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The weekly RA Podcast features an exclusive mix of electronic music from top producers and DJs around the world. Resident Advisor is an online electronic music magazine. Visit RA online at www.residentadvisor.net
October 20th, 2014
Episode 8 of 135 episodes
Unleaded techno from an UNO associate.
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Feb 13, 2017From Buyer: YoManila
Easy communication and the Item arrived as described. Overall a smooth transaction.
Dec 21, 2015From Buyer: fieldofdreams
Item was exactly as described. Communication was great. Shipping was great. Would buy from again.
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D communicated well about shipping and the item was as described. Very happy with the transaction and would buy from him again.
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transaction was smooth and quick - item was as promised.....highly recommend the seller
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Very smooth transaction. All good!
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Great suit was described. Thanks much!
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The jacket is fabulous and I couldn't be happier. Thanks again for being a great seller.
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Jacket was shipped on time, well packed. Was as described and at a great price. Thanks!
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Fast delivery, well packed and quality item. Thank you.
Apr 9, 2012From Seller: John Doe
A great buyer. Good communication and prompt payment. A+++
Feb 19, 2012From Seller: Steve Smith
Solid buyer. Highly recommended. Thanks.
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A pleasure doing business with you. Enjoy your item(s) and I hope to be of service again in the future.

subset: commoncrawl


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[PHOTO] Green Waves (close up) | Fri, 14 Apr 2000 23:50:30" />
This close-up was taken on a miserable (weather) day. It was the first
time I saw GREEN WAVES in bloom, so I excitedly snapped a few shots. Much
to my frustration I have not been able to get a substantially nicer photo
(with better focus, better light, etc.) since then. And I've tried about 2
dozen times, so I give up. Enjoy!
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Silver Lake Has Always Been Gay, Readers Say
The Coolest Family in Town
Readers thrilled to Pamela Chelin's cover story about the charmingly eccentric Morgan sisters, actress/belly dancer Molly and actress-stuntwoman-contortionist Bonnie — and their equally eccentrically charming parents, Gary and Suzy ("The Fabulous Morgans," April 19).
"This is such a delightful story of a sweet and amazing family!" Babedarla enthuses. Cb00 agrees: "Wonderful article. Great writer."
"I am blessed to know these wonderful people as friends," adds prizantproducts4u. "They are part of my spiritual family. God bless the Morgans!" From our reading of it, He already has.
Upcoming Events
UCLA Bruins Men's Baseball vs. UC Riverside Highlanders Men's Baseball
TicketsTue., Feb. 21, 6:00pm
Anaheim Ducks v. Boston Bruins
TicketsWed., Feb. 22, 7:30pm
UCLA Bruins Men's Baseball vs. Gonzaga University Baseball
TicketsFri., Feb. 24, 6:00pm
Los Angeles D-Fenders
TicketsFri., Feb. 24, 7:30pm
To Be Gay in L.A.
Tyler Trykowski's online-only piece on the merits of Silver Lake ("Move Over West Hollywood — Silver Lake Is the True Gay Mecca," April 19) stirred up plenty of buzz last week, beginning with its headline. Writes Bingoninja, "There is something peculiar about the phrase 'gay mecca.' It's either so ignorant, and so inappropriate — or so ironic, and so inaccessible. I dare say, Mecca is not a place gay people would feel welcome." Point taken.
Wendellxx isn't buying the premise. "The author is so out of it he actually believes Silver Lake is an Eastside neighborhood (it's not), and that all of West Hollywood is the same as a few blocks of Boys Town. I live across the alley from the Gold Coast, which is full of guys 50 and up, and a few blocks away from Fubar, which is full of as many alternative gays as Akbar. When my partner lost his Silver Lake apartment, he had to move to east West Hollywood because he could not afford the rents in Silver Lake, which has become totally gentrified."
Shapirobrendan writes, "The problem with young people is that they are so unaware of anything before their own time: It's all new to them, so it must be NEW, period. Having moved to Silver Lake in 1976, I can tell you that Silver Lake has always been a vibrant and wild gay mecca, the gay man's alternative to Boys Town and West Hollywood. This writer is trying to compartmentalize a group that defies compartmentalization."
CheapTrick writes, "WeHo has always drawn a younger crowd. But while 40 years ago gay men hid in the Mafia-owned clubs, and 20 years ago, gay men over 30 were either dropping dead or retreating to the suburbs, now the 40-ish crowd is big and out and going strong and not really into the glam, loud dance clubs and martini bars in WeHo. Instead we go to the more laid-back bars in Silver Lake. Not sure why that seems to upset some people."
Our April 12 GoLA section referred incorrectly to the network that first broadcast the miniseries Sybil. It was NBC. We regret the error.
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You Get an A-Plus
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Konstantin Lukin, Ph.D.
Clinical Psychologist
223 Bloomfield Street, suite 107, Hoboken, New Jersey 07030
Families are complicated. Each person in a family plays his or her own role, and the way each person behaves when the whole family is together is not necessarily the same as when they are apart. At the Lukin Center, we use emotionally focused therapy to help families understand the complex dynamics among all family members and to help each individual in the group feel heard, supported, and understood.
Walter Masterson, LCSW
Psychotherapy and Counseling
200 Rector Place, 23L, New York, New York 10280
Careers and children each place enormous stress on a couple's ability to be together in an intimate and satisfying way. Every couple has different ways of dealing with the challenges, and counseling can provide the perspective and encouragement to re-create a shared vision for the future. For something as important as this, it is surprising how little training and preparation is provided; family therapy fills in the blanks, and resolves the confusion.
Kevin Fleming Ph.D.
Coach/Change Agent/Consultant
New York City, New York 10013
Most family therapists inadvertently create more chaos through their well-intentioned efforts to assist a family move through a change process. Why? Brains are inherently deregulated (all of us, may I add) and most talking and dialoguing misses brain-centric problems that need more intensive help. Many times the emotional trauma created by years of solving problems ineffectively can wreak havoc on the brain's parasympathetic nervous system, not ever giving the family a break from flight or fight thinking. Contact us for an innovative neuroscience solution to family dysfunction. firstname.lastname@example.com or 877-606-6161.
Lois Horowitz, Ph.D, LCSW
London Terrace Gardens/ Chelsea/ West 23rd Street, New York, New York 10011
Family Therapy is not only for people with small children. I specialize in helping adult members of families to improve their communication and resolve conflicts. The goal is to teach family members skills to work through their long standing dysfunctional patterns, to deepen their family connections, and to facilitate the healing process. I work with both gay and straight families. Please visit my website lhorowitz.com for more information.
Michael Picucci, PhD, MAC, SEP
Holistic Psychologist, Author, Focalizer
44 East 12 Street, New York, New York 10003
In working with families and conflicts, I approach it more as an organic facilitation process rather than traditional therapy. Utilizing new human technologies, we come to understand that there is no right or wrong or good and bad. Yet, there are insidious unconscious dynamics that are powerful and can cause blind spots. Our process together will tease apart these dynamics while working on resolving them, thereby uncovering new fields of possibilities.
Gerald Schoenewolf, Ph.D.
Licensed Psychoanalyst
207 East 15th Street, New York, New York 10003
My approach to family therapy is to look for the source of the family conflict. After over 37 years of practice, I can usually zero in on what is going on. Sometimes one parent is out of control. Sometimes parents are in conflict over child-rearing practices; one is too permissive and one too strict. Sometimes one child is favored over another. I generally meet with the whole family over a period of time, encouraging honest and constructive communication. Through patient guidance, the problems surface and are worked out.
Carolyn AlRoy, Psy.D.
Licensed Psychologist
19 West 34th St., Penthouse, New York, New York 10001
I focus on specific relationships , rather than the entire family. Like I do in couples work, I would see the two of you both together and individually. I evaluate these cases carefully to make sure that I would be able to be of service to both of you. Usually whatever is going wrong has a long history, and we will unravel that. It may take a bit of time, but can be done if you are willing and patient.
H.C. Fall Willeboordse, LCSW-R
Fall Willeboordse, LCSW-R
244 5th Ave. Suite 9G, New York, New York 10001
When a family doesn't work well together there's little peace, contentment or fun at home. I work with families to help identify the non-working dynamics that may be repetitively occurring and causing ruptures and resentments. Family therapy can help strengthen the resiliency of a family after a crisis, as well as break long-standing destructive patterns. Sessions allow for everyone's voice to be heard and often include defining each individual's role and responsibility.
Barbara Bennett, LMFT
Licensed Marriage and Family Therapist
245 west 29th Street, suite 304, New york, New York 10001
What do you do when your family life is such a struggle…when you've read every psychology book, magazine and blog to get answers but the advice is contradictory and usually impractical? If it seems like you don't know which way to turn, then you've come to the right place. Through proven, practical techniques, I can help you work through the stress, anger and conflict that arises from issues that can prevent you from fully enjoying time with your family.
Philip Kolba, MA
New York, New York 10002
Family therapy looks at the entire family system—parents, children, extended family members—to identify strengths and problematic behaviours. I collaborate with families on strategies for utilizing their own capabilities for resolving problems, and on developing effective communication and problem-solving skills for preventing conflicts. I specialize in working with families of teenagers, which is an age that can be challenging for both the teenagers and their parents.


