth Annual Enterprise Awards
Winners of the eighth annual UCL Awards for Enterprise were announced on Wednesday 10th June, at a ceremony held at UCL Advances' new incubator space for product and creative businesses at King's Cross, BASE KX.
The awards recognise the efforts and accomplishments of UCL's most innovative and entrepreneurial staff, students, graduates and partner organisations in furthering enterprise and entrepreneurship at UCL
Find out more about UCL Awards for Enterprise 2015
4th International Early Rail Conference Honours UCL Corporate Partnerships
8 October 2010
Further to the huge success of the 4th International Early Rail Conference in 2008, and the Trevithick Bicentennial Exhibition and Commemorative Dinner, UCL has been honoured in the long-awaited book of the Conference, which was recently published.
The Conference was invited to form the fulcrum of the season of
activities, organised and hosted by the UCL Centre for Transport
Studies and UCL Business Partnerships, in collaboration with The Science
Museum and sponsored by O2 and Eurostar.
The Introduction to the volume offers high praise for the UCL Business Partnerships initiative:
success of the conference - arguably the best yet - was due in no small
part to the personal attention given by Dr Anna Clark, UCL's Director
of Business Partnerships. Her vision and drive throughout all stages of
planning smoothed away any problems or difficulties in a spirit of great
friendship. The facilities and arrangements laid on for us by the
College, and the welcome extended to us, were much appreciated."
Grahame Boyes, Editor.
Read more information about the event here.


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Top 3 management tools used by GCC organisations
Does your company use these?July 6, 2015 12:00
Register for our free newsletter
Philippines confirms second MERS case
Abu Dhabi's Dana Gas hits 9-wk high after wins Kurdistan case
Saudi Telecom Co to pay Q2 dividend of 1 riyals/share
UAE non-oil business growth slows to 22-month low in June
MIDEAST STOCKS-Gulf likely to be subdued by oil price drop, Greece
Abu Dhabi's TAQA drops 5.4 pct after saying no merger talks
Qatar's Doha Bank raises 2 bln riyal Tier 1 capital-boosting bond
Dubai's Drake & Scull increases GCC ownership limit to 100 pct
Abu Dhabi's TAQA says not holding merger talks
Gold languishes below $1,170 ahead of U.S. jobs data
International cricket is set to make its debut in the new cricket stadium in Dubai later this month. The authorities are hoping to turn the emirate into an "ideal" cricket destination.
April 13, 2009 5:05 by Aarti Nagraj
The Pakistani and Australian teams will start the tournament at 3pm on April 22 in the match that will also officially inaugurate the Dubai Sports City cricket stadium.
The second game will be played on April 24 and this will be followed by three matches in Abu Dhabi on April 27, 29 and May 3. The grand finale of the series will be a Twenty20 match on May 7 at Dubai Sports City Cricket Stadium.
According to reports, Pakistan and Australia are bringing some of their new cricketing talent for the game, which will also include players such as Andrew Symonds, Brett Lee and Shoaib Akhtar.
Ticket prices for the games range from AED50 to AED950 for the most exclusive seats.
Saudis go mad for red mercury
April 14 2009


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Similar Artists: Black Arrow | Astronomers Victorious | Hazerai | Fiction Smiles | Noella Hutton
Sorry, there aren't any upcoming shows for the druthers, +add one?
406 upcoming shows @ 92 different spots
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126 ALL AGES shows!
Simply hover over any band you're interested in and we'll pull up a bio and some links for you. Hide this


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Women and Macro Social Work Practice: Oxford Bibliographies Online Research Guide
Oxford Bibliographies Online Research GuidesThis ebook is a selective guide designed to help scholars and students of social work find reliable sources of information by directing them to the best available scholarly materials in whatever form or format they appear from books, chapters, and journal articles to online archives, electronic data sets, and blogs. Written by a leading international authority on the subject, the ebook provides
Faith-Based Initiatives and Aging ServicesGain an understanding of the increased role religious congregations now play in providing social support to the elderlyReligious congregations and faith-based organizations (FBO) from the Jewish, Christian, and Islamic traditions have worked on behalf of older adults for centuries. But the initiation of President Bush's Office of Faith-Based Initiatives has raised many questions from both the
Multiple Perspectives for Critically Understanding and Evaluating PolicyFrom formulation to implementation, an approach to the analysis of social policy through the lens of research Analyzing Social Policy prepares professionals and students to make better informed decisions related to identifying and understanding the intricacies and potential impact of social policymaking and enactment on their organization as well as their individual responsibilities, goals, and
A Guide to Understanding Human Service OrganizationsHuman service organizations are under increasing pressure to demonstrate that their programs work. Organization Practice, Second Edition helps students and professionals in human services and nonprofit management understand complex behaviors in organizations. This new edition provides a new, practical model for understanding cultural identities within organizations. Also, it is significantly
Designing, Conducting, and Presenting Your Research
Research Methods for the Social Sciences (Book #25)A comprehensive and practical guide to ethnographic research, this book guides you through the process, starting with the fundamentals of choosing and proposing a topic and selecting a research design. It describes methods of data collection (taking notes, participant observation, interviewing, identifying themes and issues, creating ethnographic maps and tables and charts, and referring to
Transformative Learning in Practice
Insights from Community, Workplace, and Higher EducationThe leading authorities in the field produced this comprehensive resource, which provides strategies and methods for fostering Transformative Learning (TL) practice in a wide variety of higher and adult education settings. The book answers relevant questions such as: What are effective practices for promoting TL in the classroom? What is it about TL that is most helpful in informing practice? How
A Guide to Engagement and InterventionPraise for the Second Edition of The Dynamics of Conflict "Mayer once again demonstrates his unique ability to weave theory and practice together with real-world examples—whether about warring nations or a family dispute—to help readers better understand both the fundamentals and nuances involved in working in the conflict arena. The Dynamics of Conflict is a very important read for anyone who
Discussion as a Way of Teaching
Tools and Techniques for Democratic ClassroomsThoroughly revised and updated, the second edition of the landmark book Discussion as a Way of Teaching shows how to plan, conduct, and assess classroom discussions. Stephen D. Brookfield and Stephen Preskill suggest exercises for starting discussions, strategies for maintaining their momentum, and ways to elicit diverse views and voices. The book also includes new exercises and material on the
Understanding and Facilitating Organizational Change in the 21st Century: Recent Research and Conceptualizations
ASHE-ERIC Higher Education Report, Volume 28, Number 4
J-B ASHE Higher Education Report Series (AEHE) (Book #155)There is a widespread discontent with the quality of education and levels of college student achievement, particularly for undergraduates preparing for the professions. This report examines the educational challenges in preparing professionals, reviews the specific types of curriculum innovations that faculty and administrators have created or significantly revised to strengthen college graduates'
The Scholarship of Teaching and Learning Reconsidered
Institutional Integration and Impact
Jossey-Bass/Carnegie Foundation for the Advancement of Teaching (Book #21)Drawing on the experience with the individuals, campuses, and professional associations associated with the Carnegie Academy for the Scholarship of Teaching and Learning and the Institutional Leadership Program, this important resource examines four critical areas where engagement with the scholarship of teaching and learning can have a significant effect. This book is intended for a broad


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Jason FongSenior Analyst, Asia Pacific Research
Since first emerging in the early 2000s, self-storage in Asia has rapidly expanded, fulfilling market demand. Although the industry faces some risks in Asian markets, high population density, robust office...
Read More
Ross MooreDirector of Research, Canada
Roelof Van DijkResearch Manager, Canada
With contributions fromJeanette Rice, Americas Head of Investment Research
Read More
In this Global In-Sights video, Serguei Chervachidze, CBRE Econometric Advisors' Capital Markets Economist, addresses important questions occupying investors' minds.
View Podcast
View more Global In-Sights


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Graduating students
Book your regalia and seat ticket online
Commercial Graduation Services
Once you complete your studies, you become part of our international alumni community, a vibrant and active network that can provide you with a diverse range of support and opportunities.
Your alumni community is never far away.
InTouch: alumni and friends portal
Academic staff
Join an academic procession
(Staff only)
- Videos of the March graduation ceremonies will remain available to stream online until the end of April at the following links:
Friday 20 March 6pm ceremony
Saturday 21 March 11am ceremony
Saturday 21 March 4pm ceremony


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Dead Island: Epidemic - Now Playing
Shaun and Erick try their luck in this Zombie fueled MOBA style game - Dead Island Epidemic!
by Dan Mihoerck on
Did you enjoy this video?Sign In to Upvote
About Now Playing
Join the GameSpot staff as they play the biggest titles and show you the coolest aspects of video games in Now Playing.
Schedule: Wednesdays at 2pm PT
2 comments
It looks good and its free :)
Really - a lane strategy game with Dead Island?


