text:
We are glad you are considering becoming part of the diverse community at Trinity Christian College! A Trinity education is a life changing experience that will challenge you academically, enrich you spiritually, and equip you for your future career and calling.
Business Department Receives Grant to Strengthen Vocational Formation
The business department has received a $50,000 2012 NetVUE Program Development Grant, which will be used to strengthen the focus on vocational calling and to develop a more integrated approach to the study and realization of vocational opportunities for students.
Trinity recognizes that, especially in today's challenging economic climate, students and parents are focused on the connection between a college education and the opportunities afforded to students to deepen their understanding of their vocational calling.
Students and parents are focused on the connection between a college education and the opportunities afforded to students to deepen their understanding of their vocational calling.
Faculty and staff from three campus departments—academic business department, Cooper Career Center, and Trinity Business Network—will work together to develop a model program for how to support students and serve alumni and business partners with regard to vocational formation.
Currently, these three distinct programs operate separately and focus on vocational calling within the field of business to varying degrees.
"By closely examining the purpose for the work we are currently engaged in and the theological underpinnings of living out our vocations, we plan to build a sustainable model for coordinating and developing these efforts," said Dr. Lynn White, professor of business.
She and her business department colleagues, Dr. Deborah Windes, Dr. Rick Hamilton, and Professor Kyle Harkema, will work over the next two summers to develop the model, which the group envisions could be helpful for other departments at Trinity.
Trinity is ensuring that veterans who are eligible for the Post 9/11 GI Bill can attend the College tuition-free, while...
Whenever computers act up, devices won't connect, or email passwords get forgotten, Trinity's IT Department is ready to...


text:
More Boards Seek Directors Who Aren't CEOs
Anne M. Mulcahy was a rarity when, as a Xerox Corp. vice president in 1997, she joined the board of Target Corp. At the time, few executives below the level of chief executive served on other public-company boards. "It was definitely out of vogue," she says.
Not anymore. Ms. Mulcahy is now Xerox's CEO, and two of her lieutenants serve on other boards. In addition, two non-CEOs have joined Xerox's board during her tenure, and...

subset: commoncrawl


text:
Question: In the previous exercise explain how the result of the
In the previous exercise, explain how the result of the 99% confidence interval in part b corresponds to the result of the decision using significance level 0.01 in part c.
View Solution:
View Solution:
Answer to relevant QuestionsA study of the effect of the gender of the tester on sex-role differentiation scores 16 in Manhattan gave a random sample of preschool children the Occupational Preference Test. Children were asked to give three choices of ...Refer to the previous exercise. For the control group, the 26 changes in weights have a mean of -0.45. From Example 8 in Chapter 9, for the therapy group the sample mean change in weight of 3.0 pounds was significantly ...A study followed patients for four years after they had a heart attack (K. Mukamal et al., Circulation, vol. 105, 2002, pp. 2476–2481). Patients were interviewed about their weekly caffeinated tea consumption during the ...In the 2008 GSS, 620 of 809 males and 835 of 978 females indicated a belief in life after death. a. Construct a 2 × 2 contingency table relating gender of respondent (SEX, categories male and female) as the rows to belief ...In an experiment on chlorophyll inheritance in corn, for 1103 seedlings of self-fertilized heterozygous green plants, 854 seedlings were green and 249 were yellow. Theory predicts that 75% of the seedlings would be green. a. ...
Post your question


text:
Brujo desnudo
IT wasn't the P350 sitting fee that I was paid upfront after an hour or so assuming, then, keeping a pose frozen for 15 minutes in puris naturalibus… what was more precious were one or two sketches in colored pencil, charcoal, or ink on art paper that a bevy of artists gifted me after the nude sketching sessions… the missus found them—maybe the angle of the dangle-- not to her liking, disposed of them, ephemeral immortality on paper.
Ah, those painters were profuse with appreciation at what they saw… veins running like root-rills on the arms, ripple of stringy flesh on torso and thighs akin to lizards about to pounce on prey… a lot of work went into such shaping of such lean and mean meat, say, a tattoo of 108 fist blows hurled by both arms… 88 kicks for each leg… 22 blows for each elbow… and odds and ends household chores throughout any day.
Get into that shape if you're a sucker for the Scriptures about the Hebrew term, musar— discipline, exclusive attainment, a genius-spirit ruling nature… probably what George Washington had in mind: "Discipline is the soul of an army. It makes small numbers formidable; procures success to the weak, and esteem to all."
So I did a stint as a nude model for artists back in the 1980s… years of hatha yoga and qigong practice made it easy for me to take on and freeze a pose for extended periods—no twitching, no movement… just easy breathing in sheer calm like an unperturbed mirror pond.
Uh, that was my body that turned up in Renato Habulan's oils on canvas depicting laborers, cane cutters, daily wage earners… the missus would recognize such a built anytime.
Nalasing sa Vino Kulafu na itinagay ng kahuntahan, nag-init ang katawan… naghubo't hubad, naglublob sa kalapit na bahagi ng dagat na nakatanaw sa mga pulong lalawigan ng Siquijor at Cebu, kalapit ng pangunahing lansangan ng Dumaguete sa gawing silangan nito… lampas na ang hating-gabi nang gawin 'yon pero nagliliyab ang mga hanay ng ilaw sa lansangan.
Sa pag-ahon, napansin na hinihimod ang lantad na katawan sa tingin ng mga banyaga sa tabing-aplaya… ni hindi naman nahimasmasan sa kalasingan, ni walang dalang tuwalya kaya ni hindi pinansin ang mga nakamulagat na mga mata… nagpantalon, pasuray ang lakad tungo sa tinutuluyang otel.
Eh ano kung sa pagkakataong 'yon, bigla akong naging—wow, Philippines-- clitourist attraction?
Bawal ang pugot at pantalon sa swimming pool ng isang otel sa Batangas na minsang tinuluyan, may dress code pa pala na dapat sundin sa paglublob… out-of-the-box thinking mode made me jump into the pool with nothing but a smile and a hard-on…
Why, if beauty is only skin-dip, skinny dipping is a sane option to turn beautiful, mwa-ha-ha-haw!
Monday, January 31, 2011
ब्रुजो देस्नुदो
Posted by DONG AMPIL DE LOS REYES at 5:01 PM
No comments:
Subscribe to: Post Comments (Atom)


text:
Denver Parks & Recreation offers six (6) venues for event rentals:
Available for reservations up to one (1) year in advance of desired rental date:
City Park Pavilion: currently available May 26 - October 15, 2017
Washington Park Boathouse: currently available April 15 - October 15, 2017
Applications ARE NOT accepted by email at any time and NO paper application is available to complete. All reservation requests are accepted online or in-person only and must be submitted at least 30 days in advance.
*Events booked less than 90 days in advance will pay 50% of total fees (non-refundable) within 24 hours of receiving reservation confirmation
Payments can be made online or in-person at the Parks Permit Office. Accepted methods of payment include Visa, MasterCard, Discover, cash, money orders, cashier checks or checks made payable to "Manager of Finance." Payment instructions will be sent via email with pending approval of reservation request.
Permit Requirements
The following requirements must be met no later than 21 calendar days prior to event:
1) All fees paid in full
2) Proof of insurance coverage.
3) Permit holder must complete a walk-through with an Event Facilitator. Event Facilitator contact information will be sent via email with pending reservation approval.
If all requirements are met by the deadline (21 calendar days prior to event date) approved permits will be sent via email no later than 14 business days prior to event date.
Other event resources
Schedule a Tour:
Free, guided one (1) hour tours are available for parties who wish to see the interior of a venue and ask additional questions before submitting a reservation application. Registered tour attendees are invited to bring one guest. View instructions on how to check availability and schedule a tour.
Schedule a Walk-through (for permit holders only):
A one (1) hour walk-through with a DPR Event Facilitator is required to obtain your approved Event Facilities Permit. The walk-through will cover day-of event logistics. Permit holders are strongly encouraged to bring any vendors, family or friends who will play a role in your event and need to see the venue prior to your reservation date. Permit holders are limited to one walkthrough. View instructions on how to schedule a walk-through.
Visit the DPR Online Service Center.
Tour and walk-through schedules are updated on the 20th of each month.
Walk-throughs do NOT need to be scheduled sooner than 60 days prior to your event date.
Tour and walk-through schedules are updated on the 20th of each month.
Walk-throughs do NOT need to be scheduled earlier than 60 days prior to your event date.
Rental Inquiries:
Event Venues
Parks Permit Office
201 W. Colfax Ave
Dept 1.G.1 (1st floor)
Denver, CO 80202
Office Hours
M-F, 8:00am - 4:00pm
Phone: 720.913.0700
Fax: 720.913-0778
For Recreation Center Rental Inquiries
Find a recreation center for information
Golf Rental Facilities
Email: email@example.com


text:
Question: What are the four components of target costing Why are
What are the four components of target costing? Why are each of these components important for target costing?
View Solution:
View Solution:
Answer to relevant QuestionsSailRight Boat Company builds and sells small sailboats. Identify at least four specific components of SailRight's value chain. For each component describe what activities might take place in that part of the value chain.Dainty Diners, Inc., produces various types of bird feeders. The following is a detailed description of the steps involved in the production of wooden bird feeders:1. Raw materials, such as wood, nails, and clear plastic are ...Dust Buster's Inc. manufactures two types of small hand-operated vacuum cleaners. Dust Busters is concerned about quality issues and has compiled the following information for the past year associated with the two ...Meiger Mining, Inc., has just discovered two new mining sites for iron ore. Geologists and engineers have come up with the estimates on the following page regarding costs and ore yields if the mines are opened:Meiger's ...Dana Martin, president of Mays Electronics, is concerned about the end-of-the-year marketing report. According to Mary O'Brien, marketing manager, a price decrease for the coming year is again needed to maintain the ...
Post your question


text:
Buffalo Horn Handled Cleaning Rod
Guns International #: 100392736 Seller's Inventory #: 234
Category: Cleaning Accessories -
Seller's Information
When emailing or calling sellers direct, please mention that you saw their listing on GunsInternational.com
Seller: Lewis Drake & Associates
Company: Lewis Drake & Associates
Member Since: 7/9/08
First Name: Lewis
Last Name: Drake
State: Kentucky
Zip: 42071
Country: United States
Phone: 270-753-7200
Fax: 270-753-2100
Seller: FFL Dealer
Return Policy: 3 day inspection and return policy on used guns.
Payment Types Accepted: AMEX, VISA, MC
* 3-piece polished rosewood have handles crafted from genuine buffalo horn OR bone & horn * Threads on jag end are 5/16"-27 T.P.I. which will fit our genuine brass jags or cleaning brushes perfectly or use with our cotton cloth rolls to make your own lint-free bore mops * Overall length (without jag)is 36 ½".
Price: $94.00