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Visit Our TONS Chapters' Websites:
We need your help!
It takes dedicated volunteers to create an exceptional SEC conference. Sign up to volunteer here.
About SEC
The Southeast Educational Conference is the premier professional development conference for the meetings industry in the Southeastern United States.
The Southeast Educational Conference:
- Provides affordable and accessible education resources and tools for the meeting professional
- Provides networking and business opportunities
- Elevates professional value within the global marketplace
Meeting Professionals International
the premier global association community for meeting and event professionals, believes in the unequaled power of events and human connections to advance organizational objectives.
Founded in 1972, MPI believes that meeting and event planners serve a critical role in the success of business and is committed to giving its members the very best in professional development, business opportunities and a vibrant industry community.
With more than 21,000 members, representing 86 countries, from 71 clubs, MPI chapters allow their members to form powerful social and professional bonds through conferences and community events.
MPI's Mission: To make our members successful by building human connections through
knowledge, ideas, relationships and marketplaces.
Who is TONS?
The SEC is a collaborative education conference that is hosted by the four MPI Florida chapters (TONS) – North Florida, South Florida, Tampa Bay Area and Greater Orlando Area
How Does TONS Guide the SEC? The TONS Advisory Council is made up of two representatives from each Florida chapter of MPI, in hopes of guiding the conference to an even greater success each year. We provide general guidelines to each year's group of chairs to assure the highest quality and value to the conference participants.
The TONS Advisory Council also acts as a steering body to provide vision, strategic planning and direction to ensure the continued success of the SEC. It is essential that the Chapters support these efforts and adhere to the standards, policies and procedures as agreed upon by TONS and the Chapters' Boards of Directors.


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Lot 1311: 20th century Japanese silk Kimono embroidered with
Stitches In Time - A Historical Collection of Important Textiles & Vintage Fashion
by Ewbank's
November 10, 2016
Woking, United KingdomLive Auction
Looking for the realized and estimated price?
Description: 20th century Japanese silk Kimono embroidered with stalks and flowers
Browse more items


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Lead Generation Specialist
This project received 5 bids from talented freelancers with an average bid price of £5000 GBP.Get free quotes for a project like this
Project BudgetN/A
Total Bids5
Project Description
A proven record of over achievement in delivering against business development sales targets in a highly competitive environment
Proven Face to Face sales skills
In depth understanding and passion for the digital medium and our on and offline brands
Experience selling digital formats, to clients or agencies is also vital
~The Role
Maintaining and growing advertising revenues across a given territory. The focus of revenue generation will be through face-to-face sales with proactive selling of digital standard and non standard advertising and sponsorship opportunities.
Emphasis on selling digital ad solutions on our established agency portfolio
Selling to consumer and B2B markets.
Key Personal Skills
-Strong business acumen.
-Highly motivated and a positive attitude
-Confidence, assertiveness and high energy
-An ability to ensure a market leading customer service experience is provided.
-Excellent verbal and written Communication and presentation skills
-Collaborative working style
-Openness to change
-Adaptable and versatile
-Focused on excellent delivery both internally and externally.
-Ability to deliver a clear and considered sales strategy and implement it.
A passion and understanding of all media platforms.
Skills Required
Looking to make some money?
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- Forbes
- The New York Times
- Time
- Wall Street Journal
- Times Online


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MANCHESTER, England (AP) — Manchester City says it has signed goalkeeper Willy Caballero to a three-year contract on a free transfer from Spanish club Malaga.
The 32-year-old Caballero, whose contract with Malaga expired at the end of the 2013-14 season, says on the club's website that "this is a new challenge and a new league for me" and that he "will try and compete for the No. 1 spot" with City goalkeeper Joe Hart.
Caballero played under City manager Manuel Pellegrini when he was at Malaga, and says "the most beautiful moments of my career were under Pellegrini."
The Argentine is City's third signing of the offseason, following Fernando and Bacary Sagna.
Copyright 2014 The Associated Press. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.


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Self-Catering near a beach in Vendee
We have found 2 properties to book, based on your search for Self-Catering Accommodation near a beach in Vendee, Western Loire, France.
Oceanian House Holiday Cottage in West France
Holiday Cottage in Lucon, Vendee, France
Oceanian House is a fully furnished one bedroom house elegantly decorated to capture the charm and the character of an oceanian style. We got the inspiration visiting Stevenson's h...
€ 90.00 - € 105.00 per night
Sleeps: 2
Les Sables d'Olonne Studio Apartments near beach
Holiday Apartment in Les Sables dOlonne, Vendee, France
This 4-floor renovated apartment complex with lift is located in a residential quarter in Les Sables d'Olonne, near the harbour and only 50 m from the main beach. self catering apa...
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Physician Salary
This site provides the physician salary and career information. You can find the following information on this site:
- Physician Salary Tool—research your local physician salary information.
- Physician Salary Data—nation-wide physician salary data
- Physician Job Outlook
- Physician Employment—physician employment conditions
- Training and Qualifications—how to become a physician
For further physician salary research, we recommend www.jobs-salary.com. This site serves real salary data.
If you are looking for physician jobs or are recruiting physicians, we recommend www.medhunting.com.
A Few Highlight
- Many physicians and surgeons work long, irregular hours; over one-third of full-time physicians worked 60 or more hours a week in 2004.
- Formal education and training requirements are among the most demanding of any occupation, but earnings are among the highest.
- Job opportunities should be very good, particularly in rural and low-income areas.
- New physicians are much less likely to enter solo practice and more likely to work as salaried employees of group medical practices, clinics, hospitals, or health networks.


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NBC Chicago
Mayor Rahm Emanuel has mandated that city employees on the clock be required to ride Chicago Transit trains and buses, with a few exceptions, in order to get reimbursed. BJ Lutz reports.
NBC Chicago
Celebrate where you came from or where you're going with city t-shirts, jewelry, and neckwear.


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Clumsy Cookie Traffic Jam
by: Run And Gun Free Android Games • 1.6K
All Android applications categories
All Android games categories
by: Run And Gun Free Android Games • 1.6K
Clumsy Cookie Traffic Jam - tasty sugar food fun. Once you play this your mouth will water for some chocolate n candy :) Tap the traffic light colored cookies as fast as possible for 1 minute to un-jam them before the time run out. A great puzzle clicker game.
★ Easy single tap game play, any age can play.
★ Beautiful, mouth watering food art work.
★ World leader boards, fight for the top score with friends.
★ Great to improve your speed time in other tap games, become a ninja.
★★★★★ "yes very sweet, simple to play. just a 60 second run to get the best score. i love the cookie pictures too. reminds me of other clicker games, big 5 star" - bird tapper - Dec 2, 2013
★★★★★ "omg this is fun, the maker is great. so easy and im getting better at it now, reached 500 points, super star :D im a cookie racer, lol" - ninja jam td - June 2, 2013
Tap the traffic colored cookie when they appear and get the best score in 1 minute. Tap red ones 1 time, yellow 2 times and green 3 times. When an arrow appears, tap the space next to the arrow. High score will get added to heyzap. Just tap the world leader boards button in the main menu to view them.
Players who love casual puzzle style games like Bejeweled Blitz and Papa Pear Saga will love this game too.
Happy Gaming :)
The Run And Gun Free Games Team
Users review
from 1.600 reviews
Download Clumsy Cookie Traffic Jam
Free - V1.0 - 11M
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Learn how to reserve a meeting room at the Pretlow Anchor Branch.
The Ocean View Station Museum is located inside the Pretlow Anchor Branch Library.
Sing-a-long FUN
Sing-a-long FUN with Amber!
- Date: 04/29/2014 2:00 PM - 3:00 PM
- Location: Mary D. Pretlow Anchor Branch Library, 2nd Floor Conference Room
111 W. Ocean View Avenue
Norfolk, Virginia 23503
Come dance and sing with us at the library! Amber will play her autoharp and lead everyone in songs and fingerplays!Return to full list >>
(Toddler & Preschool)
2nd Floor Program Room