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Canada's Insurance and Risk Magazine
The United States government has designated nearly 600 counties across 14 states as primary disaster areas due to severe drought and heat, in the first disaster designations of the new year.
The U.S. Department of Agriculture said Wednesday that farm operators across 597 counties now qualify for low-interest emergency loans. "As drought persists, USDA will continue to partner with producers to see them through longer-term recovery, while taking the swift actions needed to help farmers and ranchers prepare their land and operations for the upcoming planting season," agriculture secretary Tom Vilsack noted in the announcement. The counties have shown a drought intensity value of at least D2 (Drought Severe) for eight consecutive weeks based on U.S. Drought Monitor measurements, providing for an automatic designation. The Drought Monitor measures drought intensity on a scale from D1 to D4, or moderate, severe, extreme and exceptional respectively. The counties designated as disaster areas include: Alabama, 14; Arkansas, 47; Arizona, 4; Colorado, 30; Georgia, 92; Hawaii, 2; Kansas, 88; Oklahoma, 76; Missouri, 31; New Mexico, 19; Nevada, 9; South Carolina, 11; Texas, 157; and Utah, 17. In 2012, USDA designated 2,245 counties in 39 states as disaster areas due to drought, or 71% of the country.
DisclaimerNote: By submitting your comments you acknowledge that Canadian Underwriter has the right to reproduce, broadcast and publicize those comments or any part thereof in any manner whatsoever. Please note that due to the volume of e-mails we receive, not all comments will be published and those that are published will not be edited. However, all will be carefully read, considered and appreciated.
Learning to Share
Get things straight. Vacation rental operator Airbnb currently has more than 36,000 users in Canada. UberX, the popular app-based ride-sharing service, uses "tens of thousands" of drivers in six cities across the country, reports a spokesperson...
Drought to Deluge
For the past five years, the property and casualty insurance industry has been talking more and more animatedly about the flood peril in Canada, with a marked sharpening of focus since the Calgary and Toronto events of summer 2013.
On July 1, 2014, the anti-spam provisions of Canada's Anti-Spam Legislation, known as CASL, became enforceable law. Several months later - on January 1, 2015 - CASL's installation of computer program provisions became enforceable law. They...
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Track List
Bois Goetre-Hen a'R Cylch
Genre: Easy Listening
Label: Music Wales
Catalog No: HBIM 1090780
Format: CD
Release Date: 2013-02-26
Number of Discs: 1
UPC Code: 5060031874631
FreeFreight Is Available. Click for Details
On Sale!
List Price: $28.99 Sale Price: $22.79
Low Stock, usually ships in 3 - 5 days *
Track List


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Thanks for taking the time to report this user.
Your participation helps keep Airbnb a safe and trusted
community, and it doesn't go unnoticed!
Nederland, chillen, genieten, zwemmen fitness
Ronald has completed online and offline ID verification.
Learn more »
Well ronald was unhappy. There are 2 sides to all storirs and i know the 100% satisfaction rating we have from all guests in the same room will tell its own story. I really regret ronald found these 3 matters of concern. No one else had in 3 years. I did replace the aircon within 12 hours of when he told me, although all the staff told me it was fine. The pool is airbnb's own pic. He can see what other guests say about that. The smell? Cleaning product. The cockroach? Yes, a dying one because we treat our rooms for pests every changeover. I'm very sorry you missed the airbnb period for cancellations ronald though i did tell you in time to apply for it. But i'm still sorry ronald, and i have already offered a few ways we hoped to make it up, like free nights accommodation in the future.


text: Psychology Press Cognitive Behavior Therapy - Upcoming Conferences Conferences, exhibitions and events that Routledge and the Taylor & Francis Group will be attending. en-us 2015-02-19T16:20:55Q Copyright (c) 2015, Psychology Press Psychology Press firstname.lastname@example.com tag:,2015:12:05 5 5 British Association of Behavioural and Cognitive Psychotherapy Annual Conference tag:,2015:/resources/conferences/12.21444 2015-07-21T13:29:46Q 2015-07-21T13:29:46Z Visit our stand, where we will be showcasing our latest and key titles in the field of psychotherapy. We shall also be offering all delegates 20% discount off all titles and free delivery on all orders! ]]> American Psychological Association Conference 2015 tag:,2015:/resources/conferences/12.23328 2015-08-06T19:00:50Q 2015-08-06T19:00:50Z Visit our stand, where we will be showcasing our latest and key titles in the field of psychology and mental health. All conference attendees will receive a 20% discount on all titles and free shipping on all orders! ]]>


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FRIDAY NIGHT: Mostly clear. Low: 61. Winds: Light Northeast.
INDEPENDENCE DAY: Sunny. High: 83. Winds: Light Northeast.
SUNDAY: Mostly sunny. High: 86. Winds: Southeast 5-10 mph.
Thomas ...more »
For even more recipes, View Our Recipe Archives
• 1 1/2 pounds vine-ripened tomatoes, peeled, seeded and chopped
• Tomato juice
• 1 cup cucumber, peeled, seeded and chopped
• 1/2 cup chopped red bell pepper
• 1/2 cup chopped red onion
• 1 small jalapeno, seeded and minced
• 1 medium garlic clove, minced
• 1/4 cup extra-virgin olive oil
• 1 lime, juiced
• 2 teaspoons balsamic vinegar
• 2 teaspoons Worcestershire sauce
• 1/2 teaspoon toasted, ground cumin
• 1 teaspoon kosher salt
• 1/4 teaspoon freshly ground black pepper
• 2 tablespoons fresh basil leaves, chiffonade
Make an X with a paring knife on the bottom of the tomatoes. Drop the tomatoes into the boiling water for 15 seconds, remove and transfer to an ice bath and allow to cool until able to handle, approximately 1 minute. Remove and pat dry. Peel, core and seed the tomatoes. When seeding the tomatoes, place the seeds and pulp into a fine mesh strainer set over a bowl in order to catch the juice. Press as much of the juice through as possible and then add enough bottled tomato juice to bring the total to 1 cup.
Place the tomatoes and juice into a large mixing bowl. Add the cucumber, bell pepper, red onion, jalapeno, garlic clove, olive oil, lime juice, balsamic vinegar, Worcestershire, cumin, salt and pepper and stir to combine. Transfer 1 1/2 cups of the mixture to a blender and puree for 15 to 20 seconds on high speed. Return the pureed mixture to the bowl and stir to combine. Cover and chill for 2 hours and up to overnight. Serve with chiffonade of basil.
This recipe aired September 3, 2013.
WICS Recipe Videos
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Applications for US jobless aid rise to still-low 281,000
WASHINGTON (AP) -- More people sought U.S. unemployment benefits last week, but the level of applications is still low and points to an improving job market.
Consumer Info
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Companies that pride themselves on being eco-friendly may have conflicted
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reputation. ...
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SHELBY TOWNSHIP, Mich. (AP) -- Some Detroit-area cops are dealing with some real animals.
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PHILADELPHIA (AP) -- Philadelphia has legalized and plans to tax Airbnb and other short-term rentals ahead of Pope Francis' visit in September and next year's Democratic National Convention.
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10:00 - Newschannel 20 at 10


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The Top 10 In Action Sports
01Brick By Brick: Camping Lego Style
02Booze and Bikes: 14 Tours That Let Combine Two of the Best Things in the World
03Who Needs Hollywood? These Incredible Videos Were All Filmed on the Simplest Cameras
04Sam Pilgrim vs. The World: The Champion Aiming To Reclaim His Crown In A New Era Of Biking
05Eccentric Adventures Episode 2: Britain's Most Brutal Illegal Event is All About Cheese
06A Journey Into Darkness: We Went Mountain Biking in the Abandoned Mines of Slovenia
07After the Apocalypse: Inside The World's Most Breathtaking Ghost Towns
08Debate: Let's Stop Hating On Parents Who Want To Raise Adventurous Kids
09Guns, Roses, And Eating Insects: Adventure Cycling in Vietnam
10From Intense Trails to Intensive Care: Dicing with Death In a Mountain Biking Paradise
Snow surfing.... Binding free splitboarding
A Snowboarding video by nearls - 7,371 views
Self propelled snow surfing with a split board. Bindings get used for the hike up, then they go in the back pack and a set of traction pads are attached to surf powder down the mountain. No Helicopter, no snowmobile, no cat, no lift, and no snowshoes. this is the "greenest" and fastest way to pursue the powder without bindings. If the powder turns to hard pack or terrain gets too challenging just throw the bindings back on. Can also be adapted to a standard 4x4 snowboard. Contact email@example.com for general inquiries. Ride the white wave.
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feedind program
we are now at home for august holiday ,our kids will be back on school on 5th Sept 2013 ,so we need you support to start the food program for this need kids ,you can buy one item and another buy the other welcome AND BE BLESSED
feeding program
Status: In progress
feeding program
Status: Needs urgent help
food program started
Things needed to start the feeding program
-rice one bag of 50kgs @kshs 5,000
-cooking fat of 17kgs @kshs 3,000
-charcoal one bag rylon@kshs 2,000
-porridge flour of 50kg@kshs 3,000
-sugar one bag of 50kgs@kshs 4,500
-cups and plates 138pcs@kshs 4,000
- spoons 68 pieces @kshs 2,000
-miscellaneous budget @kshs 2,000
total budget is kshs 25,500
we as sajos academy hereby request all our friends and neighbors to come and help with ideas how we can do to start this FEEDING PROGRAM AND LEARNING MATERIALS program,we have a lot of programs but we want to start with this TWO PROGRAMS,this feed program budget is for one term only
hoping to hear a good feedback from all our friends ,you are welcome to assist with one items if your are blessed
This is worth all the support. The children will benefit from this. Such program is essential. - Flemings Ultimate Garage
Thanks for your concern and we shall apprecietes any support or sponsorship from you or the company
we are the voice for the voiceless