text:
WinRAR is a software who developed by RarLab which is would be very useful and easy to use archive manager that lets you read almost everything about archive files. WinRAR is the best and the most popular compression tool with many integrated additional functions to help you organize your compressed archives.
Main Features:
- WinRAR provides complete support for RAR and ZIP archives and is able to unpack CAB, ARJ, LZH, TAR, GZ, ACE, UUE, BZ2, JAR, ISO, 7Z, XZ, Z archives.
- WinRAR offers a graphic interactive interface utilizing mouse and menus as well as the command line interface.
- When you purchase WinRAR license you are buying a license to the complete technology, no need to purchase add-ons to create self-extracting files, it's all included.
- WinRAR is easier to use than many other archivers with the inclusion of a special "Wizard" mode which allows instant access to the basic archiving functions through a simple question and answer procedure. This avoids confusion in the early stages of use.
It can backup your data and reduce size of email attachments, decompress RAR, ZIP and other files downloaded from Internet and create new archives in RAR and ZIP file format.
Download Link:
Download Link
- WinRAR v5.40 Final ( 4Mb ) x86 Setup
- WinRAR v5.40 Final ( 4Mb ) x64 Setup
- Keygen Only ( 200Kb )
Older Version
- WinRAR v5.40 Final Full / Mirror Link ( 4Mb )
Install Instructions
- Download WinRAR 5.40 beta 4 below
Winrar32bit / Winrar64bit
- Install (Run/setup)
- Download Keygen
- Copy keygen to instalation directory
- Run Keygen
- Enjoy ..
If you like this software or you will use this software for work, or maybe you will use this software for a long time, Please support software developer by purchase or make donation their software from this site: http://www.rarlab.com/


text:
Click image to enlarge
The Beefeater Stainless Steel Side Shelf - No Burner is a well constructed and reliable shelf which is designed to provide additional storage capabilities when used in an outdoor kitchen setup. This stainless steel shelf doesn't contain a burner, but is constructed with a high strength stainless steel construction which is perfect for providing optimal resistance against heat and wear and tear applications. It is a part of the Discovery 1100s series of outdoor kitchens and is ideal for use where 4-5 burner barbecues will be in use.
This handy side shelf is part of the Discovery 1100s Outdoor Kitchen series with the option to add onto any 4 or 5 burner 1100s outdoor kitchen variations. Made from stainless steel, this one will be sure to compliment your ultimate outdoor kitchen!
Have a Question about this Product? Click Here
|Stainless Steel
|Type of Barbecue
|Outdoor Kitchen
|Limited 5 year warranty
Be The First To Review This Product!
Help other The BBQ Store Pty Ltd users shop smarter by writing reviews for products you have purchased.


text:
Assess My Needs
Choose an Edition
Watch Module Tutorials
Download a Demo
Let our team evaluate your needs and recommend a solution.
Listen to what other business owners are saying...
Watch interactive tutorials for EBMS modules.
Managing your online presence is important!
Over 25 years of serving you!
Quickly Reorder Business Forms or Checks
Eagle Business Software is useful in many different kinds of businesses and industries. Select an industry and view details on how EBMS Accounting Software is used in each industry.
"EBMS is a fully customizable inventory management/accounting software package that is affordable to the small to medium sized business."
- EBMS User
EBMS includes not only the tools listed but also a complete accounting system, profit center management tools, work order management, POS stations, and much more. The accounting modules integrate with the other modules to complete the business management toolbox of EBMS.


text:
(3 Reviews)
More Third Party Comments about this item:
Received this item fast and on time. It didn't disappoint me, both the image and material quality are pretty good! easy installation and now everything is working well! Pretty good camera! While, just my own view!
First I am very satisfied with shipment and the service. All accessories are in the box and in perfect condition! I was impressed by the price at first and did not think to get as much quality. The excellent video, very easy installation, I finished it by myself.
This product is great! I have a Spiroir that I installed it on the license plate. Works very well in pitch blackness. It allows you to know how many feet you are away from something behind your car. This way, reversing or parking my car becomes much easy. This item offers me clear pictures even at night or in rainy day. Excellent!
Product Navigation: Previous itemBack to ListNext item


text:
Things We Clicked Today: August 5, 2010
Awarding the Web's best articles, videos, and photos: Naomi Campbell's courtroom performance, Facebook's retro ads, Digg's big scandal, and Tommy Chong's political jujitsu
Best War Crimes Testimony
Naomi Campbell might have received diamonds from Liberian dictator Charles Taylor in 1997. Or not. "I get gifts given to me all the time, at any hour of the night... without notes of who they're from," she told a Hague tribunal. All she knows: they were "very small, dirty-looking stones." What a tragedy.
Best Genealogy
"1 in 200 men direct descendants of Genghis Khan" (Discover)
Best Mad Men Fact-Checking
"Domme on Don: A Dominatrix's Take on Don Draper's Prostitute" (Vulture)
Best Graphic Design
"Fake Vintage Ads For Facebook, YouTube & Skype" (Laughing Squid)
Worst Internet Scandal
"Massive Censorship Of Digg Uncovered" (Alternet)
Best Shape-Shifting Robot
(via Physorg.com)
Best Advertisement for Ukrainian Tourism
"Jessica-Jane Clement is too hot for words" (Guyism)
Best Choppers (Except for This One)
"39 Unusual Motorcycles" (Buzzfeed)
Best Animal Kingdom Showdown
"Alligators vs. Sharks -- Which Is More Deadly?" (Asylum)
Best Tommy Chong Showdown
(via TV Squad)
And Your Drink of the Day
* 2 ounces golden rum
* 2/3 ounce lemon juice
* 1/2 teaspoon superfine sugar
Shake well with cracked ice, then strain into a chilled cocktail glass (unless you happen to have a Sour glass) and give it a "lid" of 151-proof rum. No need to resist the impulse to decorate lavishly with fruit.


text:
Make Your Own Bird Treats
A birds diet that is fun and easy. Feeding your bird doesn't have to mean just seeds or nuts or commercial food. Birds love a good snack too, and many birds need treats to give them variety or additional nutrients in their diet. You can usually buy these treats from any pet store, but a … Continued


text:
Broaden Your Business Horizon At RMUC.17 In Tucson!
Spring is in full swing, and plans are well underway for the 2017 Rent Manager User Conference. We are so excited to welcome you…Read the full article »
Spring is in full swing, and plans are well underway for the 2017 Rent Manager User Conference. We are so excited to welcome you…Read the full article »
We've seen an encouraging new trend in the business world; a trend that's shifting away from the one-way communication model where corporations bark down…Read the full article »
Rent Manager® exists to make your job easier and your business more profitable. There's no shortage of ways the software can help your operation,…Read the full article »
This is a quick reminder that our next Virtual Seminar course, which focuses on Advanced Report Writer, takes place next Tuesday, August 26th at 2:00…Read the full article »
Your interest in the Virtual Classroom has been overwhelming! The Rent Manager 101 and Scripting 101 sessions have sold out every time we've…Read the full article »
Increasing Supplemental Income & Using Technology To Make It Easy Clear your calendars and get ready for a special Rent Manager webinar! At 2:00…Read the full article »
We have an abundance of opportunities for you to hone your Rent Manager know-how this month. Take a look at our upcoming training schedule…Read the full article »
Did you know we just launched a new training tool aimed at giving you even greater insight into numerous feature sets within Rent Manager?…Read the full article »
Want to learn how to create a list of renters whose leases are about to expire, as well as the different methods available to…Read the full article »
We just wanted to give you a heads-up about a scheduling change for the new year. Beginning January 9th, Rent Manager® webinars will move…Read the full article »
Helping you make the most of Rent Manager® is extremely important to us, because the more you know about the software, the more you…Read the full article »
Did you know that by using a simple Ask script, you can have Rent Manager® automatically enter a sender's name in a Letter Manager…Read the full article »
When you're looking for a way to automatically calculate a resident's security deposit refund, or any outstanding balance due, the following two-part script can…Read the full article »
One of the many great things about Rent Manager® is that it can be customized in a myriad of ways. You can tailor…Read the full article »
We probably don't have to tell you that Rent Manager® is an extremely customizable program. And you're undoubtedly aware of the many report options…Read the full article »
If you work for a fee-based management company, odds are that you realize just how important your owner statements are. Nothing can make your…Read the full article »
Reconciling your bank accounts is probably one of the most tedious, but important, tasks a bookkeeper has to do. It can often take…Read the full article »
Rent Manager® is seriously customizable software'so much so that all of you seem to use it differently. Our Implementation and Support teams marvel at…Read the full article »
When creating late payment letters or sending move-in/move-out notifications, it can be useful to specify a deadline that's a certain number of days after…Read the full article »
If your state requires you to pay interest on renters' security deposits, or if you keep security deposits in an interest-bearing account, there's a…Read the full article »


text:
It only takes a minute to get started. Fill out our uncomplicated sign up form and login to the site.
This step is voluntary but will dramatically raise your chances of getting interest from other over 40s members on the site. Let people know a bit about yourself, your pasttimes and attach any nice photographs of yourself.
Utilise our website's state-of-the-art search to locate over 40s members who match your desired requirements. Or just look through listings and locate members that arouse your interest.
Found someone who looks interesting? Send them a few words or just a wink and take it from there. With a little luck you'll be on your way to uncover your suitable match.
It's quick and simple


text:
"Thank you to the midwives and nurses"
About: Cumberland Infirmary Cumberland Infirmary Carlisle CA2 7HY
Posted by Anonymous
I have recently given birth to my first child and would like to say a big thank you to all the midwives and nurses that looked after me, my baby and my husband during our stay. They were all very friendly and put us at ease being first time parents. Our midwife was absolutely fantastic. Giving birth for the first time is a scary experience because you don't know what to expect but they really do their best to make you feel safe and cared for. Thanks again!
Response from Cumberland Infirmary 12 months ago
Congratulations to you and your husband on the birth of your first child.
I am pleased that the staff were able to help you feel cared for and at ease during this exciting but scary time. I am very happy that this went well for you.
Best wishes to your family
Alison S-D
Patient Experience
- {{helpful}} of {{total()}} people think this response is helpful
- Was this response helpful? Yes | No
Thank you for your feedback.
See more responses from Cumberland Infirmary


text:
Richard Verrall (born 1948) is a British National Front member and edited its magazine Spearhead from 1976 to 1980.
National Front career
Verrall studied History at Westfield College, now part of Queen Mary, University of London, obtaining a first class honours degree.[1] Initially a member of the Conservative Party, Verrall left in the early 1970s, along with a number of members on the right who supported Enoch Powell, to join the NF.[1] Initially a close supporter of John Tyndall, he was appointed Spearhead editor by Tyndall and used the magazine to discuss the veracity of the Holocaust.[1] He was also known for his endorsement of eugenics and biological determinism, adding to this theory that it was equally natural for members of a genetic group to sacrifice themselves for the benefit of others of the same group, thus attacking the criticism that the notion of sacrifice makes this theory inaplicable to humanity.[2]
Despite his initial support for Tyndall, Verrall did not follow him into the New National Front and indeed was appointed deputy Chairman of the NF to Andrew Brons in 1980.[3] Although appointed to this role Verrall played little further role in the politics of the NF and was aloof from the struggle between the Official National Front and the Flag Group. Instead he concentrated most of his efforts on writing about the Holocaust.
Written work
He is best known today for his pamphlet (under the assumed name of Richard Harwood) Did Six Million Really Die?, a Holocaust denial pamphlet which was the subject of the criminal action brought against its Canadian publisher Ernst Zündel. Zündel was ultimately acquitted on the basis that the crime with which he was charged was unconstitutional, but not before the trial court had found the pamphlet to be composed of fabrications and distortions: the Supreme Court of Canada ruled that the book "misrepresented the work of historians, misquoted witnesses, fabricated evidence, and cited non-existent authorities."[4]