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Country Living Storage Style
Pretty and Practical Ways to Organize Your Home
By (author) Lesley Porcelli
Format: Paperback
Normal Price: $22.99
Your Price: $20.69 AUD, inc. GST
Shipping: $7.95 per order
You Save: $2.30! (10% off normal price)
Plus...earn $1.03 in Boomerang Bucks
Availability: Available, ships in 7-10 days
Country Living Storage Style by Lesley Porcelli
Book DescriptionAway with plain plastic boxes and utilitarian metal filing cabinets! With a little inspiration in the form of unusual containers and furnishings, storage also has style. And, for an imaginative showcase of solutions, Country Living is the best place to go. Storage Style presents smart, fun ideas from the most welcoming homes anywhere - clever tricks for organizing that infuse rooms with personality, wit, and flair. For example, a vintage dollhouse attached to a kitchen wall becomes a charming, unexpected spice rack. A lift-top pine trunk topped with a cushion masquerades as a window seat - and serves as a perfect hiding place for board games, toys, and other bric-a-brac. Or try classic country baskets, armoires, and more. These space-saving, clutter-camouflaging design schemes shout 'pretty' with understated practicality.
Buy Country Living Storage Style book by Lesley Porcelli from Australia's Online Bookstore, Boomerang Books.
Book DetailsISBN: 9781588169945
(229mm x 203mm x 15mm)
Pages: 160
Imprint: Hearst Communications
Publisher: Sterling Publishing Co Inc
Country of Publication: United States
Other Editions...
» Have you read this book? We'd like to know what you think about it - write a review about Country Living Storage Style book by Lesley Porcelli and you'll earn 50c in Boomerang Bucks loyalty dollars (you must be a member - it's free to sign up!)
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For every $20 you spend on books, you will receive $1 in Boomerang Bucks loyalty dollars. You can use your Boomerang Bucks as a credit towards a future purchase from Boomerang Books. Note that you must be a Member (free to sign up) and that conditions do apply.


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|Reviews for Crash's Sneakers
| Chel127 chapter 1 . 6/1/2015
Rank but really funny XD had me laughing all the way through it
| Bandicoot Sauce chapter 1 . 2/1/2014
Crash looking less crazy? Not if I know my all-time favourite video game hero! He's crazy, he knows it and we all love it, especially me! Keep writing some good stuff!
| Athena Fall chapter 1 . 5/13/2013
LOL this is so funny! I really Like it
| BlueJay026 chapter 1 . 1/20/2013
Haha! This was awesome! It was funny, your grammar, punctuation, and spelling was nearly flawless, you kept both bandicoots well in character, and you used a nice variety of words!
I loved this line: "Crash! Get your sneakers outta mah face fool!"
Great job! Write more!


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Our Mission
To inspire and enhance a life of active existence through adventure, community and education.
Discover Axistence Today!
We are an academy for adventure specializing in fitness, nutrition, mobility, survival and other outdoor skills. At our training facility, we combine aspects of Crossfit, Olympic weight lifting, strongman, TRX, and gymnastics with MovNat, or species specific movements such as running, jumping, crawling and climbing.
"Everyone likes to think they have the best fitness system. The truth is that everything works, nothing works forever, and there's always room to improve. We take the best of everything, keep it fresh and focus on results."
- Ryan Humphries (Co-owner, instructor)
Our programming is based upon seasonal and personal adventures. We teach not only fitness classes and discuss nutrition, but also hold specialty outdoor skills training such as land navigation, backpacking, and wilderness survival. So whether it is to hike a 14er on the weekend, to backpack through Nepal, or to chase after your children or grandchildren through the creek, we will prepare you.
At the end of the day, we will get you physically, mentally and emotionally ready for adventure, but surround you with a supportive community, and satisfy your mind with education. Set up a time to stop in HERE for a complimentary mobility assessment and guided workout! We look forward to seeing you soon.
In the meantime, view the pages below to see our upcoming events and gain knowledge in one of our five specialty areas.
Why Us?
It's important to do your research before making a decision. Click on the link below for five reasons why Axistence is the right choice for you!
Get Started
Come in for a complimentary mobility assessment and guided workout.
Follow the link below to schedule an appointment today!
Check out our monthly Newsletter!
This is the place to find our Adventure, Community, and Educational events for the each month.


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Have gotten the usual plethora of catalogs in the mail. One that is new
to me is Territorial Seed Company. Has anyone dealt with them? Comments?
Am off to "balmy Colorado" today, but will monitor my mail.
Cathy, z5b, west central IL
To sign-off this list, send email to email@example.com with the
message text UNSUBSCRIBE GARDENCHAT


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Full-time PHP developer in Barnsley
This project received 15 bids from talented freelancers with an average bid price of £15 GBP / hour.Get free quotes for a project like this
Project BudgetN/A
Total Bids15
Project Description
An opportunity to work with a small dynamic web development team based in barnsley, with some exciting projects and challenges.
Key skills:
MongoDB, MySQL, SVN, MVC
This is a full time position based in an office and we're looking for potential candidates.
Skills Required
Looking to make some money?
- Set your budget and the timeframe
- Outline your proposal
- Get paid for your work
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- Forbes
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- Time
- Wall Street Journal
- Times Online