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Re: [evonline2002_webheads] Christmas greetings
Expand Messages
- What a lovely card!
Merry Christmas to you too Aiden and all the very best in the New Year :-)
----- Original Message ----
From: Aiden Yeh <aidenyeh@...>
To: firstname.lastname@example.com; email@example.com; email@example.com; firstname.lastname@example.com; firstname.lastname@example.com; firstname.lastname@example.com; firstname.lastname@example.com; email@example.com; Karena <virgokarena@...>; orlandotaipei@...; Betty Yeh <bettyyeh@...>; Amy Yeh <ymyeh@...>; linnet carungcong <linnet419@...>
Sent: Tuesday, December 25, 2007 2:25:12 AM
Subject: [evonline2002_webheads] Christmas greetings
You have a card to pick up:
http://www.riverson gs.com/christmas /carols.html# ecard
May the spirit of Christmas, love, and peace be with you all this holiday season.
Aiden Yeh
------------ --------- --------- ---
Be a better friend, newshound, and know-it-all with Yahoo! Mobile. Try it now.
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Never miss a thing. Make Yahoo your home page.
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Upload Photo
Lincoln Station Bar & Grill
Lincoln Station pulls into Bellmore -- across from the Long Island Rail Road station and on the right track. The attractive pub, with its leather banquettes and checkered cloths, offers a whistle-blower $16.95 prix-fixe every Sunday through Thursday night -- three generous courses and plenty of value.
50 Cheap Eats to try on LI The 8 best seafood restaurants on LI Pizza of the Week: Specialty pie picks 16 hidden restaurants worth the hunt Recent restaurant closures on Long Island The 10 best Italian restaurants on Long Island Recent Long Island restaurant openings 28 places to get amazing ice cream on LI 40 favorite outdoor dining spots on LI 23 must-eat lobster rolls on LI 21 new East End eateries to check out Restaurants with a water view


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Paul O'Doherty takes on the task of reviewing the year's wine books for the third year running. He has the great advantage as a book reviewer of living in Ireland and therefore, unlike us, knows none of the relevant authors personally. He writes on food and wine, and sets crosswords, for a number of publications. Here he…
Written by 26 Dec 2011
The state of wine book publishing
Further reading
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Annual membershipPerfect for wine lovers £69 annual Join Save £15
Monthly membershipPerfect for wine lovers £6.99 monthly Join
Still unsure?Find out more about membership and the Jancis Robinson Team. Find out more
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- 1/16/11
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Variety 50 Points Play as every character except ??? Medal Stats.
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Like Really Bad 5 Points Negative 20 rangs? Are you trying? Medal Stats.
Ludicrous Speed 5 Points Go as quickly as hodgemanly possible. Medal Stats.
Brass Knackles 5 Points Collect 50 rangs as Knackles. (unlocks ???) Medal Stats.
Edgy Gem Collector 5 Points Collect 7 chaos emmastones as Shadoo. (unlocks ???) Medal Stats.
You're Bad at This 5 Points Get a negative number of rangs you loser Medal Stats.
Gem Collector 5 Points Collect 7 chaos emmastones as Sonac. (unlocks ???) Medal Stats.
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VG Music Challenge Rated 4.0500 Stars Test your knowledge of video game music as you take on 50 songs in this quiz game. Puzzles - Quiz MURDER Rated 4.4023 Stars There's quite some stabbing going on in the King's castle! Action - Other Mega Miner Rated 4.1295 Stars Mine minerals and sell them for cash. Upgrade your equipment to mine deeper! Action - Other Meat Boy Rated 4.4068 Stars Extremely challenging! Best ending ever! Must Play! Action - Platformer - Other
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Evangelion in a Nutshell Rated 4.2354 Stars Every Episode in Thirty Seconds! Comedy - Parody Slippery Shower Rated 4.3226 Stars A girl wants to join her bf in the shower, but... Comedy - Original The Psychic Club Rated 3.8187 Stars What do you need to do join the Psychic Club? (NATA) Comedy - Original MK: Luigi Death Stare Rated 4.3110 Stars A short parody of MK Comedy - Parody
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Sonic Added to skins for Skincraft Aug 30, 2014. Load Level
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Join us for a track workout that will help you put some speed behind your step! We will start from the store and run to the track at Lake Shore Park. As an added bonus, every 4th Thursday will be a FREE bootcamp workout led by a different fitness instructor. For more details, vist Run lululemon Chicago.
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Open Educational Resources (OER) have created opportunities for people to learn things they may never have dreamed possible. Access to educational materials has become available through online sites across the globe. Many people may be familiar with the term "open source" as it applies to how computer programmers have shared information. However, the term "OER" may be less familiar. Within the education realm, sharing of information through OER has begun to change the process of how people learn. It has given people with little hope of receiving a higher education the ability to access information that used to only be available to people with serious financial resources.
|Scooped by Andreas Link