text:
Metadefender - Mobile Security uses four different techniques to detect jailbroken devices. If any of the four detection methods work then we consider the device to be jailbroken.
The detection methods are a mix of behavioral and signature-based techniques to increase the likelihood of finding jailbroken devices.
This article was last updated on 2015-08-28
(This article was changed to reflect the new name of Metadefender - Mobile Security on 2016-02-24)


text:
Annette's Cuisine
A Taste of Pakistan
by Annette Khan
About the Book
For distinguished palates. A concise variety of mouthwatering dishes with recipes native to Pakistan. Handy so that it fits anywhere in your kitchen for quick reference. Succulent dishes that awaken the tastebuds and are fun to prepare! First in series.
See More
Log in or create an account to post a comment.
0 Comments
Delete this comment?
See More


text:
If you want to know more about Lean Belly Breakthrough REVIEW- Does It Really Work? Is Lean Belly Breakthrough SCAM or Even The Real Deal? You've come to the right site. Product Name: The Lean Belly Breakthrough Author Name: Bruce KRAHN Customer Support: Excellent Refund Policy: 100% 60 Day Money Back Guarantee Official Website: CLICK HERE The Lean Belly Breakthrough program is designed to tackle weight loss and thereby get rid of the serious health complications that arise as a result of being overweight such as cardiovascular diseases, Gout, diabetes and so…Read More
Tag: weight loss supplements
The 3 Week Diet System Review – 21 Days To Success Best Weight Loss Diet Plan
What is the 3 week diet program of weight loss? Does it work? Is This Weight Loss Program Good? Is Brian Flatt Program Scam? Product Name: The 3 Week Diet Author's Name: Brian Flatt Price: $47 Refund Policy: 100% 60 Day Money Back Guaranteed Official Website: CLICK HERE What Is 3 Week Diet System The 3-week diet system program is a carefully researched weight loss product that is science based and promises to assist people to burn off up to 15 to 23 pounds of unwanted overweight of disgusting excess body fat in just 21 days program.…Read More
Customized Carb Cycling Solution Review by Matt Stirling
Product Name: Customized Carb Cycling Solution Product Author: Matt Stirling Bonuses: Yes Official Website: CLICK HERE ____________________________________________ Matt Stirling's Customized Carb Cycling Solution program is made for people who want to drop some unwanted fat, Performing Better in and out of the gym, and those simply looking to improve their body composition while enjoying their favorite high carb foods. What is the Customized Carb Cycling Solution? The Customized Carb Cycling Solution is the simple 3-phase power system that customizes your carbohydrate intake to your workouts, and body fat percentage targets. Here are the three phases…Read More
The Youth Method 14 Day Diet Detox By Erin Nielsen
The Youth Method 14 Day Diet Detox Review – Is The Youth Method 14 Day Diet Detox a scam or a legit one? What kind of guarantee is given? What is the product owner's credibility? Name: The Youth Method Creator: Erin Nielsen Format: Downloadable guides Refund Guarantee: 60 days refund policy Official Site: CLICK HERE What is The Youth Method 14-Day Diet Detox? The Youth Method 14 Day Diet Detox is easy to use guide including step-by-step pictures, diagrams and schematics show you how everything is done. We have now used it for…Read More
Follow Me Fitness Review By ALLISON GREEN
Follow Me Fitness Review – Is this program legit or scam? Product Name: Follow Me Fitness. Author Name: Allison Green. Official Website: CLICK HERE What is Allison Green Follow Me Fitness? Allison Green Follow Me Fitness was created by Allison Green, who you may know from the band Millionaires or from television shows like the Bad Girls Club. First Allison tried experimenting with a ton of different workout routines including Circuit Training, Bodybuilding, Yoga, and Strength Training… After bouncing around between these workouts, she finally decided to come up with ONE program…Read More


text:
Developers have the Apple iPhone as their first preference, but overall prefer Android
0. phoneArena posted on 28 Aug 2013, 12:26
Developers still prefer the Apple iPhone and the smartphone version of Apple's mobile OS remains the first preference of code jockeys around the world'; that is according to a survey conducted by research firm Forrester who spoke with 765 software developers who write mobile apps. However, more developers have the iPhone in mind to write for first...
This is a discussion for a news. To read the whole news, click here
1. PhansMuneeb (Posts: 371; Member since: 28 Jan 2012)
Temple Run : Oz arrives on wp8...
16. LetsBeHonest (Posts: 1526; Member since: 04 Jun 2013)
Good to see developers are considering WP.
2. mrfamous1 (Posts: 27; Member since: 25 Apr 2012)
Apple iPhones is the best phones period...... less issues = happier people.
3. jove39 (Posts: 1976; Member since: 18 Oct 2011)
One of best phones...not "...the best phone..."
4. Zero0 (Posts: 592; Member since: 05 Jul 2012)
My iPod touch was once wiped on update.
My iPad had issues with force closing. Maybe the developers didn't know what they were doing. The main problem apps were Safari and iBooks.
My brother is currently experiencing iMessage problems.
The only issues I've had with my Nexus 4 are the direct result of (incorrectly) installing CyanogenMod.
8. mrfamous1 (Posts: 27; Member since: 25 Apr 2012)
Sound like a personal problem.....
11. Whateverman (Posts: 3284; Member since: 17 May 2009)
The diehard Apple fanboys will never admit it, but yeah... My iPad force closes 10 times A DAY! Don't get me wrong, I love it. But the Nexus 10 or Note 12 (if the rumors are true) will be my next tablet. Maybe even a WP tablet, who knows.
12. Sauce (unregistered)
The diehard Android fanboys will never admit it, but yea... My Nexus 10 force closes 10 times A DAY! Don't get me wrong, I love it. But the iPad or Note 12 (if the rumors are true) will be my next tablet. Maybe even a WP tablet, who knows?
13. Whateverman (Posts: 3284; Member since: 17 May 2009)
It appears I have a new fan, lol! That's good info on the Nexus 10, but I'm talking about the second generation whenever it comes out. But I will ask around to see how bad the current Nexus 10 force closing issue is.
27. therealestboy (Posts: 54; Member since: 03 May 2013)
Very unlikely that you own an ipad. Force closing is extremely rare on ios.
29. DukeX (Posts: 327; Member since: 28 Aug 2013)
Im sorry but a simple Google search says otherwise. Don't fool yourself. Guess we don't breathe air too....
38. Whateverman (Posts: 3284; Member since: 17 May 2009)
Why would it be unlikely that I own an iPad? Do iPhone users have exclusive rights to purchasing I devices or something. Just because I think Android is a superior OS dosen't mean I use only Android. I like all technology and have the right to critque it if it falls short. Stability is an area, at least on the ipad, where Apple needs to improve.
Just like the good people below are saying, it happens and it happens a LOT! You can not believe me of course but it's true.
33. Shatter (Posts: 2036; Member since: 29 May 2013)
Friend iphone force closes the internet browser all the time.
20. McLTE (Posts: 922; Member since: 18 Oct 2011)
I have friends/coworkers complain all the time about their iPhones/iPads/iPods - mainly with the horrible issues with syncing music, movies, transferring filies, and anything like that.
I tell them. Hmm.. with my Android phone, you could just plug it into ANY PC USB slot and transfer to your hearts content. :P
23. tedkord (Posts: 14132; Member since: 17 Jun 2009)
My friend at work (who just last year switched to a Note II after having an iPhone since the OG) used to joke all the time about the iPhone force closes. The app would fc, and he would say that apparently the iPhone decided he was done using that app and who was he to argue with the iPhone.
I also remember him tapping an icon, then talking it repeatedly until it finally opened.
But, the reason he switched was screen size, removable battery and SD card. Secondary reasons were drag and drop/not needing iTunes to install apps and customizability and being more like a PC.
28. Napalm_3nema (Posts: 2236; Member since: 14 Jun 2013)
Not trolling you, but how did you bone flashing CM on a Nexus? I trust you have done a complete wipe and reflash?
36. Zero0 (Posts: 592; Member since: 05 Jul 2012)
I upgraded to 10.2 and thought it was okay to do a dirty flash. (Stupid, I know, but I was too lazy to backup and restore.)
Had some minor issues for a while (occasional force closes, two clock apps), and eventually it just crapped out.
42. roscuthiii (Posts: 2241; Member since: 18 Jul 2010)
I have quite a few work colleagues now that have jumped all-in to Apple... periodically throughout the day I hear them grumble, "damn iPhone/iPad", but then turn around trying to sell our manager on how great Apple is and that she should trade in her rugged flip-phone for an Apple.
Honestly, I can say I've never heard her grumble about her phone to this date, and it's worked reliably no matter where she is. Now that I have a tablet for entertainment & media consumption, she's got me thinking about giving up the smartphone altogether for a simple reliable feature phone.
7. ady_lad (Posts: 82; Member since: 17 Jul 2013)
Dream on mate...How about no issues with Android or WP8 and no Apple communism.
9. buccob (Posts: 2736; Member since: 19 Jun 2012)
did you read the article? did you understand the graphics?.
you are incorrect in your grammar and your statement... Anyway it might be the "best phone in your opinion" or the "best at some features"... but definitely not the "best period"
10. mrfamous1 (Posts: 27; Member since: 25 Apr 2012)
Go home Buccob Your drunk.....
22. WHoyton1 (Posts: 1635; Member since: 21 Feb 2013)
are you special....
15. LetsBeHonest (Posts: 1526; Member since: 04 Jun 2013)
Its personal preference buddy.
it might be best for you because its satisfy your need easily.
your best may be worse for offers and vice versa.
but true its one of the best phone available to date.
17. LetsBeHonest (Posts: 1526; Member since: 04 Jun 2013)
I mean "others"
34. Shatter (Posts: 2036; Member since: 29 May 2013)
It think you meant to say best brand brainwashing?
5. jove39 (Posts: 1976; Member since: 18 Oct 2011)
No wonder...there is just one flagship iPhone every year and devs don't have to worry about several diff versions of hardware as in Android!!!
6. davenycept (Posts: 200; Member since: 03 Jul 2012)
IPhone is very good but just not top of the line android
14. amilcarmz86 (Posts: 95; Member since: 09 Jul 2013)
The iphone is boring and simple, you cannot customize that phone as you can do with an android.
37. Commentator (Posts: 3722; Member since: 16 Aug 2011)
Thanks, phone review from 2008!
50. amilcarmz86 (Posts: 95; Member since: 09 Jul 2013)
i guess you have all the apple products, how does it feel to be ripped off ?