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i also want to know the price of new fabia in pune
which is the best driving school in jodhpur.
Well, there are many driving schools available in Jodhpur. You may click here to check the one available in your locality. Please select the city and your area from the drop down list.
i want to learn driving on busy roads..
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Home / Colleges / Applications Articles
Applications Articles
Just Released: 2017-2018 Common Application Essay Prompts
Now is the time to start thinking about how you'll approach the essay prompts – it's never too early to start thinking about college admissions!
Top 15 Mistakes to Avoid in Choosing a College
Afraid of choosing the wrong college? Avoid these 15 common blunders students often make in considers their college options.
Can Colleges Take Back Their Offers?
It's easy to lack motivation senior year, but don't let it jeopardize your future!
Maintaining Friendships during the College Admissions Process
It's not a competition when a friend applies to the same school – it just means you have similar taste, which is likely why you're friends in the first place.
February College Prep List for High School Juniors
Use this list to keep your college process on track this month.
January College Checklist for High School Juniors
January is the start of a New Year and a new semester. Which means, you have the unique advantage of a fresh start in more than one way! How cool is that?
January College Checklist for High School Seniors
January is the start of a New Year and a new semester. Which means, you have the unique advantage of a fresh start in more than one way! How cool is that?
Winter Work for High School Juniors
As a high school junior, you've got a lot going on - and even more ahead.
November Checklist for High School Seniors
November is a pivotal time of year for high school seniors - and we're not just talking turkey.
November Checklist for High School Juniors
You need to focus more than ever before on your studies, which classes you choose to take and how much you devote to preparing for standardized testing and college research outside the classroom.
33 Top Colleges Without Application Fees
Why pay to apply? There are plenty of great colleges that have free applications!
October Checklist for High School Seniors
This year, take the time at the beginning of each month to evaluate what you can do to continue your focus on the college admissions process.
College Application Checklist
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On September 15, 2008, acting Surgeon General, Steven Galson, MD MPH, noted that venous thromboembolic events (VTE) contribute to the death of at least 100,000 Americans each year. Because many of these deaths occur suddenly, the best management is prevention. In this randomized, controlled trial, Washington University researchers will test strategies to improve the safety and effectiveness of VTE prevention by using a non-profit web application (www.WarfarinDosing.org) to tailor the dose of warfarin to each person's pharmacogenetic and/or clinical profile. They also propose to test the recommendation of the American Academy of Orthopedic Surgeons (AAOS) to use low levels of anticoagulation (i.e., a target International Normalized Ratio (INR) <2.0) for VTE prevention in orthopedic patients. Thus, the overall objective of the Genetics-InFormatics Trial (GIFT) of Warfarin to Prevent DVT is to elucidate novel strategies to improve the safety and effectiveness of warfarin therapy. Washington University researchers propose a randomized controlled trial of 1500 patients having total hip or knee replacement surgery at Barnes-Jewish Hospital. By studying this high- risk population and screening for asymptomatic deep venous thrombosis by Doppler ultrasound post-operatively, the proposed trial will be appropriately powered to elucidate two novel strategies to prevent VTE. The researchers will use a 2 x 2 factorial design to randomize patients to: (1) pharmacogenetic vs. clinical dosing of warfarin;and (2) to a target INR of 2.5 vs. <2.0. The proposal has two specific aims:
Aim 1 : To determine how pharmacogenetic-based warfarin therapy affects risk of non-fatal VTE, non-fatal major hemorrhage, and vascular death. Although the FDA has approved genetic testing for variants that affect warfarin dose, no prior (or planned) trial has been powered to determine whether this strategy affects outcomes.
Aim 2 : To determine whether warfarin therapy with a target INR of <2.0 is non-inferior to a target INR of 2.5 at preventing VTE in orthopedic patients. Although the American College of Chest Physician (ACCP) recommends a target INR of 2.5, the AAOS recommends <2.0 and these conflicting goals have never been compared in a trial of primary VTE prevention.
On September 15, 2008, Acting Surgeon General Steven Galson, MD, MPH noted that blood clots (deep venous thrombosis and pulmonary embolism) contribute to the death of at least 100,000 Americans each year. Because many of these deaths occur suddenly where treatment is impossible, the best treatment is prevention. In this grant, Washington University researchers develop strategies to improve the safety and effectiveness of clot prevention by customizing blood thinners to each person's genetic and clinical profile.
|Wang, Tzu-Fei; Milligan, Paul E; Wong, Catherine A et al. (2014) Efficacy and safety of high-dose thromboprophylaxis in morbidly obese inpatients. Thromb Haemost 111:88-93
|Haas, David W; Kuritzkes, Daniel R; Ritchie, Marylyn D et al. (2011) Pharmacogenomics of HIV therapy: summary of a workshop sponsored by the National Institute of Allergy and Infectious Diseases. HIV Clin Trials 12:277-85
|Finkelman, Brian S; Gage, Brian F; Johnson, Julie A et al. (2011) Genetic warfarin dosing: tables versus algorithms. J Am Coll Cardiol 57:612-8
|Gage, Brian F (2011) Cost of dabigatran for atrial fibrillation. BMJ 343:d6980
|Johnson, J A; Gong, L; Whirl-Carrillo, M et al. (2011) Clinical Pharmacogenetics Implementation Consortium Guidelines for CYP2C9 and VKORC1 genotypes and warfarin dosing. Clin Pharmacol Ther 90:625-9
|Lenzini, P; Wadelius, M; Kimmel, S et al. (2010) Integration of genetic, clinical, and INR data to refine warfarin dosing. Clin Pharmacol Ther 87:572-8
|King, Cristi R; Deych, Elena; Milligan, Paul et al. (2010) Gamma-glutamyl carboxylase and its influence on warfarin dose. Thromb Haemost 104:750-4
|Ferder, N S; Eby, C S; Deych, E et al. (2010) Ability of VKORC1 and CYP2C9 to predict therapeutic warfarin dose during the initial weeks of therapy. J Thromb Haemost 8:95-100