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Carpentersville, IL
2153 Randall Road
Carpentersville, IL 60110
Beat the Summer Heat with Hot Membership's & Personal Training with Anytime Fitness! Don't miss out on these Summer Savings! Stop by or call today! Get to a Healthier Place with Anytime Fitness!!
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club at a glance
24-hour access
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anywhere club access
private restrooms
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personal training
cardio tvs
wellness programs
trx certified facility
functional training
cardio equipment
exercise cycles
concept2 rowers
strength equipment
free weights
plate loaded
bobby tabor
kyle piotrowski
shannon persig
My name is Shannon. I am the General Manager at the Anytime Fitness in Carpentersville Illinois. I have over 10 years of experience in the fitness industry. I am passionate about fitness, but I have not always been- like a lot of other people out there. I love being instrumental in helping others change their lives because I can relate. I was once overweight and unhealthy. Fitness chose me. It was not an easy journey for me at first, but I feel very humbled by this. In time, I fell in love with exercise and living a healthier lifestyle and now I can show others how they can love it too! I wake up every morning and feel awesome about what we do. I am so happy and proud to be a part of the Anytime Fitness Family. I love the culture of my gym because we care.
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Media figures have smeared President Obama's nominee for secretary of defense, former Sen. Chuck Hagel (R-NE), by misrepresenting Hagel's support for sanctions against Iran and his support for Israel. The media have also cast doubt on the bipartisan support for Hagel's nomination.
In a blow to his own network, Fox News host Juan Williams debunked false narratives that Fox News has frequently pushed since the attack on the U.S. Consulate in Benghazi, Libya.
In an opinion piece for The Hill, Williams offered three "corrections" for what he describes as "deliberate misinformation" from Republicans (and their conservative media mouthpieces) about the Benghazi attack.
Misinformation #1: "U.N. Ambassador Susan Rice lied to the American people in the days after the attack" when she suggested that a viral anti-Islam video sparked violence and wide-spread protests. Fox News has repeatedly pushed this narrative to make it seem like the administration was deliberately misleading the American people.
Correction: Williams debunked this false narrative, pointing to the "simple fact" that James Clapper, Director of National Intelligence, "confirmed that Rice told the truth in describing the assessment of the intelligence community at the time of her remarks." Williams went on to explain that CIA Director David Petraeus briefed the House Intelligence Committee with the same intel Rice used, as did Undersecretary of State for Management Patrick Kennedy in testimony before Congress earlier in October. Furthermore, as Media Matters has previously exposed, Williams noted that Rice "stressed that there was an ongoing investigation where conclusions were subject to change." Indeed, Rice appeared on all major networks and repeatedly stressed that there was an ongoing investigation into the attacks.
Misinformation #2: "[R]equests for extra security in Benghazi were denied by the administration," coupled with the suggestion that the "attack would have been stopped, and the ambassador still alive, if the requests had been granted." Fox News pushed this myth on multiple occasions.
Correction: Williams pointed out that requests for extra security were focused on the embassy in Tripoli, not Benghazi, and State Department officials believe that even if the requests had been granted, they would likely not have changed what happened in Benghazi because the consulate would have been ill-equipped to respond to such a large-scale assault (again echoing a previous report by Media Matters):
From the October 18 edition of Fox News' Fox & Friends:
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From the October 5 edition of Fox News' The Five:
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From the October 2 edition of Fox News' Hannity:
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Fox News host Eric Bolling complained today that members of the Obama White House have not appeared on any of his shows -- despite the fact that he has been responsible for several vicious, racially charged, and evidence-free attacks on President Obama.
On the August 22 edition of Fox News' The Five, co-host Eric Bolling complained that he has tried to book "anybody from the White House" on his shows to "explain the economic numbers" but has never been successful. Perhaps Bolling shouldn't be surprised: He has consistently been at the forefront of Fox's most vicious attacks on the president.
On May 2011, Bolling claimed President Obama was "chugging a few 40's" instead of responding to a tornado that had devastated Joplin, Missouri. Bolling's attack echoed one he made the day before on Twitter when he accused the president of "chugging 40's in IRE while tornadoes ravage MO":
On the June 11, 2011, edition of his former Fox Business show, Bolling teased a segment about a White House visit by the president of Gabon, Ali Bongo, by saying "Guess who's coming to dinner? A dictator. Mr. Obama shares a laugh with one of Africa's kleptocrats. It's not the first time he's had a hoodlum in the hizzouse." Bolling later asked, "So what's with all the hoods in the hizzy?" referring to a visit the month before by rapper Common.
Bolling was also at the forefront of Fox News' birther obsession. In April 2011, Bolling hosted anti-Muslim blogger Pamela Geller on his Fox Business show and attempted to find flaws with the long-form birth certificate Obama had recently released. During the segment, Bolling claimed "there is a legitimate question as to whether or not the president of the United States is allowed to be president of the United States."
Bolling has peddled other Obama conspiracy theories, suggesting that Obama is trying to "bring people closer to the cities" so the government can keep an eye on them and pondering whether Obama let an oil rig leak so he "could renege on his promise" to "allow some offshore drilling."
From the August 22 edition of Fox News' The Five:
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From the August 2 edition of Fox News' Fox & Friends:
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From the June 11 edition of Fox's The O'Reilly Factor:
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From the June 6 edition of Fox's The Five:
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From the June 6 edition of Fox's The Five:
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From the June 5 edition of Fox News' The Five:
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In a May 14 op-ed for The Hill, Fox News correspondent Juan Williams decried politicization of the investigation of the ATF's botched Operation Fast and Furious and labeled House Oversight Committee Chairman Darrell Issa's inquiry into Attorney General Eric Holder and the Department of Justice (DOJ) a "monstrous witch hunt."
Williams' comments are in stark contrast to his network's coverage of the issue, which over the past year has promoted Issa's investigation at every turn while also giving airtime to those who would peddle conspiracy theories about the failed operation.
In his op-ed, Williams describes Issa as a modern day Captain Ahab, hell-bent on finding fault in Holder for Fast and Furious no matter how thin the evidence is suggesting that he had any involvement in the operation. According to Williams, the ultimate goal of Issa's inquiry is to "defame Holder and hurt the president." Warning about the potential consequences of Issa's contempt proceeding endgame, he concluded:
At the moment more than 100 House Republicans have already signed on to a resolution expressing no confidence in Holder.
That kind of politics is acceptable.
But a contempt citation for the top law enforcement official is a monstrosity breaking apart public trust and dragging the nation's already polarized politics to the bottom of the sea.
While Williams may be disturbed by the direction in which Issa's investigation is going, the truth is that his employer, Fox News, has served as a clearinghouse for politicized statements about Fast and Furious for over a year. In the last month alone, Issa and two of his lieutenants (Reps. Trey Gowdy and Jason Chaffetz) have appeared on Fox News at least nine times to promote their investigation into Fast and Furious.
From the April 25 edition of Fox News' The Five:
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From the April 25 edition of Fox News' The Five:
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4. Tell them you're busy
Then immediately set up a time to talk. Schedule repeat appointments with those who need to have routine access to you.
5. Take a stand
Allowing someone to sit down inevitably means a longer interruption. If you stand up and walk to your office door or work station entrance, an interruption will take less time.
6. Look before you leap.
See what someone is doing and decide if your interruption is merited. Most conversations can be delayed without negative consequences. When someone is in "heads-down" concentration, make a proactive decision not to interrupt.
7. Do things right and on time, the first time
Handle issues correctly the first time to reduce the need for others to get clarification from you later.
8. Leave complete phone messages
Leave complete information when you are sent to someone's voice mail and request that others do the same for you. This will reduce the number of phone interruptions.
9. Plan your day
A clear daily plan helps workers stay on task and focused.
10. Don't interrupt others
If you have a habit of barging in and interrupting others, this sends a clear signal that it is OK to do the same to you.


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Justin A. Frank, M.D., is a clinical professor in the Department of Psychiatry at George Washington University Medical Center. Since 1980 he has been a teaching analyst at the Washington Psychoanalytic Institute. He is past president of the Greater Washington Chapter of Physicians for Social Responsibility. Dr. Frank lives and practices psychoanalysis in Washington, D.C.
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Once I read a quotation by the late French President Charles de Gaulle- "nations would be terrified if they knew by what small men they are in reality ruled! Read morePublished 10 months ago by T. Washington
Isn't it obvious there is something very wrong with this guy! Agree with most everything the book contends! Don't know how he ever got elected or stole the Seat! Read morePublished 17 months ago by JAMES ROVNAK
Frank did a pretty good job of exposing the stupidity, incompetence, sadism, and lack of empathy or compassion in the worst US president in its history.Published on May 1, 2013 by Larry Hoffman
A frequently absent father and a cold, stern mother produced an emotionally impaired child with learning disabilities, George W. Bush. Read morePublished on February 25, 2012 by Cecile Farber
Justin A. Frank, M.D., is a psychiatrist and psychoanalyst. As a psychoanalyst, he follows Melanie Klein's approach to psychoanalysis, using her model of our psychological... Read morePublished on January 13, 2012 by Thomas J. Farrell
Bush on the Couch: a first-rate psychoanalytical hatchet job. It took me less than 60 seconds to find a list of Dr. Read morePublished on October 31, 2011 by Zach B.
This book is way too long, it could have been written as an article. And it really doesn't validate it's claims, with documentation within the text. Read morePublished on July 7, 2010 by Gr8gazoo
This book explains so many things and is written so the reader can understand it without a M.D. in psychoanalysis. Read morePublished on December 12, 2009 by H. Amado
I'm sure it's done a great deal of good for others, but I've always found psychoanalysis to be the ultimate in senseless navel-gazing (maybe that's my own psychosis talking!). Read morePublished on July 23, 2009 by Tom Dupree