text:
The average dog is a nicer person than the average person
-Andy Rooney-
I might watch, not sure yet. Honestly this whole franchise has gotten pretty boring the past few seasons. I don't know how much longer they can keep trying to pump new life into it - for *the most dramatic season evah!*.
Of course getting an unknown lead might help.
Boob Wrinkles - it is not a myth
Well enough that a lot of people made judgements about him pretty early on, well before the show aired. That is why I say someone we've never met might be just the shot of invigoration this show needs.
Boob Wrinkles - it is not a myth
The casting promos have Chris saying "would you like to DATE our next bachelor or bachelorette?" not "do you know someone who might be a good bachelor or bachelorette" or whatever. I too am sick of retreads. Would have liked to see Renee.
The average dog is a nicer person than the average person
-Andy Rooney-
Does Andi really dance that badly or was she camping it up? She was really channeling Elaine from Seinfeld's dancing:Elaine Benis Dances from Seinfeld - YouTube
Who knows with Andi - she might have been acting her way to the B'ette from the get go.


text:
Ganache Using Trader Joe One Pound Plus Milk Chocolate Bars
Decorating By savannahquinn Updated 19 Aug 2013 , 12:58pm by savannahquinn
Has anyone made ganache using Trader Joes one pound plus milk choc. bars? Last time I made it - it was too soft....I'm wondering if the 2:1 ratio I normally use to make ganache is too much...(obviously it was)....
AI haven't used Trader Joes but I've heard it's very good. With Milk or White Chocolate I use less cream b/c they tend to be softer 3:1
thank you so much for replying....I'm going to try that... the 2:1 just was way too soft .
AYour welcome! I love using ganache
ADoes Trader Joe's also have white chocolate available?
I have not seen white chocolate available there....but they sell pound plus bars of dark, semi (I think) and milk chocolate and are very reasonable


text:
RNA Sequencing
Webinar: Discover More in Transcriptome Research with the Unprecedented Read Coverage, Sensitivity and Resolution of Targeted RNA Sequencing
Why RNA-Seq
The transcriptome is comprised of all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNA transcribed in one cell or type/population of cells. Transcriptome analysis often aids in the identification of genes that are differentially expressed in distinct cell populations and the relative abundance at which they are expressed. Recently, long noncoding RNAs (lncRNAs) have been described that are a large and diverse class of transcribed RNA molecules, thought to play important regulatory functions, with a length of > 200 nucleotides.
Why Targeted RNA-Seq
Researchers have been able to perform whole-transcriptome sequencing (RNA-Seq) of cDNAs derived from RNA to evaluate expression levels, variant splicing events, SNPs, and InDels throughout the transcriptome. The standard whole-transcriptome RNA-Seq, however, may not provide the highest efficiency and cost-effectiveness to a researcher. No matter if you're conducting gene expression profiling or novel transcript discovery, enrichment by SeqCap is recommended. We created the SeqCap RNA enrichment system for exactly this purpose.
The benefits for the SeqCap RNA system include:
- Decreased sequencing requirements, allowing the use of MiSeq for confident expression profiling — for example, for a panel of 250 genes, you may need 50x less sequencing compared to standard RNA-Seq to achieve the same accuracy and sensitivity
- Eliminating the need for Poly A selection or rRNA depletion in your workflow — Instead, it selects directly for coding or noncoding regions, allowing for any design of your choosing
- Increased flexibility — this system is suitable for a wide range of designs, from small panels up to whole transcriptome analysis, or other designs like lncRNA
- Improved quantitation and detection efficiency for low abundance transcripts and isoforms —by focusing your sequencing reads on genes of interest and not on housekeeping genes
Genome-wide discovery of human splicing branchpoints.
Transcriptomics of the bed bug (Cimex lectularius).
The primary transcriptome of the major human pathogen Helicobacter pylori.
Recurrent mutation of the ID3 gene in Burkitt lymphoma identified by integrated genome, exome and transcriptome sequencing.


text: Hi! My first post here... I recently went back to Verizon/HTC Eris after a two year exile with T-Mobile/Blackberry. Service with T-Mobile constantly involved dropped calls, disappearing calls, lost voice mails, emails, blah, blah, blah. Haven't had any of that with the new service/Droid so who knows if it was the phone or service prior. I have scoured this and other websites for help with my dreadful Eris and while the solutions worked initially, I suddenly am getting the error "Low on space", again. I click on that message, open app manager, and attempt to clear cache but nothing needs it's cache cleared. I have cleared my browser history. I have no text messages. I have deleted all my email in all folders (hotmail and gmail) and yet in the app manager it shows 'Mail' with 101MB Total and 101MB Data. I have no idea how to clear this and it grows daily. Otherwise most of my apps are in the low 2MB range with Maps at 8.9MB. Additionally, I am interested in purchasing a new phone and getting rid of this Eris. I don't have the time or interest to spend trying to fix the problems, I just need a phone that works and works well! I need a phone with app capability, excellent camera with flash and HD video, email, web, texting, etc.... I use the phone for personal use. Can you suggest a good phone that won't break my bank (I'm not due to upgrade for another 15 months and am satisfied with Verizon). Also, I'd like to purchase a new phone for my 10 year old for Christmas. I'd like to find a touchscreen (same situation, 15 months to upgrade). Thanks for your advice, Lis