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Previous clinical studies have documented a close relationship between cerebrovascular disease and risk of Alzheimer's disease. We examined possible mechanistic interactions through use of experimental stroke models in a transgenic mouse model of β-amyloid deposition (APPswe/PS1dE9). Following middle cerebral artery occlusion, we observed a rapid increase in amyloid plaque burden in the region surrounding the infarction. In human tissue samples, however, we were unable to detect a localized increase in amyloid burden adjacent to cerebral infarcts. To resolve this discrepancy, we generated cerebral microstrokes in amyloid precursor protein mouse models with the photosensitive dye Rose bengal, and monitored plaque formation in real time using multiphoton microscopy. We observed a striking increase in the number of new plaques and amyloid angiopathy in the area immediately surrounding the infarcted area; however, the effect was transient, potentially resolving the discord between mouse and human tissue. We did not detect changes in candidate proteins related to β-amyloid generation or degradation such as β-amyloid-converting enzyme, amyloid precursor protein, presenilin 1, neprylisin or insulin-degrading enzyme. Together, these results demonstrate that strokes can trigger accelerated amyloid deposition, most likely through interference with amyloid clearance pathways. Additionally, this study indicates that focal ischaemia provides an experimental paradigm in which to study the mechanisms of plaque seeding and growth.
Virtually all risk factors for vascular disease are also risks for dementia: midlife hypertension (Launer et al., 1995; de Leeuw et al., 2002; Reitz et al., 2007), midlife cholesterol (Kivipelto et al., 2002; Pappolla et al., 2003), diabetes (Luchsinger et al., 2007), hyperinsulinaemia (Luchsinger et al., 2004), plasma homocysteine (Seshadri et al., 2002), tobacco use (Ott et al., 1998), metabolic syndrome (Razay et al., 2007), and an overall composite measure of arteriosclerosis (Hofman et al., 1997; Honig et al., 2005) have each been implicated as risks for cognitive impairment. Epidemiological studies have shown that vascular factors predisposing to cerebrovascular disease or stroke are associated specifically with Alzheimer disease and that cerebrovascular disease increases the presence and severity of the clinical symptoms of Alzheimer's disease (Breteler, 1994, 2000; Ellis et al., 1996; Kalaria, 2000; Launer et al., 2008; Helzner et al., 2009).
At a minimum, these associations between vascular risk factors and dementia reflect the additive effects on cognitive function of vascular brain injury and amyloid-related brain injury, a major pathological hallmark of Alzheimer's disease (Hyman, 1997). Beyond that, however, it remains possible that cerebrovascular disease interacts with the β-amyloid cascade at a mechanistic level. Such a relationship might occur if cerebral ischaemia potentiated one of the steps leading to β-amyloid deposition, such as by accelerating β-amyloid production or impairing β-amyloid clearance (Stone, 2008; Hiltunen et al., 2009). It has been suggested that one method by which brain levels of β-amyloid are reduced (clearance along perivascular drainage pathways) may be impaired by thromboembolic occlusion of the artery feeding a particular brain territory (Yow and Weller, 2002; Weller et al., 2008).
We used transgenic mouse models, including APPswe/PS1dE9 and tg2576, to test the effects of cerebrovascular lesions on β-amyloid deposition in brain parenchyma in the form of senile plaques and in small superficial vessels as cerebral amyloid angiopathy. Our hypothesis was that such lesions would interfere with local clearance of β-amyloid and thereby promote deposition in the brain territory perfused by the lesioned blood vessels. We also examined human tissue samples from patients diagnosed with both Alzheimer's disease and stroke.
Materials and methods
APPswe/PS1dE9 mice (Jankowsky et al., 2001) aged 6–7 months old were obtained from Jackson Laboratory, and Tg2576 mice (Hsiao et al., 1996) aged 11–13 months were used. All studies were conducted with approval of the Massachusetts General Hospital Animal Care and Use Committee and in compliance with National Institutes of Health guidelines for the use of experimental animals.
Human tissue
All studies were performed using autopsy brain tissue collected through the Neuropathology Core of the Massachusetts Alzheimer Disease Research Centre with approval from the Partners Human Research Committee. All subjects had both a clinical and neuropathological diagnosis of Alzheimer's disease according to the accepted criteria. Paraffin embedded sections of inferior parietal lobule were examined, with four subjects having no evidence of ischaemia in this region and three with an area of remote ischaemic infarction involving a portion of the sampled cortex.
Texas Red® dextran of 70 000 D molecular weight was obtained from Molecular Probes; methoxy-XO4 was a gift from Dr Klunk (University of Pittsburgh); Ketamine HCl and xylazine were obtained from Phoenix Pharmaceuticals, anti-β-amyloid antibody (10D5) (developed in mouse) was a gift from Elan Pharmaceuticals; anti-β-amyloid-converting enzyme (BACE) antibody (developed in rabbit) was obtained from ProSci Incorporated; anti-amyloid precursor protein (APP) antibody (developed in rabbit) was obtained from Calbiochem; anti-insulin degrading enzyme (developed in goat) was obtained from Santa Cruz; and anti-neprilysin (developed in rat) was obtained from R&D Systems. Secondary Cy-3-conjugated antibodies were obtained from Jackson Immunoresearch Laboratories Inc.; Rose bengal, anti-presenilin 1 antibody (developed in goat), thioflavin S, Mayer's haematoxylin, eosin and common chemical reagents were obtained from Sigma.
Yellow fluorescent protein expression in layer 1 dendrites of the neocortex
Yellow fluorescent protein was expressed in a subset of mice using genetic transfer via an adeno-associated virus (serotype 2). The yellow fluorescent protein was part of a larger cassette that was prepared and injected as previously described (Kuchibhotla et al., 2008). We used the construct pAAV-CBA-YC3.6-WPRE. The plasmid contained the AAV inverted terminal repeats, the only remaining feature of the wild-type AAV genome. Flanked by the repeats, the expression cassette included the following components in 5′–3′ order: (i) a 1.7 kb sequence containing hybrid cytomegalovirus (CMV) immediate-early enhancer/chicken β-actin promoter/exon1/intron; (ii) yellow cameleon 3.60 complementary DNA; and (iii) woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). The virus titre was 1.1 × 1012 viral genomes/ml
For intracortical injections of YC3.6-AAV2, 6–7 month old APPswe/PS1dE9 mice were anaesthetized with isoflurane and placed in a custom made, heated stereotaxic apparatus. A 2–3 mm incision was made in the scalp along the midline between the ears. Burr holes were drilled in the skull, 1 mm lateral to bregma bilaterally. Using a Hamilton syringe, 4 μl of virus (titre 4.2 × 1012 viral genomes/ml) was injected 1.2-mm deep in somatosensory cortex (targeting layer 5 neurons) at a rate of 0.2 μl/min. After one injection in each burr hole, the scalp was sutured and the mouse was allowed to recover from anaesthesia on a heating pad. After a > 4 week incubation period to allow expression of YC3.6 in neurons, cranial windows were implanted.
Cranial window implantation and microstroke production
APPswe/PS1dE9 mice with and without YC-AAV2 injections, and Tg2576 mice were anaesthetized and cranial windows were implanted as previously described (McLellan et al., 2003; Garcia-Alloza et al., 2010). To produce targeted strokes using photothrombosis, Rose bengal was administered by tail vein injection (100 μl, 25 mg/kg in sterile phosphate-buffered saline) as previously described (Zhang and Murphy, 2007) with minor modifications. An Olympus Fluoview 1000MPE with pre-chirp optics and a fast acousto-optical modulator mounted on an Olympus BX61WI upright microscope and Olympus ×20 dipping objective (numerical aperture 0.95) were used to provoke the photothrombotic microstrokes and determine the velocity of red blood cells. Rose bengal was activated using 543 nm laser (100%) directed to the selected arterial segment (∼20 × 20 μm) from 2 to 3 vessels, 20–50 μm in diameter, located under the right hemisphere of the cranial window. Red blood cell velocity was assessed using repeated line scans of the same area at 860 nm (×6 zoom) through the horizontal part of the selected region of interest as previously described (Zhang and Murphy, 2007). Twenty-four and 48 h after the blockage animals were anaesthetized and the blood flow was checked again in the same spots using Texas Red dextran (70 000 D), intravenous red blood cell velocity was measured as previously described (Zhang and Murphy, 2007) using ImageJ software. Vessel diameter was measured in ImageJ at three points along each vessel segment and the average diameter was taken from these three measurements.
In vivo imaging
β-Amyloid was visualized with methoxy-XO4, a Congo red derivative that binds fibrillar β-amyloid, that was injected intraperitoneally the day before each imaging session. Angiograms were performed with 12.5 mg/ml intravenous injection of Texas Red® dextran preceding each imaging session. As previously described, two-photon fluorescence was generated with 750 nm excitation and fluorescent emission was collected in the range of 380–480 nm for methoxy-XO4, and 560–650 nm for Texas Red® dextran or Rose bengal. A ×20 (0.95 numerical aperture, Olympus) water immersion objective (615 × 615 μm, z/step 5 μm, depth ∼200 μm) was used on a Fluoview 1000MPE microscope (Olympus). Maximum intensity projections of z-series were generated using ImageJ software. Stacks were used to measure plaque size and number (Garcia-Alloza et al., 2010). Senile plaque size was measured by thresholding, segmenting and measuring using the blue fluorescence channel and ImageJ software. Cerebral amyloid angiopathy burden was also analysed in maximum intensity projections as previously described (Robbins et al., 2006; Garcia-Alloza et al., 2009a). Autofluorescent signal from the dura was removed by deleting the uppermost slices of the z-series. Vessels were outlined and the cerebral amyloid angiopathy deposits, appearing as bright areas circumferential to the vessels, were manually thresholded so that cerebral amyloid angiopathy-positive regions were set white and cerebral amyloid angiopathy-negative regions were set black. The number of pixels in white clusters composed of >6 contiguous pixels was counted. The cerebral amyloid angiopathy burden was calculated as the percentage of the vessel area affected by cerebral amyloid angiopathy. The vessel area from the initial imaging session was used for calculation of cerebral amyloid angiopathy burden because of the tendency for vessels to dilate in subsequent weeks. New growth of cerebral amyloid angiopathy deposits was measured from week to week. Vessel diameter was measured in ImageJ at three points along each vessel segment and the average diameter was taken from these three measurements. In a subset of experiments, yellow fluorescent protein-filled dendrites were imaged alongside plaque deposition to confirm the impact of photothrombotic lesions on nearby neuronal structure.
Focal cerebral ischaemia
Middle cerebral artery occlusions were performed in APPswe/PS1dE9 and age-matched non-transgenic littermates as previously described (Shin et al., 2008) with minor modifications. Animals were anaesthetized with isoflurane, placed in a stereotaxic frame (David Kopf) and allowed to stabilize for 30 min after preparation. The temporalis muscle was separated from the temporal bone and removed. A burr hole (2-mm diameter) was drilled under saline cooling in the temporal bone overlying the middle cerebral artery just above the zygomatic arch. The dura was kept intact and distal middle cerebral artery was occluded for 1 h using a microvascular clip. Mice were allowed to recover, and were sacrificed 7 days later. Mice were perfused with 4% paraformaldehyde. Whole brains were paraffin embedded and cut in 16 μm coronal sections.
Haematoxylin–eosin staining
Standard haematoxylin–eosin staining was performed in 1 in every 10 sections (16 μm) from APPswe/PS1dE9 mice in order to identify the stroke areas after middle cerebral artery occlusion in APPswe/PS1dE mice as well as in human tissue (6 μm) to identify stroke areas in tissue from patients with Alzheimer's disease. Tissue was deparaffinized and incubated for 8 min in Mayer's haematoxylin solution and washed in warm water for 10 min. Sections were dipped in 95% ethanol and counterstained with eosin for 30 s.
Thioflavin S staining and plaque counting
Senile plaque size was measured in post-mortem tissue in the stroke areas from APPswe/PS1dE9 mice after middle cerebral artery occlusion. Stroke areas were delimited by haematoxylin–eosin staining and the selected area in the ipsilateral hemisphere was used as a phantom area in the contralateral hemisphere for comparison. One in every 10 sections from stroke areas was stained with thioflavin S (0.1%) for 20 mins and carefully rinsed in water before mounting an aqueous coverslip. Micrographs of stained tissue were obtained on an upright Olympus BX51 fluorescence microscope with a DP70 camera using DPController and CPManager software (Olympus). Senile plaque size was measured by thresholding, segmenting and measuring using the blue fluorescence channel and ImageJ software. Similarly, in human tissue from patients with Alzheimer's disease, stroke areas were delimited by haematoxylin–eosin staining. Thioflavin S staining was used to plot plaque number versus distance to the stroke area, in order to assess a possible gradient effect of the strokes on senile plaque deposition up to 2000 μm from the stroke area boundaries. Selected areas in tissue from patients with Alzheimer's disease with strokes were used as phantoms in non-stroke Alzheimer's disease tissue as control, and the number of plaques as a function of distance was also plotted.
Immunohistochemistry with anti-β-amyloid, anti-BACE, anti-presenilin 1, anti-neprilysin and anti-insulin degrading enzyme antibodies was performed in tissue from APPswe/PS1dE9 mice after middle cerebral artery occlusion. Briefly, tissue was deparaffinized and sections were washed in Tris-buffered saline for 30 mins, blocked with normal goat serum (5%) for 1 h and incubated overnight at 4°C with primary antibodies [1:50 for anti-Aβ (10D5) and anti-BACE antibodies, and 1:100 for anti-presenilin 1, anti-APP and anti-insulin degrading enzyme antibodies and 1:20 for anti-neprilysin]. After washing in Tris-buffered saline, sections were incubated in conjugated Cy-3 secondary antibodies (1:100–200) for 1 h, rinsed and aqueously coverslipped.
Senile plaque deposition is increased after middle cerebral artery occlusion
In order to evaluate the relationship between focal ischaemia and amyloid deposition characteristic of Alzheimer's disease, we performed transient unilateral middle cerebral artery occlusion in APPswe:PS1dE9 mice at an age where amyloid plaque deposition was detectable. The animals survived the insults, and were sacrificed 1 week later. The brains of the animals were probed for amyloid deposits with histo- and immunohistological approaches in coronal sections of post-mortem tissue. Adjacent sections were processed with haematoxylin and eosin following standard protocols to allow delineation of the stroke volume within the brain. Although variable, stroke volumes showed a tendency to be enlarged in transgenic mice when compared with wild-type animals (∼9.80 versus ∼4.56 mm3, Student t-test for independent samples, P = 0.086) as has been previously described in other APP mouse models (Zhang et al., 1997; Koistinaho et al., 2002). Fig. 1 shows an example of haematoxylin–eosin staining, and thioflavin S staining in the middle cerebral artery occlusion territory of the lesioned hemisphere, and the contralateral territory of the same mouse, which was used as a control for the variability in plaque density between APP mice of the same age. There were significantly more senile plaques detected in the ipsilateral versus contralateral hemisphere and media (minimum–maximum) were as follows [83 (28–156) versus 75 (74–123)/mm2, **P < 0.009 Wilcoxon test for paired samples, four mice, 4–5 sections per mice]. The average size of the plaques was not significantly different between hemispheres (223 ± 10 versus 199 ± 9 μm2, P = 0.08, n = 961–1233 plaques from four mice, 4–5 sections per mice). To test whether the increased plaque deposition was restricted to dense-core versus diffuse amyloid aggregates, we used 10D5, an N-terminal specific anti-β-amyloid antibody for immunostaining in adjacent sections from the same mice. We found complete co-localization of immunostaining with thioflavin S staining suggesting that the rapid appearance of dense core plaques was not associated with diffuse amyloid plaques (data not shown).
Effect of stroke on senile plaque deposition in patients with Alzheimer's disease
The ability to detect increased amyloid burden within the ischaemic territory of experimental stroke in the animal model motivated us to examine human tissue in a complementary fashion. We obtained samples of post-mortem human tissue with a diagnosis of both Alzheimer's disease and moderate stroke. Using haematoxylin–eosin staining to delineate the stroke volumes, we probed for amyloid deposition with thioflavin S, and compared the amyloid burden in the areas of the brain immediately adjacent to the lesion. The regular distribution of the plaques was not affected by strokes up to 2000 μm from the stroke volumes as shown in Fig. 2. The data also indicate that no differences in plaque size were observed in the surround of the stroke areas when compared with non-stroke patients (235 ± 7 versus 251 ± 5 μm2, n = 627–1444 from 3 to 4 patients).
The lack of a detectable difference in β-amyloid burden surrounding stroke volumes in human tissue is consistent with literature reports, but surprising in the context of our experiments eliciting strokes in experimental animals. We postulated that the discrepancy could be related to the duration of a stroke-mediated effect on amyloid deposition, prompting us to examine the kinetics of the interaction between ischaemia and β-amyloid deposition.
Photothrombosis-mediated ischaemia increases β-amyloid deposition locally
To examine the timing of β-amyloid deposition following ischaemic stroke, we made longitudinal measurements of amyloid burden using multiphoton microscopy and systemic labelling of amyloid deposits with methoxy-XO4 as we have done previously (Garcia-Alloza et al., 2009a,b, 2010). In tandem, we generated ischaemic lesions by eliciting photothrombosis using Rose bengal as a photosensitizing agent within individual vessels as previously described (Zhang and Murphy, 2007). This approach allows targeting a single vessel to study thrombosis and β-amyloid deposition in a time-dependent manner. The efficacy of induction of ischaemia was examined by measuring the instantaneous velocity of red blood cells in affected and nearby vessels. This allowed us to demonstrate that the photothrombosis method resulted in complete cessation of blood flow in the targeted vessel that lasted 1–2 days, and substantial alterations in blood flow in the immediate surround of the occlusion (data not shown). Severe morphological changes to neurites in the affected areas were detected with yellow fluorescent protein-expressing neurons, as described previously (Zhang and Murphy, 2007) and shown in Fig. 3. This allowed us to define the affected volume from the point of photothrombosis as having a radius around the occluded vessel of ∼250 µm (a 'microstroke'). Thus, this single vessel photothrombosis approach allowed focal ischaemic lesions in targeted areas that were readily imaged both acutely and longitudinally with multiphoton microscopy.
We imaged amyloid deposits at weekly intervals in the area of the brain with occlusions, and in adjacent areas within the same mouse where blood flow was unperturbed. We detected the appearance of many new plaques within the volume of the microstroke after 7 days [expressed as media (minimum–maximum), stroke regions 13 (0–79) versus non-stroke regions 0 (0–52)/mm3 *P = 0.040 Mann–Whitney test for independent samples, 24–53 fields from 11 to 15 mice; Fig. 4]. At 14 days, there was no difference in the number of new plaques in the stroke volume versus the adjacent area compared with the previous week [stroke regions 7 (0–13) versus non-stroke regions 0 (0–39)/mm2P = 0.327 Mann–Whitney test for independent samples, 14–42 fields from 11 to 15 mice]. No differences were observed in non-stroke regions between Days 7 and 14. (Wilcoxon for paired samples P = 0.877). Importantly, the appearance of new plaques was also observed in another transgenic mouse model (Tg2576) demonstrating the broader significance of the observation. Seven days after the microstroke, we measured 26 (0–39)/mm3 new plaques in stroke regions versus 0 (0–26)/mm3 new plaques in non-stroke regions, P = 0.048 using Mann–Whitney test for independent samples, 6–7 fields from five mice (Fig. 4). These results demonstrate that plaque formation was significantly accelerated in ischaemic areas, but only transiently; a result that could explain why the middle cerebral artery occlusion study in mice led to an increase in the number of plaques 7 days later, whereas the human tissue analysis (where the timing of the stroke was unknown) did not show a difference. The increase in the amyloid burden was the result of the generation of new plaques, and not the growth of existing plaques in concordance with previous observations (Garcia-Alloza et al., 2006; Meyer-Luehmann et al., 2008).
The vessels targeted in these mice had small amounts of vascular amyloid deposition representing cerebral amyloid angiopathy (Masuda et al., 1988; Ellis et al., 1996). The presence of cerebral amyloid angiopathy insured that we were targeting arterioles (which are affected by cerebral amyloid angiopathy) rather than veins, and also allowed us to evaluate whether the rate of cerebral amyloid angiopathy progression (Robbins et al., 2006; Garcia-Alloza et al., 2009a) was affected by restricting blood flow. We found (Fig. 5) that cerebral amyloid angiopathy progression was accelerated nearly 2-fold in the occluded vessels (progression rate = 0.54% per day) compared with cerebral amyloid angiopathy affected vessels that were not targeted within the same mouse (progression rate 0.28% per day). While the trends suggest that the increased rate of progression of cerebral amyloid angiopathy is also transient (first week rate faster than the second week rate), our statistical model for cerebral amyloid angiopathy accumulation does not allow for comparison of slopes using only two time points so our analysis is limited to the overall rate of progression over both weeks.
β-amyloid production and degradation
We examined possible mechanisms for the rapid appearance of new plaques and cerebral amyloid angiopathy in the stroke volume. We postulated that the rapid appearance of new amyloid deposits could result from either an increase in β-amyloid production or a decrease in β-amyloid clearance. We used immunohistochemistry in brain sections from the mice with middle cerebral artery occlusion to probe for alterations in proteins in the stroke versus non-stroke area of the brain. We determined that the lesions did not alter the distribution or amount of presenilin 1 or BACE, which would reflect an increase in β-amyloid production. We were also unable to detect differences in the levels of neprilysin or insulin degrading enzyme, which would demonstrate a change in capacity for β-amyloid degradation. There were also no detectable differences in the levels of APP, arguing that an effect on the level of transgene expression was not responsible for the generation of new plaques (Fig. 6). The failure to find effects on factors related to either production or breakdown of β-amyloid suggests that the normal clearance pathways for β-amyloid along interstitial fluid drainage routes might be responsible for the increased β-amyloid deposition.
In this report, we have demonstrated that ischaemic lesions in the brain led to rapid deposition of β-amyloid in two models of stroke and two transgenic mouse lines. Whereas previous studies showed correlations between cerebrovascular disease and β-amyloid deposition (Yow and Weller, 2002; Weller et al., 2008), our data suggest a direct causal link. Our transient middle cerebral artery occlusion studies are in accordance with previous studies where increased stroke volumes were observed in transgenic mice with Alzheimer's disease, when compared with wild-type mice (Zhang et al., 1997; Koistinaho et al., 2002), supporting the possibility of increased susceptibility to ischaemia as a result of neurotoxic effects of β-amyloid in the post-ischaemic brain (Zhang et al., 1997). Our middle cerebral artery occlusion studies showed a significant increase in senile plaque deposition as soon as 1 week after the procedure. We were not able to image the middle cerebral artery territory longitudinally in these mice, so we used an alternative model of stroke to follow the kinetics of this effect. We used intravital multiphoton microscopy and Rose bengal microstrokes to monitor the kinetics of the effect in real-time. We observed that the increased rate of amyloid deposition was transient. Newly deposited amyloid remained indefinitely, but the increased rate of deposition returned to the baseline level within 1 week. These surprising results demonstrate that stoppage of blood flow in the brain leads to rapid and robust deposition of fibrillar amyloid.
The transience of the effect of ischaemia on accelerated β-amyloid deposition might explain the observed absence of association between infarcted brain tissue and plaque deposition in post-mortem human tissue. Post-mortem studies in humans have generally not reported a tight association between cerebrovascular lesions and regions of extensive plaque and cerebral amyloid angiopathy formation, suggesting that cerebrovascular injury is at most just one of many contributors to β-amyloid deposition. This aspect is further blurred by the fact that many confounding factors may contribute including type 2 diabetes (Ott et al., 1999; Schrijvers et al., 2010), ApoE haplotype (Chartier-Harlin et al., 1994; Bickeboller et al., 1997) or previous pathologies. We examined a small number of cases, which does not represent an exhaustive evaluation that takes all these factors into consideration. Likewise, in our studies of human tissue with clinical diagnosis of both Alzheimer's disease and stroke, the strokes occurred well before patient death, making it very difficult to determine the precise timing of the events, since microstrokes are largely asymptomatic. A transient increase in amyloid deposition in the lesion area would be masked over time as the patient aged and amyloid deposition continued throughout the brain. Thus, our results in animal models confirm the majority of the clinical reports of the risk of Alzheimer's disease following stroke (Leys et al., 2005; Rastas et al., 2007; Helzner et al., 2009), and may explain the minority of reports where a risk of Alzheimer's disease was described if the interval between cerebral ischaemia and death was short. Even a transient increase in plaque deposition may nonetheless be relevant to an individual's risk of dementia and might thus contribute to the observed associations between vascular risk factors and dementia in humans (Elias et al., 1993; Breteler, 1994, 2000; Launer et al., 1995; Kalaria, 2000; Roher et al., 2003; Luchsinger and Mayeux, 2004; Honig et al., 2005). Vascular disease may also contribute to the inaccuracies in diagnosis of Alzheimer's disease, as an additional brain insult may exacerbate the cause or causes of the underlying dementia. In clinical trials, for instance, cerebrovascular disease is now commonly used as an exclusion criterion (Dodge et al., 2011; McKhann et al., 2011).
While the mechanism of rapid amyloid deposition is unknown, it appears not to be related to changes in protein levels related to production (APP, presenilin 1 and BACE) or degradation (neprilysin and insulin degrading enzyme) of β-amyloid. Although we cannot exclude that alternative methods could detect differences in BACE, as previously shown in ischaemic Sprague-Dawley rats (Tesco et al., 2007), the use of standard immunohistochemistry approaches to detect differences between the ischaemic and non-ischaemic areas in animals has been demonstrated to be effective (Wen et al., 2004). These studies showed an increase in BACE levels after 24–48 h after middle cerebral artery occlusion in rats. We found no differences in BACE levels after 1 week, suggesting that if there is a modest increase in BACE levels acutely, that it resolves within a few days, as predicted in a model where an increase in the rate of β-amyloid deposition following ischaemia is transient. In certain hypoxia models, it has been shown that hypoxia facilitates APP processing by an upregulation of BACE1 gene transcription (Sun et al., 2006) and that it may also facilitate β- and γ-cleavage (Li et al., 2009). On the other hand, degradation enzymes have also been reported to mediate Alzheimer's disease progression. Insulin degrading enzyme has been shown to increase in a rat middle cerebral artery occlusion model (Hiltunen et al., 2009) and reductions in neprilysin may increase β-amyloid formation (Iwata et al., 2001). Moreover, it seems that neprilysin activity is independent of other β-amyloid degrading enzymes, such as endothelin-converting enzymes (Eckman et al., 2006). Our data related to neprilysin levels are in accordance to the previous studies in a middle cerebral artery occlusion rat model (Hiltunen et al., 2009), where no changes were observed in neprilysin levels up to 30 days after the procedure. A limitation of our study is that we examined protein levels and not activity of these enzymes. While generally correlated, it is possible that changes in activity can occur independently of total protein levels.
Likewise, as ischaemia is always accompanied by neuroinflammation, it is possible that a direct or indirect consequence of an immune response plays a role in amyloid deposition. However, in the absence of observable changes in the mediators of β-amyloid production or degradation, it is likely that locally impaired clearance results in the accumulation of amyloid deposits, although no specific experiments were carried out to test this explicitly. Our data are consistent with the model of reduced amyloid clearance along interstitial fluid drainage pathways (Yow and Weller, 2002; Weller et al., 2008). According to this model, β-amyloid clearance depends on counter current drainage along arterioles. Pulsatile intravascular blood flow drives clearance in the perivascular space; when this is disrupted by vascular occlusion, there is a transient loss in clearance of soluble parenchymal materials. The reduced clearance leads to a high local concentration of β-amyloid, and this may facilitate aggregation. While this hypothesis is difficult to test in vivo, it remains the most likely candidate for the transient increase in β-amyloid deposition that is restricted to the volume affected by reduced blood flow. However, we cannot completely rule out the contribution of alternative mechanisms. As a note of caution, these experiments were performed in mouse models that overexpress mutant human APP, and it is possible that these models do not accurately reflect the human condition.
Although other studies have suggested a link between ischaemia and amyloid production (van Groen et al., 2005; Makinen et al., 2008), the current work is the first to demonstrate the deposition of β-amyloid as plaques and cerebral amyloid angiopathy in living animals. We used real-time imaging in living animals to show increased plaque deposition in areas local to the occluded arteriole, as well as an accelerated rate of progression of cerebral amyloid angiopathy in vessels that were already partially affected. An important additional implication of these results is the demonstration of an experimental approach to initiate plaque and cerebral amyloid angiopathy deposition in vivo in a precisely defined volume of the brain. This technique should allow discrimination of the molecular basis of seeding amyloid deposition, since plaques and cerebral amyloid angiopathy can be induced to form within a small spatial and temporal window.
Finally, identifying cerebrovascular lesions as even a partial contributor towards β-amyloid deposition would have potential implications for preventing or slowing the progression of Alzheimer's disease and cerebral amyloid angiopathy. Additionally, the increased susceptibility of the APP mouse brain to ischaemic insults suggests a feed-forward cycle where ischaemia and amyloid deposition exacerbate each other. Although many candidate approaches have been suggested for inhibiting the β-amyloid pathway (McLaurin et al., 2000; Bacskai et al., 2001; Townsend et al., 2006; Prada et al., 2007; Rajendran et al., 2008; Bateman et al., 2009; Elvang et al., 2009; Garcia-Alloza et al., 2009b) none has yet been shown to block progression of the human diseases. Vascular disease, conversely, has a large number of established treatments, and therefore further studies to assess this possibility might set the basis for future attempts to prevent dementia.
National Institutes of Health (EB00768 to B.J.B.); (AG021084, AG005134 to M.P.F.); (RYC-2008-02333; L'oreal-UNESCO 2010; PN I+D+I 2008–2011: ISCIII Subdirección General de Evaluación y Fomento de la Investigación PS09/00969 to M.G.-A.).
amyloid precursor protein
β-amyloid-converting enzyme