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This invention is an algorithm for estimating the topology of a protein by determination of the free energy from a global entropy evaluation model combined with local correction methods.
1. A method to predict the topology of the spatial arrangement of an amino acid sequence using an entropy evaluation model that takes into account the global contributions of entropy to the folding of a biopolymer (herein referred to by the name cross linking entropy (CLE) and described in the literature) combined with other thermodynamic potentials as a protein-folding model.
2. A method according to
A. inputting an amino acid sequence of a protein,
B. preparing information on the secondary structure of the said amino acid sequence by way of at least one theoretical or experimental estimate,
C. applying the CLE method to the said amino acid sequence and secondary structure information to evaluate the free energy of a combinatorial number of β-strand and α-helix arrangements as rapidly as polynomial time: c(n−1)(n+1) wherein c is a constant and n is the number of secondary structure elements found in the said amino acid in 2A and prepared in 2B,
D. applying the CLE method in conjunction with other thermodynamic potentials that approximate hydrophobic, electrostatic and polar interactions, but not limited to these aforementioned thermodynamic potentials stated herein, in a thermodynamic calculation to account for both short and long range folding interactions and predict a minimum free energy and corresponding topology of the said amino acid sequence,
E. applying the CLE method to predict the global folding kinetics of the said amino acid sequence, and
F. storing the information in a data file or in other form of digital memory.
3. A method according to
4. A method according to
5. A method according to
6. A method according to
7. A method according to
8. A method to predict the topology of the spatial arrangement of an amino acid sequence comprising following steps:
A. inputting an amino acid sequence of a protein,
B. preparing information on the secondary structure of the said amino acid sequence by way of at least one theoretical or experimental estimate,
E. applying the CLE method to approximate the global folding kinetics of the said amino acid sequence,
G. applying the CLE method to the said amino acid sequence and secondary structure information to reduce the combinatorial number of β-strand and α-helix arrangements to a computationally manageable number, and
H. applying the CLE method to optimize the free energy to find the most thermodynamically favorable topology for the said amino acid sequence,-
wherein the global free energy (FE) contribution from the CLE between two distinct amino acid residues, herein labeled i and j, is calculated by equation (1):
wherein, i and j represent the indices of two distinct residues in the said amino acid sequence, and j>i, ΔNij=j−i +1 expresses the number of residues separating i and j, ΔGij is the difference in the free energy contribution to the CLE from residues i and j transitioning from the denatured (random flight) state to the native state, ΔSij is the corresponding entropy loss, ξ is the persistence length, γ is a dimensionless weight parameter describing the self-avoiding properties of a polymer chain, kB is the Boltzmann constant, T is the temperature, and λij (the bond gap) expresses the amino acid separation distance between the center of mass of residue i and the center of mass of residue j when both are treated as isolated molecules.
9. A method according to
wherein, ΔGij is defined in equation (1), i′ and j′ are indices specifying two secondary structure elements (α-helices or β-strands) that are joined together by the corresponding set of bonds i and j, fi′j′(ξ) is a positive definite penalty function used to enforce topology constraints on the minimum allowed sequence length of a loop connecting two elements of secondary structure i′j′ and is a function of the persistence length ξ, and ΔGξ o is a renormalization correction and is an integral function of ξ as shown by equation (3):
wherein, ξ, γ, kB, and T mean the same as defined in
10. A method according to
11. A method according to claim any one of
12. A method for building a 3D structure of a protein for MD simulation from the topology obtained by the method according to any one of claims 1, 2 and 8-10.
13. A method according to
A. obtaining an amino acid sequence of a protein,
B. preparing information on the secondary structure of the said amino acid sequence by way of at least one theoretical or experimental estimate,
E. applying the CLE method to approximate the global folding kinetics of the said amino acid sequence,
I. using the global folding kinetics to predict the optimal topology of the said amino acid sequence, and
F. storing the information in a data file or in other form of digital memory.
1. Field of the Invention
This invention is concerned with a novel algorithm for determining the spatial-arrangement topology of a protein from its secondary structure. The invention is an essential aid in finding the topology and ultimately the three dimensional (3D) structure of unknown protein structures. The invention also employs the algorithm to estimate the structural stability of a protein with a given secondary structure and sequence using a global entropy evaluation method combined with thermodynamic parameters obtained from experimental data of binding free energy (FE). The method also predicts the dominant folding pathway of a protein as a result of using this global entropy evaluation method.
2. Description of the Related Art
Determination of the three dimensional structure of a protein remains a very difficult process (1-5). The most successful approach is x-ray crystallography, which involves fitting diffraction data obtain at very large government-funded synchrotron facilities (1). Although academic professionals are able to apply for government funding to measure and analyze protein structures at such facilities, the costs of maintaining and operating such facilities that provide the beam time to support these experiments are prohibitively expensive and not accessible for commercial enterprises to fund. Moreover, biology related commercial enterprises need this information; particularly pharmaceutical companies where new drugs are always under development.
Furthermore, protein structures obtained by x-ray crystallography require skilled techniques to express and crystallize a given protein before such a measurement can be made (7, 8). It remains questionable whether all proteins can be crystallized and whether the crystalline structures fully represent the in vivo features of many biologically relevant proteins. Whereas many enzymes remain active even in this crystalline geometry (9), the true dynamics of these structures and the range of conformations can only be inferred in the x-ray data because the protein structures are rigidly locked in a crystal. The in vivo structures of protein subunits are even more difficult to assess as crystals.
A second approach is NMR spectroscopy (10, 11). NMR spectroscopy is cost efficient for a company to carry out. However, this technique is often fraught with difficulties due to the time resolution of NMR experiments, the effects of solvent exchange and other complex coupling effects (7, 8). In addition, the same problems that hamper x-ray crystallography research—protein expression, isolation, and characterization—also render this approach costly.
The easiest information to obtain accurately with NMR spectroscopy is the protein secondary structure (11). However, the protein secondary structure carries insufficient information to unambiguously identify the topology of a given protein (12).
The most important topological information gained by NMR experiments is the nuclear Overhauser effect (NOE) constraints (10-12, see also U.S. Pat. No. 6,512,997). One must first obtain many unambiguous NOE-constraints to obtain a successful prediction. However, many proteins have highly ambiguous NOE-constraints or the NOE signals are too weak and broad banded to properly assign. In such cases, the protein structure cannot be resolved by NMR and the only remaining option is to turn to x-ray crystallography.
A third approach is protein threading (13-20, see also U.S. Pat. Nos. 6,512,981; 5,878,373; 5,884,230 and 6,377,893). However, many proteins still have less than 25% homology with known protein structures in the protein data bank (PDB). To find a plausible template structure for a protein of 25% homology, considerably more information is needed to insure the accuracy of the prediction (13, 17) and there is no objective method for deducing which structures make acceptable threads.
A remaining option is to carry out a molecular dynamics (MD) simulation (21-26). Currently, molecular dynamics however, the time frame for a full protein refolding experiment remains intractable because of the long calculation times required from even the fastest computers (thousands of years even on a parallel processor supercomputer to achieve one ms of biological simulation time). Moreover, the uncertainties and ambiguities of even the state of the art MD simulation program render whatever conclusions can be made from such a long simulation questionable (21, 23-26).
Combinatorial folding models of secondary structure alone (27, 28) yield an intractable number of structural topologies to test in an MD simulation in explicit water (15, 26) If the correct topology can be obtained, the computational cost of an MD simulation is drastically reduced and the confidence level of the predictions improved to a root mean square (RMS) deviation of no more than 3 Å (29).
What is needed is an intermediate cost effective and objective approach that can infer the topology without having to wait several millennia for the answer to be produced, applying for large grants to budget synchrotron machine time, spending long hours in the lab searching for ways to isolate proteins, or utilizing subjective methodologies to infer the protein structure. The topology indicates how the secondary structure of a protein is arranged spatially and is the main juncture between the secondary structure and the full 3D structure. The topology (spatial arrangement) cannot be obtained from the three-state secondary-structure alone.
The invention is a semi-dynamic thermodynamic model of protein folding that we developed from RNA research (30) to account for the entropy of folding. Once the topology is known, a protein can be tested for its 3D structure with only a small fraction of the computer simulation time required for a complete protein refolding MD simulation. The invention is intended to aid the NMR and x-ray crystallographer in finding the 3D structure of an unknown protein based upon partially determined structural information, specifically the protein secondary structure.
The importance of gaining a foothold on protein topology cannot be emphasized enough. First, the experimental conditions that complicate the NMR experiments on proteins are generally the norm. Highly flexible proteins may have marginally stable secondary structures that make their structures difficult to determine experimentally with high precision by NMR. Second, functional proteins are dynamic entities, not static crystals (31, 32). For regions of structure that exhibit a high degree of flexibility and polar-regions where there is rapid solvent exchange, NMR spectroscopy is limited by its time resolution (10, 11). X-ray crystallography can obtain the structure of a protein that can be crystallized; however, the overall dynamics of the protein in solution are less clear. Topology prediction offers an independent tool to guide the structural determination and improve our understanding of the physics of protein structure and folding dynamics.