text:
Opening Statements
House Judiciary Committee Hearing on U.S. Attorney Firings
Wednesday, May 23, 2007; 11:27 AM
REP. JOHN CONYERS JR., D-MICH. CHAIRMAN: Good morning, Madam Witness, and to the members of the committee.
Today, the House Judiciary Committee continues its investigation into the controversy surrounding the United States attorneys and related matters.
Our sole witness today is Ms. Monica Goodling, who served as senior counsel to the attorney general and White House liaison until she resigned on April 7th, 2007.
As the members know, two months ago Ms. Goodling informed us that she would assert her Fifth Amendment right not to incriminate herself if she was called to testify.
As a result of that assertion, this committee, after careful consideration, authorized, by a vote of 32-6, a grant of immunity to Ms. Goodling, which we have secured from the chief judge of the United States District Court for the District -- the D.C. District.
The committee also authorized a subpoena to compel her testimony, as well as the production of documents.
Now, these are steps that I did not take likely, but only after consultation with the ranking member, Lamar Smith, and my colleagues on both sides of the aisle with the Justice Department.
I believe that this is an important and necessary step to help us get to the bottom of the U.S. attorney matter and other concerns regarding possible improperly politicalization of the Justice Department.
As the White House liaison and one of the attorney general's most trusted advisers, Ms. Goodling may well have information that will help us in our inquiry.
Let me observe that the fact that we are granting limited immunity to Ms. Goodling for her testimony should not be taken as any indication that the committee believes that she is guilty of a crime. Nor does the fact that she is testifying today mean that she necessarily has a greater role than some of the other individuals involved in the firing whom we have already interviewed or who have testified.
CONYERS: She is before us today in a public hearing simply because we have no other means of obtaining her testimony in a timely manner.
We appreciate your cooperation.
I would hope that the fact of her testimony would encourage others to come forward and cooperate with our inquiry. And that includes personnel in the White House itself, whose role in these firings appear to grow more central every day, every time another Department of Justice official denies recommending putting any particular prosecutor on the firing list.
The issues we are examining -- which include possible obstruction of justice, misleading the Congress, violations of the Hatch Act -- are obviously serious. If we cannot trust the Department of Justice to fairly and impartially enforce our nation's laws, if we cannot trust the testimony of our most senior officials in the administration, if it appears that the U.S. attorneys are merely pawns in a game of politics, then we will have suffered the loss of one of our nation's most fundamental principles, the rule of law.
And I would now recognize our ranking minority member -- whom I commend for the cooperation that has existed between all of us on this committee -- the distinguished gentleman from Texas, Lamar Smith, the ranking minority member.
REP. LAMAR SMITH, R-TEXAS RANKING MEMBER: Thank you, Mr. Chairman.
Ms. Goodling, welcome. You are apparently the last witness we're going to hear from who's worked within the Department of Justice.
And let me say at the outset, I know this is not an easy process for you to go through, and I know you've never had to endure anything like this hearing before, which I think makes us all particularly appreciate of your being here. And thank you for being here.
Mr. Chairman, we, our staff, the public and the media have pored over reams of documents, heard from nearly 20 witnesses, issued a plethora of subpoenas and taken the extraordinary step of immunizing your testimony.
The Senate has received and examined evidence as well.
For some time, many have been on the edges of their seats, waiting for the moment in which you, Ms. Goodling, as the department's former White House liaison, might link the U.S. attorney dismissals to any appropriate action by Karl Rove or Harriet Miers.
I understand that the majority staff already has learned that you never had any contact with Mr. Rove or Ms. Miers. I do not share their disappointment.
When this investigation began, the department defended its actions on the ground that the dismissed U.S. attorneys served at the pleasure of the president.
When pressed, the department volunteered that the dismissals were mainly performance-related.
When examined, the evidence of performance problems was in some cases well-documented, and in other cases not.
SMITH: The questions about the differing degrees of proof as to whether this or that U.S. attorney really was dismissed for performance reasons have helped fuel the speculation.
With regard to the dismissals, the right interpretation may just be that the dismissals were in fact for performance reasons or, in one case, to offer an opportunity to another qualified candidate.
Finally, with regard to this investigation itself, we and our investigators see ever more clearly that the accusations of wrongdoing in these eight U.S. attorney dismissals don't seem to have legs.
But we won't stop. Instead, some try to graft on new legs, whether with regard to other U.S. attorneys, or with hearings in which we hear about the attorney general's activities as White House counsel or with new allegations against you, Ms. Goodling.
We want to hear from you today so that we can find out the truth, clear the air or take other appropriate steps.
We have not reached any final conclusion. And we should not until we know all of the relevant facts.
Thank you, Mr. Chairman. I'll yield back.
CONYERS: I thank the gentleman.
And without objection, all other members' opening statements will be included in the record.
We'll now hear from Ms. Monica Goodling.
Ms. Goodling served in a variety of capacities in the Department of Justice, beginning in the Office of Public Affairs, then for a short period in the U.S. attorney's office in the Eastern District of Virginia, then in the Executive Office for U.S. Attorneys and most recently in the Office of the Attorney General, where she was senior counsel to the attorney general and White House liaison.
She received her law degree from Regent University School of Law in 1999.
CONYERS: Ms. Goodling is accompanied by counsel. And we would please ask that he introduce himself for the record.
JOHN DOWD: Mr. Dowd.
CONYERS: Good morning, Mr. Dowd.
DOWD: Mr. John Dowd and Jeff King and Jim Sherry, Akin Gump Strauss Hauer Feld, on behalf of Ms. Goodling.
CONYERS: Thank you very much. We'll welcome -- we welcome you and are happy that you are here.
And under our House rules, Ms. Goodling is entitled to have counsel present for the purpose of advising her as to her constitutional rights.
And we thank you very much, sir.
I want to make sure you understand that your authorized role is to advise Ms. Goodling, and more specifically to advise her as to her constitutional rights. And with that understanding, we have furnished you with your own copy, counsel, of the notebook that we have given Ms. Goodling containing a number of documents that she may be questioned about today, to facilitate you and her memory and cooperation.
Have you received those documents?
Thank you.
And have you received them, Ms. Goodling?
GOODLING: Yes, thank you.
CONYERS: The documents are tabbed to make it easier to locate them if and when there is any necessity to use them.
We have also provided copies of the notebook to our friends up here for the same purpose.
REP. DAN LUNGREN, R-CALIF.: Mr. Chairman, point of personal privilege.
CONYERS: What is it?
LUNGREN: You just referred to some documents. We don't have them at our place here.
CONYERS: We gave a number of them...
LUNGREN: Are those the ones that were just given to us beforehand that...
CONYERS: Yes.
CONYERS: Yes, sir. They're being copied now, Mr. Lungren.
One housekeeping matter before we proceed.
Ms. Goodling, our subpoena requires that you furnish us documents in your possession, custody or control relating to matters we're investigating. And although we have mentioned this to your counsel, we believe the committee is entitled to receive the documents without delay.
We understand from counsel that you wish to confer with your former employer, the Department of Justice, before they're released to us. And we will work with you to give you a reasonable time to take care of that, reserving the right to take further action if necessary.
Oh, you do have them? Thank you. Thank you very much, Counsel Dowd.
Ms. Goodling, given -- yes, sir?
DOWD (?): We're already corresponded and communicated about the balance of them with both you and the Department of Justice.
CONYERS: Exactly.
DOWD (?): Thank you, sir.
CONYERS: I thank you, sir.
Ms. Goodling, given the gravity of the issue we're discussing today and your role in the hearing, and to help ensure that there's no misunderstanding about your obligation here, we would ask that you be sworn in before we proceed.
Would you kindly raise your right hand? If you would please stand and raise your right.
Do you solemnly swear or affirm under penalty of perjury that the testimony you are about to provide the committee will be the truth, the whole truth and nothing but the truth, so help you God?
MONICA GOODLING: I do.
CONYERS: Thank you.
Ms. Goodling, let us begin.
I will ask you, who made the recommendations to place the list -- place on the list of the United States attorneys to be fired each of the nine U.S. attorneys who were in fact terminated in 2006?
GOODLING: Upon the advice of my counsel, I respectfully decline to answer the question, based upon my Fifth Amendment right not to be a witness against myself, and my Sixth Amendment right to rely on my counsel's advice.
CONYERS: Ms. Goodling, I am hereby communicating to you an order signed by Chief Judge Hogan of the United States District Court for the D.C. District.
The clerk is bringing to you now a certified copy of the order to you. And we've made a copy for your counsel. And without objection, the order will be placed in the record.
The order provides, in substance, that you may not refuse, on the basis of your Fifth Amendment privilege against self-incrimination, to provide testimony or other information to this committee under compulsion.
The order also provides that testimony or other information obtained from you under compulsion pursuant to the order may not be used against you in any criminal proceeding, nor may information derived from what you provide us under compulsion be used against you as long as the testimony and other information you provide us is truthful.
As I am sure your counsel has no doubt advised you, you are obligated to answer each question completely and truthfully. And failure to do so could subject you to prosecution for perjury or for giving false statements to Congress.
So I want to be careful about how you answer each question.
CONYERS: And if you occasionally need to confer with your counsel, Mr. Dowd, before answering a question, we will be happy to accommodate you in that regard. And if a member has that happen, the clock will be suspended so that our time won't be running while she might be conferring with her counsel.
With that said, Ms. Goodling, pursuant to the order you now have in front of you, I direct you to answer the questions that will be put to you regarding our investigation as I have just described it.
This, Ms. Goodling, completes the procedure for conferring use immunity on you pursuant to the order.
Now, before we begin questioning, Ms. Goodling, I appreciate that you have a statement in writing that you would like to make. And we welcome it at this time. And we will include your statement in the record, and invite you to begin whenever you would like.
I ask the clerk to distribute copies of Ms. Goodling's written statement to every member of the Judiciary Committee.
You may proceed whenever you'd like.
GOODLING: Thank you, Mr. Chairman.
Good morning, Chairman Conyers...
CONYERS: Pull the mike up just a little closer to you, please.
Nice loud voice.
GOODLING: Good morning, Chairman Conyers, Ranking Member Smith and members of the committee. Thank you for allowing me to make this statement.
With the committee's permission, I would like to submit lengthier written remarks to be entered into the record.
CONYERS: Without objection, so ordered.
GOODLING: My written remarks will address four topics that I expect will be of interest to the committee.
GOODLING: First, I wish to set the record straight regarding what I understood to be the deputy attorney general's allegation to Senator Schumer that I withheld information from him prior to his public and private testimony before the Senate Judiciary Committee.
The allegation is false. I did not withhold information from the deputy.
To the contrary, I worked diligently to compile and provide the deputy with dozens of pages of statistics and other information that I thought he was likely to need, based on the questions that were being asked at that time.
Despite my and others' best effort, the deputy's public testimony was incomplete or inaccurate in a number of respects.
As explained in more detail in my written remarks, I believe the deputy was not fully candid about his knowledge of White House involvement in the replacement decision, failed to disclose that he had some knowledge of the White House's interest in selecting Tim Griffin as the interim U.S. attorney in the Eastern District of Arkansas, inaccurately described the department's internal assessment of the Parsky commission, and failed to disclose that he had some knowledge of allegations that Tim Griffin had been involved in vote- cadging during his work on the president's 2004 campaign.
After the deputy's public testimony, I continued to work to assemble information that the deputy had promised to provide in a future, private session.
On February 14th, 2007, the deputy attended a private briefing with the Senate Judiciary Committee. That afternoon I rode with him to the Senate building, intending to observe the session and support the deputy by providing any information that I had.
GOODLING: However, a few minutes before the private Senate briefing was to take place, the deputy made clear to me that he did not think I should attend. The deputy suggested that if someone recognized me as the White House liaison, the members would be more likely to ask questions about the White House.
As a result of that conversation, I waited outside the room while the deputy briefed the Senate committee.
During a break, Richard Hurtling told me the briefing was not going well, and recommended that I return to the department immediately. Like the deputy, Mr. Hurtling suggested it could complicate matters if I was recognized as the White House liaison. As a result, I returned to the department in a taxi.
In light of these events, I was surprised to learn that the deputy had blamed me for his incomplete or inaccurate information.
Second, I wish to clarify my role as White House liaison.
Despite that title, I did not hold the keys to the kingdom, as some have suggested. I was not the primary White House contact for purposes of the development or approval of the U.S. attorney replacement plan.
I have never attended a meeting of the White House Judicial Selection Committee. The attorney general and Kyle Sampson attended those meetings.
To the best of my recollection, I've never had a conversation with Karl Rove or Harriet Miers while I served at the Department of Justice. And I'm certain that I never spoke to either of them about the hiring or firing of any U.S. attorney.
Although I did have discussions with certain members of their staffs regarding specific aspects of the replacement plan, I never recommended to them that a specific U.S. attorney be added to or removed from Mr. Sampson's list. And I do not recall that they ever communicated any such recommendation to me.
Third, I wish to address my role in selecting U.S. attorneys for replacement.
I first learned that others more senior to me were discussing the possibility of replacing some U.S. attorneys at some point in mid- 2005. And I believe I first saw a list of candidates for replacement in January 2006, when Mr. Sampson showed me a draft memorandum he was preparing for Harriet Miers.
GOODLING: At that time, I recommended that two of the U.S. attorneys Mr. Sampson had listed be retained in office and that certain other U.S. attorneys be considered for replacement.
Paul Charlton and Daniel Bodgen were two of the U.S. attorneys that I recommended considering for replacement. However, it appears from the documents produced to Congress by the department that Mr. Sampson did not initially accept that recommendation. Mr. Bogden and Mr. Charlton did not appear on iterations of the list sent to the White House in January, April or May, and first appeared on the list in September 2006, presumably for reasons unrelated to my initial recommendation.
Although I'm prepared to tell the committee what I know about the eight replaced U.S. attorneys, the truth is that I do not know why Kevin Ryan, John McKay, Carol Lam, Paul Charlton, Daniel Bogden, David Iglesias and Margaret Chiara were asked to resign in December of 2006.
I can describe what I and others discussed as the reasons for their removal, but I just can't guarantee that these reasons are the same as those contemplated by the final decision-makers who requested these resignations.
However, I'm not aware of anybody within the department ever suggesting the replacement of these U.S. attorneys to interfere with a particular case or in retaliation for prosecuting or refusing to prosecute any particular case for political advantage.
Fourth, I wish to clarify my role in career hiring at the department.
During my five years at the department, I believe that I interviewed hundreds of job applicants, and the vast majority of these were applicants for political appointee positions. But some were applicants for certain categories of career positions.
Specifically, I interviewed candidates who were to be detailed into confidential policy-making positions and attorney general appointments, such as immigration judges and members of the Board of Immigration Appeal. I also interviewed requests for waivers of hiring freezes imposed on districts with an outgoing U.S. attorney or interim or acting U.S. attorney.
In every case I tried to act in good faith and for the purpose of ensuring that the department was staffed by well-qualified individuals who were supportive of the attorney general's views, priorities and goals.
GOODLING: Nevertheless, I do acknowledge that I may have gone too far in asking political questions of applicants for career positions, and I may have taken inappropriate political considerations into account on some occasions. And I regret those mistakes.
In conclusion, I'd like to give the committee a little better sense of who I am, because the person that I read about on the Internet and in the newspaper is not me.
At heart, I'm a fairly quiet person. I try to do the right thing, and I try to treat people kindly along the way. I always knew that I wanted to grow up and do something to serve or help other people. I went to public schools growing up, but I chose Christian universities in part because of the value that they place on service.
I've seen in my life what violent crime can do to its victims. And I knew that at some point I wanted to do my part to seek justice on their behalf. And that's why I've loved the Department of Justice, particularly my time as a prosecutor.
For the five years that I spent there, I worked as hard as I could at whatever task that was put before me. And I hope that's the reason that I was promoted five times during my time at the department.
I considered the people that I worked with to be my family. And I care about them deeply.
I have no desire to say anything negative about anyone that I worked with, including the leadership team or the U.S. attorneys who are the subject of my testimony. But I'm here to be a fact witness to what I heard, saw, did or know, and I'll do that to the best of my recollection.
Thank you for allowing me the time to make this statement. I'm prepared to answer your questions.
CONYERS: And I thank you for your statement.