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The Office of the Comptroller of the Currency, the Board of Governors of the Federal Reserve System, the Farm Credit Administration, the National Credit Union Administration, and the Federal Deposit Insurance Corporation (collectively, the Agencies), recently issued an announcement regarding the implementation of new flood insurance laws under the new federal flood insurance regulations. Specifically, the statement sets forth the Agencies' expectations for supervised institutions regarding the availability of increased flood insurance coverage under the National Flood Insurance Program (NFIP) for "Other Residential Buildings."
The announcement provides guidance to financial institutions on the following issues:
While the Agencies did not mandate full portfolio reviews, they noted existing guidance in the Interagency Questions and Answers Regarding Flood Insurance may be necessary for safety and soundness purposes. It appears that financial institutions should give serious consideration to portfolio reviews.
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When strawweight Felice Herrig stepped into the Octagon at Chicago's United Center in July to the poppy chorus of Rachel Platten's "Fight Song," she did so as an MMA veteran with everything to prove. read news >>
We found out about the rematch between Muhammed Lawal and Quinton Jackson at Bellator 175 earlier this month. Now we know a venue. Filed under: Bellator, News read news >>
Stream or download Friday's edition of MMAjunkie Radio with guests Renzo Gracie, Sammy the Squid and Hal from Chicago. Filed under: News, Radio, UFC, WSOF read news >>
MMAjunkie Radio kicks off at 1 p.m. ET (10 a.m. PT) with guests Renzo Gracie, Sammy the Squid and Hal from Chicago.Filed under: News, WSOF read news >>
If Curtis Blaydes is on top of you landing elbows, chances are you're in big trouble. Filed under: News, UFC, Videos read news >>
Fast rising lightweight Will Brooks journeys to Chicago to revisit his harrowing past where he battled his family's struggles and homelessness. read news >>
UFC on Fox 20 hit Chicago, and the main event featured another bantamweight shake-up. Valentina Shevchenko up-ended Holly Holm by unanimous decision. Ever since Holm's stunning KO of Ronda Rousey, th read news >>
Pressure can crush you, or it can make diamonds. The MMA world found out what kind of carbon object Holly Holm was Saturday night at UFC on Fox 20. I'm sorry for the sports cliche, but it's a perfect read news >>
Bantamweights Eddie Wineland and Frankie Saenz headlined the UFC on Fox 20 preliminary card in Chicago on Saturday. Check out the fight highlights. read news >>
The Ultimate Fighting Championship returned to Chicago on Saturday for UFC on FOX 20: Holm vs. Shevchenko. read news >>
At UFC on Fox 20 in Chicago, No. 6 lightweight Edson Barboza (18-4) took a unanimous decision over former Strikeforce lightweight champion Gilbert Melendez (22-6). Barboza's vicious kicks to the legs read news >>
Watch the post-fight press conference for UFC on Fox: Holm vs. Shevchenko streaming live from Chicago shortly following tonight's main event. read news >>
The UFC returns to Chicago for UFC on Fox 20 on Saturday evening. Former UFC bantamweight champion Holly Holm headlines the card against Valentina Shevchenko. The 135-pound bout has big implications read news >>