The folding model considers the direction in which biological proteins are synthesized and transported through the cell as a basis for considering the step-by-step thermodynamics of folding.
This invention is a method implemented as a program for estimating the topology of a protein based on the combined information of a global entropy evaluation model and local thermodynamic potentials that express hydrophobic, polar and electrostatic interactions as well as other corrections associated with size, shape or chemical properties. Parameters and models for these local thermodynamic potentials can be obtained from either theoretical sources, or from experimentally obtained data. The local interactions are modeled to help align individual protein secondary structure elements. Using a given amino-acid sequence and an obtained secondary structure estimate, the method is used to predict the best topology for the protein and to determine the dominant global folding kinetics of a protein in a biologically relevant fashion as described by the order and change in the protein's topology during optimization of the free energy where the synthesis of a protein is expected to be from the amino terminal (N-terminal) end toward the carboxy-terminal (C-terminal) end.
More concretely, the invention of this application is relating to a method to predict the topology of the spatial arrangement of an amino acid sequence using an entropy evaluation model that takes into account the global contributions of entropy to the folding of a biopolymer (herein referred to by the name cross linking entropy (CLE) and described in the literature) combined with other thermodynamic potentials as a protein-folding model.
Further, the invention of this application may comprise the following steps: inputting an amino acid sequence of a protein,
In the invention of this application, the cross linking entropy (CLE), which is an entropy evaluation model that takes into account the global effects of entropy in the folding of a biopolymer, can be used to evaluate the entropy loss of a protein due to folding into a particular topology given a known secondary or estimated secondary structure.
Further, the invention of this application is relating to above-mentioned inventions, in which loss of biological activity of the protein can be further predicted.
Further, in the invention of this application, a initial theoretical estimate of the secondary structure can be obtained from either a theoretical source, an experimental source such as an NMR experiment or x-ray crystallography, or both.
The invention of this application further relates to above mentioned method, in which the theoretical estimate can be further supplemented with sequence alignment to find regions in which conserved segments remains essentially unchanged by differences in the aligned sequences.
In the invention of this application, the amino acid sequence and secondary structure-information can be used to evaluate the free energy of a combinatorial number of β-strand and α-helix arrangements as rapidly as polynomial time: c(n−1)(n+1) wherein c is a constant and n is the number of secondary structure elements found in the said amino acid and obtained.
Further, the invention of this application is relating also to a method to predict the topology of the spatial arrangement of an amino acid sequence comprising following steps:
Here, the total CLE contribution to the free energy (Δ Gcle) can be calculated by equation (2):
In the invention of this application, the optimal β-sheet alignments can be obtained by using thermodynamics.
Further, the CLE method is applied in conjunction with other derived or constructed thermodynamic potentials that approximate hydrophobic, electrostatic and polar interactions, in a thermodynamic calculation to account for both short and long range folding interactions and predict a minimum free energy and corresponding topology of the said amino acid sequence.
The invention of this application is also relating to a method for building a 3D structure of a protein for MD simulation from the topology obtained by one of the above-mentioned method.
The invention is relating to a method to predict the topology of the spatial arrangement of an amino acid sequence using the entropy evaluation model, comprising the following steps:
The file of this patent contains at least one drawing executed in colour. Copies of this patent with colour drawings will be provided by the Office upon request and payment of the necessary fee.
The present invention may be more readily described with reference to the accompanying drawings, in which:
Definitions and Background on Biopolymer Theory
We first present a brief summary of the concepts used within this work. Special words used in other parts of this work are given in double quotation marks " " when first encountered in this section. It is hoped that by seeing these words used in this context, the nearly interchangeable meaning of such expressions as 'entropy loss' and 'global entropy' will become clear. For further details and clarification on the theoretical concepts of biopolymer folding, reference 48 (although somewhat dated now) is very strongly recommended. Additional supplementary material can be found in such sources as A. Yu. Grosberg and A. R. Khokhlov, Statistical Physics of Macromolecules, AIP Press, Woodbury (N.Y.), 1994; and M. Doi and S. F. Edwards, The Theory of Polymer Dynamics, Clarendon Press, Oxford, 1986; and P. G. deGennes, Scaling Concepts in Polymer Physics, Cornell University Press, Ithaca, 1979. Another supplementary text important for understanding the concept of "renormalization theory" as used in this presentation can be gained from the first six chapters of J. J. Binney, N. J. Dowrick, A. J. Fisher, and M. E. J. Newman, The Theory of Critical Phenomena: an Introduction to the Renormalization Group, Clarendon Press, Oxford, 1992.
The word "cross link" is used in a very broad sense in this work to describe any type of bond (35). In proteins, typical bonding effects include hydrophobic interactions, covalent bonds such as the disulfide bonds, hydrogen bonds, and salt bridges. Each bonding effect is considered a cross-link in this model.
The entropy associated with the folding of a biopolymer is a statistical function associated with the number of conformations that are available to the biopolymer. For example, if we restrict ourselves to the three regions of the Ramachandran plot that correspond to the right-handed alpha helix region (αR), the β-sheet or extended region (β or ε), or the left-handed α-helix region (αL), then an N residue peptide has 3N conformations available to it (excluding corrections for excluded volume). Even for such a grossly over-simplified model for the conformations, the number of possible conformation becomes astronomical for any reasonable sized protein.
Therefore, the problem is usually reduced to a much simpler model, the simplest and most transparent being the "random flight model" also known as the "Gaussian polymer chain" (GPC) model. In the GPC-model, the individual monomers (referred to here as "mers" for short) are reduced to mere formless particles akin to "beads on a string". Although very abstract and seemingly not highly representative of any real monomers, such models are able to approximate some important features of polymer dynamics.
The GPC-model expresses the statistical probability of finding a given end-to-end separation distance r for the first and last mer in the polymer sequence and is expressed as follows
The GPC model is too simple a model for a real polymer because it does not even consider that the polymer chain is "self-avoiding": a property in which no two mers of the polymer sequence can occupy the same spatial position at the same time (conservation of matter). The first approximation of a self-avoiding polymer chain consists of a variant of the "Gamma function" that has the form
For a system in which the size of the beads is the same as the mers, these pdfs permit us to express the entropy as follows
The formation of cross-links between the ends of the chain, r is compressed to some distance λb where λ is a dimensionless proportionality constant we refer to as the "bond gap".
The end-to-end correlation effects are not restricted to the terminal ends of the sequence. For a given monomer i and j (where i≠j and i<j ), this weak coupling leads to a dependence in which rij 2∝(j−i+1)b2. In the current embodiment, we use this to help estimate the entropy loss caused by the formation of bonds (or cross-links) between i and j. Furthermore, because we assume weak coupling between mers in the polymer chain separated by a distance ξ or greater, the coupling between a cross-link (bond) formed between mers i1-j1 and that of i2-j2 is sufficiently weak that they can be treated as independent permitting us to approximate the "global entropic contribution" by a summation. The summation of all the global entropic contributions is what we call the "total (global) entropic contribution". For mers separated by a distance less than ξ, we apply "renormalization group theory" in which these mers are grouped as though they behaved as a single monomer of fractional size 1/ξ. Approximations and corrections that account for this grouping are developed from this theory. Again, if the beads and mers exactly correspond in number, the global entropic contribution formers i and j is similar to Eqn (0c)
The "free energy (FE) contribution" from this "global CLE contribution" for the interaction of mers i and j is therefore ΔGij=−TΔSij, and this FE, when viewed from the perspective of folding a biopolymer from its entropy maximum (ro) to some small distance (r=λb), can be seen to yield a positive value. Since the free energy must be negative to be considered spontaneous, a positive entropic contribution to the FE can be understood to be a "free energy cost", since some other interaction must make up the difference in the free energy to insure that the reaction remains spontaneous.
In general, a "global entropy evaluation" of the effects of correlation between mer i and mer j is a function of the allowed conformations of the polymer change due to its chemical and mechanical properties. It is not a fully settled issue as to what extent complex systems such as a heterogeneous biopolymer can be simplified to a mere summation of the respective global entropic contributions corrected over a persistence length ξ. More forms of correlation might be anticipated than are currently addressed with the Γ-pdf. Therefore, in its most general form, the global entropic contribution or CLE between residue i and j of a protein is currently an unknown function. In the current embodiment, this function has been approximated by the Γ-pdf subject to corrections for ξ>1 mer. Other local interactions such as hydrophobic, charged and polar conditions are also assumed to be sufficiently weakly coupled so that they can be resolved at the monomer or at most the dimer level.
In summary, the general form of the CLE should not be assumed to be a Γ-pdf. However, for practical reasons of computation, the Γ-pdf is used to evaluate the CLE in the embodiment and examples given here. As long as one can find some way to write some description of the CLE in terms of either the beads or the mers, the method for evaluating the CLE, the assumptions, the approximations, and the procedures used in evaluating the free energy of the biopolymer ("the CLE method") will amount to the same procedure.
Theoretical Secondary Structure Predictions
Prior to use of this invention to determine the topology of the protein, information on the secondary structure is required.