text:
Emily Blair Wareham was born and raised on beautiful Prince Edward Island but has lived, studied and worked in Halifax since 2004 Since graduating in April 2010 with her BFA in Jewellery Design and Metalsmithing she has worked as a technician, first at the Nova Scotia Centre for Craft and Design, and then at NSCAD University - in the very same department where her passion was ignited. After 4 years of working in her field, producing a body of jewellery works for her solo show at the Mary E Black in Halifax, and becoming a mother, she has begun to pursue her Masters in Fine Arts at NSCAD University.
Contact us:nscad.ca T 902 494-8185
Mailing Address: 5163 Duke Street Halifax, NS B3J 3J6
Office Address: 5163 Duke Street 4th Floor, Student Accounts Halifax, NS
Office Hours: Monday to Friday, 9:30am-4pm


text: Popeyes® Louisiana Kitchen, a division of AFC Enterprises, Inc. (NASDAQ: AFCE), launches its redesigned company website today, at www.popeyes.com. (Photo: Business Wire)Popeyes flavor, environment and energy are deeply-rooted in its Louisiana heritage and its new website reflects those attributes. The homepage highlights eye-catching food photography announcing current products and promotions available in restaurants and New Orleans themed background images. The new interactive menu also features new food images as well as enhanced functionality. A clean design and moving interchangeable images illustrate additional offerings from Popeyes such as its catering service, beverage platform, and loyalty program. And, for those consumers fascinated by recommendations, the website features PopCulture, a social media portal sharing recent posts by brand zealots. "Popeyes is excited to unveil this new website to our guests," said Dick Lynch, chief global brand officer at Popeyes. "A few years ago, we began a brand transformation that included a new brand identity, menu, advertising and restaurant reimage. Today, we are taking the next step by launching our newly redesigned website, Popeyes.com. Guests will find the new site to be consistent with who Popeyes is today, easier to navigate and much more interactive."


text:
- Abbreviation of Authorized Version, which is the King James Bible.
Ballentine's law dictionary. Anderson, W.S.. 1998.
Ballentine's law dictionary. Anderson, W.S.. 1998.
A/V — abbreviation audio/video … New Collegiate Dictionary
A. V. Ramanan — is a Tamil television host, singer and an actor. He became a household name in Tamil Nadu, during his hosting of the famous Sun TV program Saptha Swarangal . His cheerful style and bass voice endeared him to the audience.A stage singer on the… … Wikipedia
A.V. Roe — may refer to:*Alliott Verdon Roe, British industrialist *A.V. Roe and Company (also known as Avro), British aircraft manufacturer founded by Alliott Verdon Roe **A.V. Roe Canada its Canadian subsidiary … Wikipedia
A. V. Phibes — A.V. Phibes (Short for Alia Vulnavia Phibes) is an illustrator known for her slick, retro inspired images, cartoons and pinups. Born in Baltimore, Maryland, USA, she also lived in New Mexico and Colorado before settling in Brooklyn, New York in… … Wikipedia
A. V. Kuttimalu Amma — A.V. Kuttimalu Amma was a top ranking Congress Party leader of Kerala, India and a fearless freedom fighter. Born in Anakkara Vadakkath family of Ponnani taluq,S. Malabar, in 1905, Amma was drawn into the freedom movement in her youth. She and… … Wikipedia
A. V. Ilango — (born March 17, 1950) is an Indian painter. He was born in Gobichettipalayam, a small town in Tamil Nadu.Painting styleThe rural background has left an indelible impression in his formative years. In India, art, culture, religion and rituals are… … Wikipedia
A.V.C. College — Infobox University name =A.V.C. College native name = latin name = motto = established =1955 type =Autonomous men s college endowment = staff =221 faculty =119 president = principal = Dr. M. Varadharajan rector = chancellor = vice chancellor =… … Wikipedia
A. V. Alexander, 1st Earl Alexander of Hillsborough — Albert Victor Alexander, 1st Earl Alexander of Hillsborough, KG, CH, PC (1 May 1885 ndash;11 January 1965) was a British Labour and Co operative politician. He was three times First Lord of the Admiralty, including during the Second World War,… … Wikipedia
A. V. Dicey — Albert Venn Dicey Born 4 February 1835 (1835 02 04) Died 7 April 1922 … Wikipedia
A V star — An A V star is a main sequence (hydrogen burning) star of spectral type A and luminosity class V. These stars have spectra which are defined by strong hydrogen absorption lines. [ [http://hyperphysics.phy astr.gsu.edu/hbase/starlog/staspe.html… … Wikipedia
A. V. Bellarmin — Infobox Indian politician name = A.V. Bellarmin caption = birth date =birth date and age|1954|5|5|df=y birth place =Kanyakumari, Tamil Nadu residence =Kanyakumari death date = death place = office = Member of Parliament constituency = Nagercoil… … Wikipedia