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Jan 2016
Love Inspired Suspense,
My Casa series,
"The Defenders",
Book #5 (the final one)
"Back home in Serenity,
Jamie Lynn Henderson's determined to prove her
brother was framed for the town sheriff's
hit-and-run death years ago. But as she encounters
dead end after dead end, Jamie Lynn discovers
someone will go to any length - even murder - to
bury the evidence. Her only hope of staying alive
long enough to uncover the truth is Shane Colton,
the late lawman's wary son. Shane's world was
shattered by one senseless act that he can't
forgive, yet somehow he is drawn to protect the
lovely woman trying to free her brother from prison.
If they don't work fast, Jamie Lynn's single-minded
quest might lead them both into the real killer's
Read an Excerpt!
July 2016
Love Inspired Suspense
"Maggie Morgan was once
madly in love with Flint Crawford and assumed they
would be married as soon as she graduated. Instead,
partly due to a feud between their families, Flint
left town to join the military. Maggie was left to
pick up the pieces of her life, including giving
birth to the child Flint never knew about. By the
time he returns to Serenity as a game warden, his
son is five years old. Maggie has returned to her
Christian faith and is supporting herself by running
a rehabilitation program for wild animals. She knows
it's inevitable that father and son will eventually
meet but wants to delay as long as possible. When it
begins to look as if someone is trying to kill both
Maggie and Flint, they must work together to figure
out who and why and stop the attacks. Is the old
feud still alive? Is someone on the Crawford side
after Maggie? Could it be a person related to her
who is aiming to eliminate Flint or a poacher who
resents his job of enforcing the law? Either way,
their son could be caught in the cross-fire. They
are both willing to risk everything to protect the
September 2016
Love Inspired Suspense
"Rookie K-9" series
Book #6
"The small Arizona town of
Desert Valley has been the scene of murders dating
back five years, beginning with the shooting death
of the wife of current Police Chief, Ryder Hayes.
Ryder is raising his daughter, Lily, and continues
to search for the killer, who was finally identified
but then escaped. The town boasts a highly
successful K-9 training facility headed by Sophie
Williams. When someone begins to repeatedly target
Sophie, Ryder is worried that the same serial killer
may be involved. If so, they know who to search for.
If not, who in Sophie's turbulent law enforcement
past might still hold a grudge?" (note to readers -
I can't tell you more or I might spoil the earlier
books in this series. Sorry!)
(no excerpt at this time because it would give away
too much plot)
And, at Christmas time, 2016, watch for a follow-up
novella set in Desert Valley featuring authors
Valerie Hansen and Lenora Worth.
December 2016
Love Inspired Suspense
"Rookie K-9 Unit" series
Novella with Lenora Worth
Surviving Christmas
by Valerie Hansen...
"When single dad Sean Murphy returns from a war zone
and discovers someone is following him, he turns to
his old friend K-9 officer Zoe Trent for help.
But as the threat escalates, can Zoe and her police
dog find whoever is menacing Sean and his son...and
make sure this Christmas
isn't their last?"
Holiday High Alert by Lenora
"After a cryptic note appears on the playground at
Josie Callahan's daycare center, rookie K-9 officer
Dalton West vows to protect Josie and the kids she
loves - especially his daughter, Maisy. And with a
stalker closing in, the widower and his four-legged
partner are all that stands between Josie and a
deadly Christmas."
|| Home|| News
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Excerpts ||
Links ||