Determination of the secondary structure can come from a variety of sources. Theoretical estimates can be obtained from such sources as PredictProtein (33-37), Jpred (38, 39), the secondary structure predictions from 3Dpssm (40, 41), NNpredict (42, 43), or PSIpredict (18-20). These theoretical estimates can be further supplemented with sequence alignment obtained from BLAST (and PSI-BLAST) to find regions in which conserved segments such as the hydrophobic core (in particular) remains essentially unchanged by differences in the aligned sequences. Secondary structure information can also come from an NMR experiment or x-ray crystallography.
The Protein Folding Model: Cross-Linking Entropy (CLE)
For questions on algorithms discussed in this section, the reader is referred to the following reference book. G. F. Luger, Artificial Intelligence: Structures and Strategies for Complex Problem Solving, 4th ed. Person Education Ltd, Essex, 2002 and P. A. Pevzner, Computational Molecular Biology: an Algorithmic Approach, MIT Press, Cambridge (Mass.), 2000.
Levinthal's Paradox points out that if a protein must search all the possible conformations available to it, even a simple protein would take much longer than the lifetime of the universe to fold (44). To eliminate this large conformation space, nature must reduce the number of degrees of freedom in the folding process and the free energy should be such that folding has a funnel shape (44-47). In the model, these degrees of freedom are expressed in the persistence length (ξ) (48). A very stiff structure would have a long persistence length, would tend to remain correlated over several amino acids and would fold as a unit rather than as individual residues (30, 47, 48). In the model, it is assumed that there are no significant kinetic traps (5, 9) and that the denatured state of a protein (4, 49) can be approximated by a random flight approximation (30). We have shown that the CLE successfully models the global folding of RNA in conjunction with the thermodynamic parameters that are currently in use for these structure calculations (30). Whereas the detailed packing rules of protein folding are fundamentally different from RNA (50), the entropy associated with the global folding conformations of a biopolymer can be treated as independent of the details of the particular system in the first approximation (48).
The CLE method helps to simplify the problem of calculating the topology of the protein. There are several ways in which this is accomplished.
First, the amino acids are grouped in units of length ξ, where ξ could be treated as a variable if more information is known about the flexibility of the biopolymer. Units of length ξ are of the same order as the length as the secondary structural units. Hence, the problem of handling N weakly coupled mers is reduced to one of handling approximately 2�NSS weakly coupled beads. This drastically reduces the problem of solving a 3N conformation problem to a much simpler one of beads on a string.
Second, in applying the CLE, it is assumed that the three state secondary structure (i.e., α-helix, β-strand, coil) is precisely known and we have only to consider how it is to be arranged in the folding process. At least 70% of the secondary structure can be predicted based on primary sequence data alone, and can be increased up to 80% by using sequence alignment techniques (33).
However, if no topological restrictions are applied in the folding problem, the number of ways in which NSS secondary structure elements can combine is factorial (2N
Therefore, a third aspect of this method is the way in which folding is carried out. In the RNA problem, we have found that a branch and bound algorithm is sufficient for describing the folding of RNA. The reason is that RNA folds from the 5′ to the 3′ end and therefore, it is actually folding during synthesis or as it is transported through a biological membrane. A similar situation occurs in a protein where the synthesis and transport proceed from the N-terminus to the C-terminus. Therefore, in the limiting case, the branch and bound approach is sufficient to solve this protein-folding problem within the CLE model. However, the branch and bound method still exhibits exponential growth in computer resource. The theory developed from the CLE shows that for such structures as pleated β-sheets are strongly favored entropically compared to other conceivable structures such as a jellyroll fold. Protein folds such as the Greek-key or the jellyroll fold are rare compared to β-meanders. The reason is due to entropy. (A similar situation can be posited in the folding of adjacent α-helices in such proteins as cytochrome C.) A picture emerges in which the folding of a protein is quite rapid, and the dynamics are such that a protein effectively "grabs" the closest neighboring secondary that exhibits a reasonably favorable FE. Since the closest neighbor is likely to be the next adjacent strand, it should be no wonder why the β-meander structure is so common in protein folds (refs 1-3, and although quite dated, we also recommend G. E. Schulz and R. H. Schirmer, Principles of Protein Structure, Springer-Verlag, New York, 1979). Evolution has further fine-tuned this folding so as to usually produce a thermodynamically stable structure that can also be obtained through refolding (where N-terminus to C-terminus folding should not be assumed but rather nucleation). The length of many proteins is often in the range of 200 amino acids or less. Secondary structure elements are typically on the order of 5 amino acids in length (similar to ξ), and most often, only half of the protein sequence forms secondary structure. This means that for a 200 amino acid protein, there are about 20 secondary structure segments. The jellyroll fold, one of the most complex in protein folding groups is composed of only six β-strands. This means that the scan region for folding can be limited to about 8 elements in the first step. A recursive search (without replacement) can then be employed that preserves the dominant order in which the secondary structure units combine. Unlike the time it takes for the protein to fold, which is an exponential function of the global entropy (see example 5, Eqns (4) through (7)), the recursive search (without replacement) is done in polynomial time. If the secondary structures are all of the same size, then the time to align individual mers can be treated as a constant (At) times the recursive search (without replacement) of the secondary structure elements or tsearch∝AtB(NSS+1)(NSS−1)∝NSS 2 where B is a constant and max{At}∝ξ3 (depending on the procedure and assumptions used to evaluate At). This is a substantial gain over other methods. The underlying assumption in the CLE model is clearly that proteins don't wait a long time to find their fold. In the event that much longer folding times are thought to occur, a branch-and-bound approach is also an option.
We now discuss how the cross linking entropic contribution to the free energy (FE) is evaluated as it pertains to using the Gamma polymer chain equation with no loss of generality for other methods that can express the entropy relationship between two residues i and j in an additive fashion. The total FE refers to the summation of the individual contributions of bonds (cross-links) i and j. In the current embodiment, ξ is treated as though it were a constant. However, the expressions can be modified to consider a variable ξ, if an experimental or theoretical estimate is available.
The global entropic free energy cost for folding a denatured protein into a β-sheet structure is expressed as follows. Let i and j represent the indices of two distinct residues in a protein sequence, where j>i. The number of residues separating i and j is ΔNij=j−i+1. The global FE contribution from the CLE of residues i and j can be approximated by the following expression
The total CLE contribution becomes the sum of each ΔGij contribution to the native state,
The global CLE is a function of the likelihood that a polymer would spontaneously adopt a specified configuration represented by Eqn (2). Each cross-link adds to the global configuration entropy according to Eqn (2). This results in a cumulative (integral) effect that grows as ΔNij ln (ΔNij). Ultimately this limits a domain or loop size because the CLE grows logarithmically {ln (ΔNij)} with each cross-link whereas the individual hydrophobic interactions are local and independent of ΔNij. The CLE has shown considerable progress in addressing the folding of RNA (30).
In our current global CLE evaluation strategy, we assume the hydrophobic effect is the dominant feature that leads to attraction between different secondary structure elements of the protein sequence (50, 52). Thermodynamic parameters are dealt with based upon thermodynamic potentials in the local regime (52, 56-59). Structure is considered from standard models (3, 60-66). To help to orient the secondary structure, we use the polar, non-polar and hydrophobic interactions between neighboring β-sheets to help align the secondary (3, 56). Hydrophobic parameters can be obtained from such sources as Ref 45). Other parameterizations can be either resolved based on approximations from molecular dynamic simulations, or various theoretical or experimental sources.
To help to orient the β-strands, we use the polar, non-polar and hydrophobic interactions between neighboring β-sheets to help align the β-strands since these are likely to help stabilize the structure (3, 56). Scales of hydrophobicity can be obtained from such experimental sources as Y. Nozaki and C. Tanford, J. Biol. Chem. 246; 2211-2217 (1971) and references therein to name one. Theoretical sources could be for example T. Lazaridis, J. Phys. Chem. B, 102:3531-41 (1998). Other parameterizations can be either resolved based on approximations from molecular dynamic simulations, or constructed from some first principle approach.
This later approach follows a similar strategy first used by Cohen et al. (27, 28) to find ways to arrange β-sheets together: termed β-sheet alignment. As Cohen et al. reasoned, the alignment of residues should follow sensible relationships for neighboring residues along a pleated β-sheet region such as similar hydrophobicity, complimentary acid/base interactions, etc. (56-59). The improvement here is that we use this entropy to eliminate the multitude of combinations to a single topology or a finite set of solutions if dominant suboptimal structures are considered. Moreover, in this invention, the optimal β-sheet alignments are obtained by using derived (or estimated) thermodynamic potentials rather than statistical relationships between different proteins. A statistical relationship approach would be a possible alternative in a tuned potential; however, mere statistical relationships provide limited insight on the origin of the effects or what contextual aspects influence the uncertainty whereas physical models provide a conceptual framework that can be improved upon with better understanding.
A similar strategy is applicable in the case of α-helices. The major difference is that the residue alignments between two secondary structures much coincide with the cylinder shape of an α-helix. Thus the contact points of residues along this face have gaps {i, i+1, i+4, i+5, i+8, i+9} where i is a reference position of the beginning of an α-helix (27, 28).
The protein folder works on the principle of crawling along the sequence from the N— to C— terminus searching for the nearest acceptable β-strand that permits the minimum entropy loss, balanced with favorable hydrophobic and ionic interactions. In this respect, the protein folder assumes that a protein folds as it is extruded from such biological structures as the ribosome rather than assuming that the protein specifically folds from the denatured state.
The algorithm currently is primarily concerned with fitting all β-sheet proteins of approximately 70 amino acids in length. Longer sequences can in principle be solved using this approach with varying degrees of success in its current form.
Explanation of Practical Example is Shown as Follow.