text:
|Home | About | Journals | Submit | Contact Us | Français
Elimination of autoreactive CD4+ T cells through the death receptor Fas/CD95 is an important mechanism of immunological self-tolerance. Fas deficiency results in systemic autoimmunity, yet does not affect the kinetics of T-cell responses to acute antigen exposure or infection. Here we show that Fas and TCR-induced apoptosis are largely restricted to CD4+ T cells with an effector memory phenotype (effector memory T cells (TEM)), whereas central memory and activated naïve CD4+ T cells are relatively resistant to both. Sensitivity of TEM to Fas-induced apoptosis depends on enrichment of Fas in lipid raft microdomains, and is linked to more efficient formation of the Fas death-inducing signaling complex. These results explain how Fas can cull T cells reactive against self-antigens without affecting acute immune responses. This work also identifies Fas-induced apoptosis as a possible immunotherapeutic strategy to eliminate TEM linked to the pathogenesis of a number of autoimmune diseases.
The TNF-family receptor Fas/CD95 can directly induce apoptotic programmed cell death and is essential for maintaining immunological self-tolerance. Humans with dominant-negative Fas mutations in the autoimmune lymphoproliferative syndrome (ALPS) and mice deficient for Fas or Fas ligand are highly predisposed to autoimmunity.1, 2, 3 Recent findings have shown that expression of Fas on T cells is important for the function of Fas in maintaining immunological self-tolerance.4 In vitro, apoptosis induced by the TCR, termed restimulation-induced cell death (RICD), is dependent on Fas in CD4+ T cells.5 However, it has been difficult to demonstrate defective deletion of Fas-deficient T cells in response to acute infections, immunization or superantigen stimulation in vivo.6 Rather, Fas appears to have a role mainly in deletion of T cells that are chronically or repeatedly exposed to TCR stimulation.6
T cells previously exposed to antigen acquire phenotypic and functional characteristics of memory T cells, including the ability to self-renew more efficiently and respond to antigen with minimal co-stimulation.7 These properties are important for maintaining immunity to pathogens but may also allow the perpetuation of pathological autoimmune responses. Memory T cells exist in functionally distinct subsets based on expression of CCR7 and CD62L, which allow lymph node homing,8 and the TNF-family member CD27. Memory T cells expressing CD62L, CD27 and CCR7 are termed 'central memory' (TCM) because they efficiently home to lymph nodes and self-renew. By contrast, effector memory T cells (TEM) migrate to extralymphatic sites and have immediate potential to produce effector cytokines on TCR restimulation. Although the precise interplay between effector and TCM is not fully understood, TEM have been implicated in the pathogenesis of a number of T-cell-dependent autoimmune diseases and animal models of autoimmunity.9, 10
Here, we have found that TCR and Fas-mediated apoptosis in primary human and mouse CD4+T cells is largely restricted to cells with an effector memory phenotype, and have linked this sensitivity to Fas localization to lipid raft microdomains and more efficient assembly and activation of the Fas-associated death-inducing signaling complex (DISC).
We observed a wide variation in sensitivity to Fas-induced apoptosis between donors in samples of total CD4+ T cells from healthy volunteers, and reasoned that this may correlate with the variation in the percentage of CD45RO+ memory T cells. To assay the apoptosis sensitivity of different T-cell subsets defined by surface markers, we developed a multi-parameter flow cytometric assay to determine the percentage of annexin V-positive early apoptotic cells simultaneously with surface markers for memory T cells (CD45RA−) and the chemokine receptor CCR7 and the TNF-receptor family member CD27, which distinguish effector and central memory CD4+ T-cell subsets.11, 12 As previously observed,12 naïve CD45RA+ T cells were predominantly CCR7+/CD27+, while CD45RA− memory T cells could be divided into TCM (CCR7+/CD27+) or TEM (CCR7−/CD27−) and a small subset of CCR7−CD27+ 'transitional' memory cells, (Figure 1a, bottom left). Naive CD4+ T cells did not express detectable Fas as previously reported,13 while TCM and TEM CD4+ T cells expressed identical levels of surface Fas (Figure 1b). Correlating with their lack of surface Fas, naïve T cells were highly resistant to apoptosis induced by crosslinked anti-Fas antibodies (Figure 1c). Strikingly, TCM phenotype CCR7+CD27+ T cells were also almost completely resistant to apoptosis induced by Fas crosslinking, whereas TEM contained nearly all the Fas-sensitive T cells in the memory T-cell pool (Figure 1c). TEM CD4+ T cells were also the most sensitive to apoptosis induced by a stabilized form of FasL (FasL-LZ), the physiological ligand for Fas (Figure 1d). Taken together, these data show that within freshly isolated CD4+ T cells, TEM are virtually the only cells with a functional Fas receptor.
In order to test whether Fas-induced apoptosis is restricted to cells with a TEM phenotype also in activated T cells, we purified populations of naïve or memory CD4+ T cells from normal donors, activated them with anti CD3/CD28 followed by expansion in IL-2, conditions extensively used to define and study RICD.14, 15 We observed progressive conversion of memory cells to a TEM phenotype, downregulating CD27 and CCR7 and gaining CD70 expression (Figure 2a, data not shown). Naïve CD4+ T cells gained CD45RO and lost CD45RA expression, but maintained a CCR7+/CD27+ TCM phenotype for up to 2 weeks of culture in IL-2 (Figure 2a). After activation, naïve T cells also acquired nearly equivalent levels of Fas to that expressed on activated memory T cells (Figure 2b). When activated T cells derived from naïve T-cell precursors were treated with anti-Fas antibody or FasL, they remained relatively resistant to Fas-induced apoptosis compared with memory T cells (Figures 2c and d). Memory T cells retaining TCM-like phenotype were as resistant to Fas-induced apoptosis as naïve T cells. Apoptosis of TEM cells accounted for nearly all of the Fas sensitivity of activated T cells similar to cells analyzed directly ex vivo (Figures 2c and d). As cells with a TCM and TEM phenotype were cultured together in the experiments, these data indicate cell-autonomous differences. These results show that a TEM phenotype is the best predictor of sensitivity to Fas-induced apoptosis in CD4+ T cells, both directly ex vivo and after in vitro expansion.
In activated CD4+ T cells, much of the apoptosis triggered by restimulation of the TCR (RICD) is mediated through Fas–FasL interactions.16 T cells sorted initially for a naïve and TCM phenotype and then activated and expanded in vitro were significantly more resistant to RICD than memory and TEM cells (Figure 3a). TCM had significant residual sensitivity to TCR-induced apoptosis, which may be due to conversion of these cells to a TEM phenotype during in vitro expansion. Indeed, when activated T cells were assayed for RICD simultaneously with surface markers, TCR-induced apoptosis in TCM with a CD27+CCR7+ phenotype at the time of restimulation was reduced (Figure 3d). To determine the extent to which TCR-induced cell death was dependent on Fas–FasL interactions, we used blocking antibodies against FasL (Figure 3b) and also studied CD4+ T cells purified from the blood of ALPS patients containing Fas mutations that dominantly interfere with Fas apoptosis (Figures 3c and d). Anti-FasL (Nok-2) antibodies efficiently blocked RICD in restimulated naïve and memory subsets, indicating that memory and TEM cells undergo increased apoptosis induced by Fas-specific RICD mechanisms (Figure 3b). ALPS patient T cells were significantly protected from Fas-induced apoptosis, with the greatest differences occurring in T cells from ALPS patients harboring Fas mutations in the death domain (DD), consistent with previous data showing that Fas DD mutations cause the most severe impairment of Fas-mediated apoptotic signaling.17 TEM contained the most Fas-sensitive cells, which were also dramatically resistant to Fas-induced apoptosis in ALPS patients (Figure 3c). Much of the TCR-induced apoptosis seen in the TEM cells was dependent on Fas–FasL interactions, as TEM from ALPS patients were significantly resistant to RICD compared with healthy donors (Figure 3d).
TCR triggering also results in synthesis and secretion of FasL. To determine whether differences in FasL production could contribute to the differential apoptosis of T-cell subsets, we measured FasL after TCR restimulation. Naïve and memory T cells produced comparable amounts of FasL whereas TCM and TEM produced slightly less (Supplementary Figures S1a and b). Interestingly, despite the similar levels of FasL as measured by ELISA, FasL produced by restimulated naïve T cells was less cytotoxic for the Fas-sensitive cell line SKW6.4 (Supplementary Figure S1c). This suggests that differences in posttranslational processing of FasL may contribute to the reduced TCR-induced apoptosis of naïve restimulated T cells.
Previous studies have identified murine central and effector CD4+ memory T cells based on surface expression of -selectin (CD62L) and CD44.18 We tested naïve (CD62Lhi/CD44lo), central memory (CD62Lhi/CD44hi) and effector memory (CD62Llo/CD44hi) CD4+ T-cell subsets for apoptosis in response to TCR crosslinking in cells from wild-type C57Bl/6 mice as well as Fas-deficient lpr/lpr mice on the B6 background. Unlike human T cells, murine naïve T cells did express some surface Fas (Figure 4e). Nevertheless, TEM phenotype cells were the most susceptible to RICD and FasL-induced apoptosis (Figures 4a and c), and experiments Fas-deficient Lpr T cells confirmed that FasL and RICD were completely dependent on Fas (Figures 4b and d). After activation and expansion in vitro with IL-2, naïve and TCM T cells downmodulated CD62L and the majority acquired an effector memory (CD62Llo/CD44hi) phenotype (Supplementary Figure S2a). In accordance with these phenotypic changes, naïve T cells acquired sensitivity to FasL-induced apoptosis that was higher than the apoptosis observed in activated TEM or TCM, but lower than that of TEM in resting T cells (Supplementary Figure S2d). In response to RICD, in vitro expanded TEM remained the most sensitive to apoptosis (Supplementary Figure S2c). In Lpr Fas-deficient activated T cells, there was slight upregulation of surface Fas that is likely because of the known 'leakiness' of the lpr mutation in the Fas gene19 (Supplementary Figure S2b). Despite this small amount of Fas expression, Lpr T cells remained totally resistant to FasL-induced cell death (Supplementary Figure S2d). These data indicate that Fas and RICD in mouse CD4+ T cells is largely confined to TEM phenotype CD4+ T cells directly after isolation, but that conversion to TEM takes place more freely in mouse T cells after activation through the TCR and expansion in IL-2.
The differential sensitivity of CD4+ T-cell subsets to Fas-induced apoptosis may be regulated by cell-intrinsic survival factors, such as bcl-2 family members, or more proximally, at the Fas signaling complex. Flow cytometry revealed similar levels of bcl-2 and bcl-xL, two pro-survival members of the bcl-2 family,20 in activated human naïve, memory, TCM and TEM cells (Supplementary Figure S3a). The BH3 protein Bim triggers apoptosis of effector T cells after cytokine deprivation and is critical for the contraction phase of T cells after an acute immune response.21, 22 We tested the ability of naïve and memory T-cell subsets to undergo apoptosis after deprivation of IL-2 and found that all died with similar kinetics (Figure 5a). A previous study implicated reduced activity of AKT and higher levels of Bim in the increased sensitivity of TEM to apoptotic stimuli.23 However, we did not find reduced phosphorylated AKT or FOXO3a in TEM, and found slightly lower levels of Bim in TEM compared with other subsets (Supplementary Figure S3b). Additionally, apoptosis in response to anti-Fas (Figure 5c) or anti-CD3 (Figure 5d) was not dampened in the absence of Bim siRNA, which produced a 60–80% knockdown of Bim protein and mRNA (Figure 5b). The PI3-kinase inhibitor LY294002, also did not sensitize cells to Fas-mediated apoptosis (Supplementary Figure S4a) or spontaneous cell death (Supplementary Figure S4b) in the different CD4 subsets. Thus, the resistance of TCM and activated naïve T cells to apoptosis triggered by Fas or the TCR does not appear to depend on the activity of the PI3-kinase or AKT kinase pathways or the expression of Bim, which is regulated by these pathways.
To determine whether efficient Fas-induced apoptosis induction in TEM is coupled to early events in signal transduction, we studied the recruitment and processing of FADD and caspase-8, components of the Fas DISC,24 in activated T cells derived from memory or naïve CD4+ T cells. Recruitment of FADD to the DISC was severely reduced in naïve compared with memory T cells (Figure 6a). Recruitment of the p51/53 caspase-8 proenzyme was also significantly reduced in naïve T-cell preparations, and cleavage of caspase-8 into the p41/43 forms was impaired in the DISC of naïve T cells. Importantly, even weak bivalent uncrosslinked anti-Fas mAb (APO1-1) treatment results in FADD recruitment and significant caspase-8 cleavage (Supplementary Figure S5a). The lack of caspase-8 cleavage in the APO1-1-treated cells can be seen more clearly in the overexposure (Supplementary Figure S5a, lower inset). In cells derived from purified TCM and TEM, FADD recruitment and caspase-8 cleavage was reduced in TCM, especially at later time points (Supplementary Figure S6).
c-FLIP, a protein containing tandem DED domains and a pseudocaspase domain, can compete with caspase-8 for recruitment to the Fas DISC and inhibit activation of caspase-8 in the signaling complex when present at high levels relative to caspase-8.25, 26 Analysis of the Fas signaling complex for the presence of c-FLIP revealed increased levels of processed c-FLIPL (long isoform) as well as the short isoform, c-FLIPS in the DISC of memory versus naïve and TEM versus TCM subsets. Total levels of c-FLIPL and c-FLIPS were also higher in these Fas-sensitive subsets (Supplementary Figure S7). Therefore, c-FLIP is unlikely to regulate the assembly of the DISC in these experiments.
Measurement of caspase-8 p18 (cytosolic active caspase-8) and poly-ADP ribose polymerase (PARP) cleavage, which depends on mitochondrial permeability transition,27 revealed that Fas-resistant naïve T cells failed to produce the p18 subunit or cleaved PARP after Fas stimulation. Additionally, there was delayed production of these cleavage products after treatment with crosslinked anti-Fas mAb (Supplementary Figure S8). Thus, the reduced DISC activity seen in naïve T cells after Fas stimulation is likely the limiting factor in production of active caspase-8 and subsequent activation of effector caspases and resultant cell death.
We have previously shown that efficient Fas-induced apoptosis depends on the localization of Fas to glycosphingolipid and cholesterol enriched, low-density lipoprotein fractions of the plasma membrane, termed 'lipid rafts'.28 Cells in which Fas is prelocalized to lipid rafts, such as 'type I' cell lines and T cells after stimulation through the TCR, are sensitive to apoptosis induced by bivalent anti-Fas, APO-1-1. Type II T-cell lines have little or no Fas present in lipid rafts and require stimulation with at least a dimer of Fas trimers or membrane-bound FasL to undergo apoptosis.28, 29 To test this paradigm, we stimulated activated naïve and memory CD4+ T cells with anti-Fas APO-1-1 or crosslinked anti-Fas (APO-1-3 plus anti-IgG3), and determined Fas-induced apoptosis. Naïve or TCM cells were consistently resistant to crosslinked and bivalent anti-Fas as well as FasL-LZ, although naïve T cells began to be sensitive to higher doses of crosslinked anti-Fas (Figure 7a). Density gradient fractionation of detergent lysates revealed a prominent amount of approximately 20% of the total Fas localized to fractions 3 and 4 containing lipid rafts in activated T cells derived from memory, but not in naïve T cells (Figure 7b). Enrichment of Fas in lipid rafts was specific, because the kinase lck, which localizes constitutively to lipid rafts, exhibited a similar partitioning into lipid rafts in T cells derived from purified naïve and memory precursors (Figure 7b). Additionally, use of filipin, a polyene antibiotic known to bind cholesterol,30 revealed no differences between naïve and memory T cells (Supplementary Figure S9a). Therefore, Fas appears to preferentially traffic to lipid raft microdomains in memory T cells and in TEM, and this correlates with higher sensitivity to Fas-induced apoptosis.
Localization of Fas to lipid raft microdomains requires palmitoylation of cysteine 199 in the juxtamembrane region of Fas, and treatment with the palmitate analog 2-bromopalmitate (BrPA) reduces the fraction of Fas in lipid raft microdomains and the efficiency of Fas-induced apoptosis in cell lines.31, 32 To determine if Fas localization to lipid rafts is required for efficient Fas signaling, we treated T cells with BrPA followed by stimulation with bivalent or crosslinked anti-Fas mAb. Naïve activated T cells, which were relatively resistant to apoptosis induced by either bivalent or crosslinked anti-Fas, were not further protected from Fas-induced apoptosis by BrPA, consistent with the low amounts of Fas localizing to lipid raft microdomains in these cells. Interestingly, in naïve cells, BrPA treatment increased sensitivity to apoptosis induced by anti-Fas mAb (Figure 7c) but not FasL (Supplementary Figure S9b). This may indicate that in naïve T cells, under certain conditions, Fas outside of lipid rafts may preferentially activate an apoptotic pathway, as occurs with TNFR1.33
BrPA was able to significantly protect memory T cells from apoptosis induced by bivalent, but not crosslinked anti-Fas (Figure 7c). This difference may be due to extrinsic crosslinking overcoming the reduced efficiency of Fas apoptosis signaling outside of lipid rafts. Importantly, BrPA protected T cells from FasL-induced apoptosis, with the greatest protection occurring in the highly Fas-sensitive TEM (Supplementary Figure S9b), indicating that lipid raft localization may be important for apoptosis induced by the physiological ligand for Fas. Taken together, these data show that localization of Fas to lipid raft microdomains accounts for at least part of the enhanced sensitivity of memory T cells and TEM to Fas-induced apoptosis.
These results show that Fas-dependent apoptosis is highly restricted to cells with an effector memory phenotype in both mouse and human CD4+ T cells. Restriction of Fas-induced apoptosis to TEM could be demonstrated both with anti-Fas antibodies and a preparation of FasL stabilized with an isoleucine zipper (FasL-LZ). Apoptosis triggered through the TCR was also restricted to TEM and was dependent on Fas, as TEM from ALPS patients and Lpr mice were protected from RICD. Remarkably, TCM were as resistant to Fas and RICD as cells harboring Fas mutations from patients with ALPS. Once activated, the sensitivity of these T-cell subsets to Fas-mediated apoptosis correlated with their surface phenotype. Human T cells with a central memory (CCR7+/CD27+) phenotype remained relatively resistant to TCR and Fas-mediated apoptosis, while murine T cells largely converted to a TEM phenotype and became sensitive to Fas. The relative resistance of activated naïve and TCM cells to TCR and Fas-induced apoptosis may allow expansion of these cells, which is critical for efficient T-cell immune responses. Restraining expansion of TEM through Fas-mediated apoptosis may be important in preventing immunopathology and expansion of T cells that can provide help for autoreactive B cells.
We have found a similar hierarchy in sensitivity to anti-Fas-induced cell death as Riou et al.23 However, under these conditions, neither Bim levels nor phosphorylation of AKT and FOXO3a contribute to apoptosis sensitivity induced by Fas, TCR or cytokine deprivation in activated T cells. Other studies have found Bim making little or no contribution to Fas-mediated apoptosis in CD4+ T cells.6, 34, 35 Instead, we have found that sensitivity to TCR and Fas-induced apoptosis is regulated by the efficiency of Fas signal transduction, namely the assembly and activation of the Fas-associated DISC. FADD and caspase-8 recruitment was reduced in naïve versus memory and TCM versus TEM and the recruited caspase-8 was also processed inefficiently in Fas-resistant T cells. Despite the known role of c-FLIP in regulating caspase-8 recruitment and activation in the DISC, we did not find increased total or DISC-associated c-FLIP in Fas-resistant T cells. Recent work has described a novel mechanism of caspase-8 activation via polyubiquitination in the TRAIL DISC.36 Whether a similar mechanism regulates Fas DISC efficiency in TEM is not known. Overall, susceptibility of TEM to the extrinsic cell death pathway may help regulate these potentially dangerous cells in a number of environments.
A number of findings reported here support the notion that the presence of Fas in lipid raft microdomains enhances Fas apoptosis signaling in primary T-cell subsets. A significantly higher fraction of Fas occurs in lipid rafts of memory compared with naïve T cells. TEM resemble T cells that have been restimulated through the TCR in their sensitivity to apoptosis even with low levels of Fas oligomerization.28 However, the fact that naïve T cells, unlike type II tumor cells such as Jurkat, were resistant to apoptosis induced even by highly crosslinked anti-Fas mAb indicates that Fas resistance in these T-cell subsets involves mechanisms beyond simply localization to lipid rafts.
These results also have a number of diagnostic and therapeutic clinical implications. Multi-parameter gating, used here, allows measurement of cell death in samples directly ex vivo, dispensing with T-cell expansion in culture. In samples with complex mixtures of cell types, this method should enable more accurate diagnostic tests for cell death defects especially in ALPS or other diseases linked to defective T-cell apoptosis. Elimination of antigen-specific T cells through administration of antigen or antigen-bearing cells in a non-immunogenic manner has been proposed as a therapeutic strategy for a number of autoimmune diseases and allograft rejection.37, 38 In spontaneous autoimmune diseases, particularly systemic lupus erythematosus, TEM have been reported to be abnormally expanded,10 perhaps because TCR signaling that normally triggers RICD is altered.39 The hierarchy of apoptosis sensitivity we have described here might be disrupted in these patients, allowing pathological expansion of TEM. Understanding the mechanisms that underlie the more efficient assembly of the Fas DISC in TEM may allow the development of therapeutic strategies that sensitize T cells to antigen and TCR-mediated apoptosis in diseases mediated by autoreactive T cells. This work also predicts that Fas ligand might be used therapeutically to specifically eliminate TEM in diseases in which these cells cause pathology.
Antibodies were obtained from Kamiya (Seattle, WA, USA) (Apo1-1), Alexis Biochemicals, Enzo Life Sciences (Plymouth Meeting, PA, USA) (Apo1-3, cFLIP NF6), eBiosciences (San Diego, CA, USA) (anti-CD3 OKT3), Santa Cruz (Santa Cruz, CA, USA) (FasC20, Lck) BD Pharmingen (San Diego, CA, USA) (FADD), Cell Signaling (Danvers, MA, USA) (p-FoxO3a, FoxO3a, AKT, p-AKT, PARP, C-8 1C12) and Stressgen, Enzo Life Sciences (Plymouth Meeting, PA, USA) (Bim). C-8 (C15 clone) was from Alexis and anti-FasL (Nok2) was from BD. FasL-LZ is derived from overexpression of the extracellular region of FasL tagged with isoleucine zipper and Flag tag construct in 293T cells and purification of the protein on a Flag column. Stealth select siRNA (medium GC control and Bim) was obtained from Invitrogen (Carlsbad, CA, USA) and targets all isoforms of human Bim.
Peripheral blood leukocytes (PBLs) were obtained from normal donors in the Department of Transfusion Medicine, Clinical Center, NIH, under the Institutional Review Board (IRB) approved protocol. Peripheral blood from patients with ALPS Type Ia germline Fas mutations were obtained from NIAID clinical center under the IRB approved protocol number: 93-I-0063. For direct ex vivo apoptosis assays, CD8+ T cells were depleted by MACS separation and the PBL were incubated with Fas agonists, anti-Fas antibodies or FasL-LZ for 6–8h. A multi-parameter FACS analysis was used to delineate the levels of Fas-induced apoptosis within various CD4+ T-cell subsets. Cells were stained with CD4 and CD45RA to differentiate between naïve (CD4+CD45RA+) and memory (CD4+CD45RA−) CD4+ T cells (Figure 1a). Surface receptors CCR7 and CD27 on memory T cells demarcate central (TCM, CD4+CD45RA− CCR7+/CD27+) or effector memory (TEM, CD4+CD45RA− CCR7−/CD27−) cells (Figure 1a). Co-staining was done with annexin for 20min and analyzed by FACS. Cell death was calculated using the formula ((1–% live-treated cells)/(% live-untreated cells)), wherein cells that remain annexin negative are considered as live cells. For assays on activated T cells derived from ALPS patients, PBMCs were activated with concanavalin A (2.0μg/ml, SIGMA, St Louis, MO, USA) for 48h and expanded in RPMI medium containing IL-2 (100IU/ml) for 3–5 days. Apoptosis assays were done as above. Mouse CD4+ T cells were isolated from 6- to 8-week-old wild-type (C57BL/6) or lpr mice using a CD4+ T-cell isolation kit (Miltenyi Biotec, Auburn, CA, USA) and sorted for naïve (CD62Lhi/CD44lo), central memory (CD62Lhi/CD44hi) and effector memory (CD62Llo/CD44hi) cells. For the ex vivo assays, cells were directly incubated on anti-CD3-coated plates or treated with FasL-LZ for 6–8h and analyzed for apoptosis using annexin/PI staining. Sorted cells were activated on plate-bound CD3/28 (5μg/ml) for 3 days and expanded in RPMI medium containing IL-2 (50IU/ml) for another 5 days for the in vitro activated cell assays. All cell death assays were done in triplicates for each sample.
For lipid raft separation Optiprep density centrifugation preparations were used as described previously.28 For depletion of palmitoylated Fas, activated naïve and memory cells were cultured for 18h in the presence or absence of 100μM 2-bromopalmitate, then treated with anti-Fas antibody and multi-parameter flow cytometry was used to detect apoptosis as described above. For analysis of the DISC, 107 activated naïve, memory, TCM or TEM cells were stimulated with either uncrosslinked (1μg/ml) or crosslinked (0.2μg/ml) anti-Fas antibodies and lysed in 1ml of DISC lysis buffer. Immunoprecipitation was done with protein G beads (Pierce, Rockford, IL, USA) O/N at 4°C. The beads were lysed with SDS buffer and immunoblotted against Fas, FADD, C-8 and cFLIP.
We would like to thank Jagan Muppidi for construction of the FasL-LZ vector, Mike Lenardo, John Ashwell and Josh Farber for critical reading of the paper, Andy Snow for providing Bim siRNA, Jim Simone and Jeff Lay for assistance in cell sorting. This research was supported (in part) by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on Cell Death and Differentiation website (http://www.nature.com/cdd)
Edited by H-U Simon
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