text: Dozens of cars have been reported off the road as a major winter storm brings heavy snow and gusty winds across Maine. The National Weather Service says Friday's storm will drop between 4 and 14 inches of snow, with sleet and rain mixing in some coastal areas. Maine State Police say dozens of cars have been reported sliding off the Maine Turnpike, Interstate 295 and Interstate 95. The most serious accident was in York, where a man was critically injured when his pickup truck overturned on the Maine Turnpike. Police say a state police cruiser was struck on the turnpike in Portland when another car slid into the rear of the cruiser while the trooper was out of the car investigating a crash. Nobody was injured. (AP)


text:
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Rates in USD (taxes & fees included), shown as:
Nightly Average
Cabina Arsol
Avenida 333, Calle 464, La Fortuna CR, Costa Rica
Find Hotel Rates
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Wego searches multiple travel sites so you can find and compare the best rates.
Cabina Arsol Hotel Reviews
Based on reviews from across the web.
Cabina Arsol Hotel Details
Check-in: 15:00
Check-out: 11:00
Cabina Arsol is a great choice for a stay in La Fortuna. For a bite to eat, guests can check out Flying Tomato Cafe, which serves Vegetarian cuisine and is open for breakfast, lunch, and dinner.
Avenida 333, Calle 464 La Fortuna CR, Costa Rica


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