When the invention is applied to a standard β-meander protein such as IL-8 (melanoma growth stimulating activity: 1 MGS), the correct topology is obtained. Table 1 shows the best residue alignment and topology found in the calculation (67).
In the top section of Table 1, the left most column indicates the index of the given β-strand segment of the amino acid sequence. The next two columns indicate the position at the N-terminus (N) and the C-terminus (C) of the β-strand. The next three columns indicate the determined topology of the protein sequence. The middle column labeled f_i (forward strand i) contains all the β-sheet indices. The column immediately to the left (r_j: reverse strand j) indicates the β-strand immediately adjacent to f_i whose index is less than i. Likewise, the column immediately to the right of f_i (f_j: forward strand j) indicates the β-strand immediately adjacent to strand i whose index is greater than i. The final column indicates whether the two β-strands that form the loop from antiparallel or parallel β-sheets (b—means antiparallel beta).
For a β-meander, the middle row (1 2 3) indicates that strand #2 has strand #1 to one side and strand #3 on the other. (In the topology, the axis of symmetry is two fold degenerate) The bottom part of the table indicates the detailed topological arrangement of the mers in the β-strands relative to each other. The amino acids associated with strand i are denoted by ik where k specifies an individual amino acid in secondary structure i and similarly for jk. The expression ik-j1 indicates that the residues (ik and jl) from each strand (i and j) are directly adjacent to each other.
The topology and physical alignment of the β-strand residues is shown graphically in the
Similar to example 1, the structure of an undetermined protein LECT2 is also calculated using this model (68, 69). Again, the topology is indicated in Table 2 in a similar way and a cartoon of the alignment and topology is shown in
For LECT2, there are 6 β-strands and the alignment also has the form of a pleated β-strand structure. The pleated β-sheet fold and the β-meander fold are some of the most common structural motifs in the protein β-strand architecture (1-3). Examples 3 and 4 show how this topology can be used to find the 3D structure of the LECT2 protein. The topology of LECT2 is shown graphically in
Combining NMR Chemical Shift Data with Topology Information
NMR chemical shift data can now be used to evaluate the topological prediction by utilizing torsion angles estimates using such software as Talos (72, 73) and weighting the predictions relative to the certainty of the Ramachandran angles using a power law such as xn10n−2, where x is the ratio of matching predictions to the best 10 predictions in the Talos database and n>6. An example of a prediction using this combined method is shown in
Building a Trial 3D Structures for MD Simulation
Once a topology is obtained from this invention, the 3D structure can be built and refined through dynamical simulations using a number of approaches, for example Refs 29 and 47.
To fold the amino acid sequence into the correct secondary structure and topology, the structure is first built in extended form to mimic the approximate configuration of the PII structures found in denatured proteins (31, 70, 71).
To introduce the secondary structure, torsion constraints are applied over the regions of secondary structure forcing out the secondary structure in the sequence using relatively strong torsion constraints on the order of kθ=10 kcal/mol�rad2 with a maximum potential energy (PE) of 100 kcal/mol, where kθ is the torsional force constant. To make the proposed contacts, weak distance constraints are then applied: kr≦2.0 kcal/mol�Å2 (max. PE<10 kcal/mole), where kr is an effective spring constant. These distance constraints are typically applied to the C═O . . . H bonds. The structure is then allowed to relax during an MD simulation (either in explicit water or in vacuo) using simulated annealing starting from a high temperature (at least 400 K for at least 10 ps). The simulation time is doubled with each decrease in the temperature in a fashion akin to Newton's law of cooling. High temperature runs (T>600 K) required kr>1 kcal/mol�Å2, but annealing at lower temperatures can be done with kr<1 kcal/mol�Å2. To help minimize the effect of large fluctuations in the structure at high temperatures, the time increment should be set to 0.125 fs and strong torsion constraints should be placed on all the amide bonds to restrict the orientation in a trans configuration. For temperatures below 410 K, the time increment can be set to 0.5 fs. This annealing process should be applied over a period of 0.5 to 1 ns. The approach differs from Refs. 29 and 45 in that we recommend using small distance constraints (kr<2. kcal/mol�Å2 versus 100 kcal/mol�Å2) and applying them over long and graduated simulated annealing times (preferably in excess of 200 ps) so that the structures have considerable time to explore the local conformation space. We also recommend starting with the PII conformation or an extended structure.
The very high temperature permits sufficient thermal energy to help fix some of the mis-oriented residues in the turns. At 300 K, the distance constraints are reduced to 0.5 kcal/mol�Å2 (max 1.0 kcal/mol) and the simulation is run for 100 ps to help the structure further relax. After annealing in the water bath, all topological constraints can be removed and the structure is allowed to relax at 300 for 100 ps.
Folding Principles of Various Structures
To help illustrate the folding model, in
The β-strands 1, 2, and 3 are already decided in the structure. However, one must chose how to arrange the fourth strand. If the coil residues between the strands is very long, then λij will be large in Eqn (1) and the structure can fold to the left (depending on-the composition of the β-strand at 1). On the other hand, if the sequence length is short, then the structure is likely to fold to the right. The possibilities are depicted in the triangle graph by the yellow arrows. The topological constraint function (fi′j′)(ξ)) will force β-strand 4 to link with strand 3 if the adjoining sequence is too short to permit crossing over to strand 1. In this case, stretching of the Gamma pdf (r>ro) will result in a large positive entropy as well.
Protein folding can be largely limited to short range binding covering at most 10 secondary structure elements before settling into a structure. This is unique part of our invention. The CLE sets a limit on domain size due to the heavy weight of entropy loss. Topological restrictions such as the length of the loop region further restrict the allowed structures eliminating pointless combinatorial alignments when a connecting strand is shorter than a specified length.
This can be taken to other structures. In
The form of plotting used in this
A Qualitative Description of Protein Folding Based on CLE Model
Here we provide a qualitative description of how the cross-linking entropy affects protein folding according to Eqns (1) through (3). Because proteins tend to fold in a sequential manner (from N to C terminus), one must visualize this process in as dynamic rather than static. Nevertheless, it should be remembered that the folding of a complex structure requires at least as much sequence as the shortest loop of the domain. The entropy loss should be minimized and this usually favors short loops according to Eqn (1). Only a very special type of amino acid sequence can produce a protein where a segment at its N-terminus actually waits while the rest of sequence folds and then hooks on as a β-sheet at the C-terminus.
The hydrophobic effect is a strongly local phenomenon. Secondary structure may form with dominantly extended-like features (70) or even much of the basic secondary structure in tact (5), but the effects of attraction between the two β-strands is only possible when the two strands are in proximity of each other.
Qualitatively, the CLE helps discriminate between different folding processes. In
The cross-links between residues in an α-helix are at every fourth residue so ΔGij (Eqn (1)) will be essentially a constant for all α-helix contacts and the formation rate will depend strictly on the local characteristics of the residues. This means the folding rate should be approximately simultaneous over the length of the α-helix in a refolding experiment.
The folding of each loop in the β-sheet of
It is known that the rate of folding is much different between α-helices and β-sheets (74) where the rate for α-helices is much faster. This is used in the method developed in the CLE model.
Assuming the folding proceeds directly from the denatured state to the native state with no significant kinetic traps along the reaction coordinate, the rate of folding can be expressed as
Qualitatively, the structure in
Since, these contributions are separable from the rest of the expression in ΔGtotal we can estimate the protein-folding rate to be
The CLE provides a quantitative physical model with solutions that resemble the contact order model (75), but more than that, the CLE shows why the contact order is a reasonable heuristic in protein and RNA folding. The CLE also predicts that α-helices can fold to any conceivable length (76), and that they fold cooperatively because the contact points and their relative distance along the protein chain are uniform (
Effect of the Invention
Finding the topology of a protein is an essential intermediate step between the simple three-state secondary structure prediction and the final 3D structure of a protein.
Secondary structure in proteins only refers to whether the amino acid sequence has α-helices, β-sheets or coil. Some programs also can indicate whether there is a high probability of a turn. Since there are 2n−1n!/2 possible ways to arrange this secondary structure (where n is the number of secondary structures), merely assuming all configurations are allowed yields an astronomically large number of structures to test for any real sequence. It costs considerable resource to test them all. Therefore, a powerful approach that can weed out the useless solutions is needed to help drastically reduce unnecessary ways of arranging the secondary structure. The invention has the strong capabilities to assist in research in this direction.
This algorithm is expected to aid the protein structure researcher to find the topology of a protein quickly and effectively. For RNA structure calculations, we have already developed an approach that significantly improves the performance of these calculations over existing standards (30). We have applied it to protein structure calculations here because of the universality of this theory of biopolymer folding. To account for the difference between proteins and RNA, different parameterization and a different folding approach was used.
The invention also helps grant insight into the dominant folding pathway of a protein and can be helpful in understanding the effects of mutations on the topological stability of a protein.
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AudioScore Ultimate 7 from Sibelius is software that allows you to open a CD track or MP3 file and transcribe it to a score. By using the most intelligent technology available, it is possible to convert up to 16 instruments/notes playing at a time into multiple staves, with up to 4 voices per staff. Immediately after transcribing your music, you are able to send it directly to Sibelius, or save MusicXML, NIFF and MIDI files for use in most other music programs.
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Bean, J. C. (2001). Engaging ideas: The professor's guide to integrating writing, critical thinking, and active learning in the classroom. San Francisco: Jossey-Bass, A Wiley Imprint.
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