text:
Endothelial nitric oxide synthase gene polymorphisms and cardiovascular damage in hypertensive subjects: an Italian case-control study
- Authors
- Journal
- Immunity & Ageing 1742-4933
- Publisher
- Springer (Biomed Central Ltd.)
- Publication Date
- Volume
- 5
- Issue
- 1
- Identifiers
- Source
- Legacy
- Keywords
- Disciplines
1742-4933-5-4.fm ral ss BioMed CentImmunity & Ageing Open AcceResearch Endothelial nitric oxide synthase gene polymorphisms and cardiovascular damage in hypertensive subjects: an Italian case-control study Daniela Colomba1, Giovanni Duro2, Salvatore Corrao1, Christiano Argano1, Tiziana Di Chiara1, Domenico Nuzzo2, Federica Pizzo2, Gaspare Parrinello1, Rosario Scaglione*1 and Giuseppe Licata1 Address: 1Department of Internal Medicine, University of Palermo, Italy and 2Institute of Biomedicine and Molecular Immunology CNR, Palermo, Italy Email: Daniela Colomba - [email protected]; Giovanni Duro - [email protected]; Salvatore Corrao - [email protected]; Christiano Argano - [email protected]; Tiziana Di Chiara - [email protected]; Domenico Nuzzo - [email protected]; Federica Pizzo - [email protected]; Gaspare Parrinello - [email protected]; Rosario Scaglione* - [email protected]; Giuseppe Licata - [email protected] * Corresponding author Abstract Background: Nitric oxide (NO) synthesized by endothelial nitric oxide synthase (eNOS) plays an important role in regulation of endothelial function and in the control of blood pressure. However, the results from some studies on the association between three clinically relevant eNOS gene polymorphisms (G894T, T786C and intron 4b/a) and essential hypertension are unclear. We designed a case-control study to evaluate the influence of eNOS polymorphisms on target organ damage in 127 hypertensives and 67 normotensives. Clinical evaluation, biochemical parameters, Urinary Albumin Excretion (UAE) and echocardiogram were performed to characterize target organ damage. eNOS polymorphism were recognized by PCR method. Results: The distribution of eNOS genotypes was similar in hypertensives and normotensives but 4aa was present in the 2.5% of hypertensives and completely absent in normotensives. Subjects with 4bb, G894T, and T786C genotypes showed an increased prevalence of target organ damage. Moreover prevalence of G894T and