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County News
Freeholders Host Fourth of July Freedom Festival at the Camden Waterfront
(July 7, 2015) - The Board of Freeholders helped celebrate America's independence at the 2015 Camden Waterfront Freedom Festival, which featured n...Read More
Mosquito Spraying in Five Towns
(July 7, 2015) - The Camden County Mosquito Commission regularly checks several thousand suspected mosquito breeding sites across the county. Spraying...Read More
Check Out Over 800 New Titles at Camden County Library System
(June 2, 2015) - The public can find hundreds of new titles that have been added to the Camden County Library System's collections by going...Read More
Holtec International Breaks Ground in Camden City
(June 2, 2015) - Creating the bedrock for a rebirth in Camden City and County, elected leaders from the region and state gathered at the South Je...Read More
Construction Completed on Broadway Bridge
(July 2, 2015) - The reconstruction of the Broadway Bridge (CR 551) connecting Gloucester City and Brooklawn is completed and all lanes of the sp...Read More
Mosquito Spraying in Four Towns
(July 1, 2015) - The Camden County Mosquito Commission regularly checks several thousand suspected mosquito breeding sites across the county. Spraying...Read More
New Residential Development Transforms Blighted Commercial Site
(July 1, 2015) - The Camden County Improvement Authority (CCIA) and Conifer Real Estate Development and Management Company celebrated the opening and ...Read More
Reading Skills Instructor Wins Top Teaching Award at CCC
(June 30, 2015) - A professor whose career has been devoted to helping underserved students learn literacy skills has won the top instructional award ...Read More
Freeholder Board Keeps Cooling Centers Open to the Public
(June 26, 2015) - While the cleanup continues in Camden County the Office of Emergency Management and the Health Department still have cooling centers...Read More
Closures Continue, Freeholders Exploring Disaster Proc
(June 25, 2015) - Over the last 48 hours Camden County crews continue to cleanup and assess damage from Tuesday night's super storm. The Freehol...Read More


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TweakTown Posts - Page 5
Save 65% On A Nexus 5 With Unlimited Talk and Text
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Continue reading 'Save 80% on Two Years of Lightning-Fast Internet' (full post)
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Celebrate Star Wars Day 2015 in true Jedi fashion with an exhilarating 'May the Fourth Be With You' gamer bundle. The four-game pack is just $12.50 at TweakTown Deals and includes:
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Bigger isn't always better... but in the case of Apple's gorgeous Thunderbolt display, it definitely is. So you'll be glad to hear that we're giving one away at TweakTown Deals.
The Thunderbolt allows you to turn your MacBook Air or Pro into a seriously powerful desktop, complete with a microphone, quality speakers, and an HD camera. Your laptop experience will never be the same, as you explore a much wider visual spectrum.
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"Did an excellent job, was able to see 3 hours after the first surgery of the right eye. Everyone was very helpful! Had to change the 2 surgery dates, no problem," was able to accommodate different arrival times from the original schedule. I have a pair of sun glasses from when I was in the
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Local Articles in Delta Junction
Health Care Facilities And Service reviews in Delta Junction
I think they are pretty good hospital. I am not sure they want to take a lot of drugs. I did not take them. I got out of the hospital and then I mowed the lawn which I was not supposed to do and I got really weak. They have a rehab thing where you walk on treadmills and they check your blood pressure. You do that until you get your strength back. My hemoglobin was way down and they gave me blood transfusions. That was a month after I had the heart attack. Our family decide to use that one because it as a good one. They have different clinics in conjunction with them so they have your record which is nice because you "
We trust his expertise and he has always been accurate, professional and thorough. We trust him with our lives. We wish all physicians were as good as he is. He is an MD, not DO, and I do not have knowledge of anyone else in the practice."
I would say they were all a 'B' experience. It's fantastic staff but the doctors seem to be rushed. They seemed to be rushed and some of the staff seem to be rushed. The professionalism is great, I think it is fine it is just like a lot of facilities, understaffed."
People come from all over the country to go to this hospital. They are just fantastic. They extended the life of my mother, who was a cancer patient there. They are very kind and positive people. They also have a lot of health and wellness activities available."
Considering a stroke, she was taken immediately and cat scanned immediately. I was amazed about how many people they had conferring. They had anywhere from 6 to 12 others conferring in another room. She only waited 4-5 hours to get a room. It was a small room but I was pleased with it overall. The nurses were very friendly and their assistants all had great bedside manner."
They have everything, every department were imaginable, he does have to go here and there I mean it's a good hospital. They're clean, friendly, the nurses seemed to know their jobs, they worked hard and their very nice and I liked it. I was knocked out totally and they had to have heavy dry I don't remember anything about it. I liked them, everyone there has always been very nice and all of my doctors are there and I got through. I think they're good doctors and they were personable, they're all our family. If you have a problem they fixed it. They send you this monthly statement, this is not a bill okay? and they would tell you how much like I'm on medicare and I have that private insurance so they pay what you just paid. They let you know about what you're going to pay.
I have been visiting since 2008.They connect with you well while speaking to them. My niece also visits and most of my experience with the hospital is through her for her medical problems. The staff here has helped her out tremendously. One of the nurse practitioners suggested my niece have gastric bypass surgery and she is a whole new person now. Her life has turned around and she has fewer issues now because of their help. The entire staff is very friendly and makes you feel welcome. I have not really had any difficulties with scheduling."
My first encounter with the "bad booby gods". "
Health Care Facilities And Service Providers in Delta Junction, AK
Companies below are listed in alphabetical order. To view top rated service providers along with reviews and ratings, Join Angie's List Now!
A Place For Mom
701 Fifth Avenue
Advante Members Only
140 E 28 th St
409 Senna Ave
Allegiant Group Inc
189 W Clarkston Rd
Allsup Inc
300 Allsup Pl
530 7th Ave, Suite 3
ARCI Enterprises, LLC
1313 St Regis Ct
Bassett Army Community Hospital
4076 Neely Rd
Fort Wainwright
Fort Wainwright
Best Medical Alert LLC
8310 Allison Pointe Blvd
Blais, Dr. Kendrick D.
1867 Airport Way
Bruno N. - Health Insurance Broker
1600 N Saba St
Careington Care 500 Plan
7400 Gaylord Pkwy
Chena Obstetrics & Gynecology
1919 Lathrop St
CoPatient Inc.
1505 SW 6th Ave
Costa Rica Medical Tourism Services
5025 Marina Cove Dr
Delford Roth DO
N8069 County Rd F
Embrace Pet Insurance
4530 Richmond Rd
Employee Screening Management
104 Colt square Ste 2
EMS Sentry, Inc.®
435-B Applegrove St.,NW
Fairbanks Memorial Hospital
1650 Cowles St
Fairbanks Sam's Club
48 College Rd
1998 Ruffin Mill Rd
Fred Meyer Pharmacy
3755 Airport Way
1568 Spring Hill Rd
510 North AVE
Health Benefit Consultants LLC
20275 Honeysuckle Dr.
JG McGinness Foot Orthotics
1445 Dekalb St
PO Box 342108
Lifenet Alert Systems LLC
15 Bryant Ave
LifeWatch USA
266 Merrick Rd
Medical Care Alert
43334 W. Seven Mile Road
1818 Market Street
1055 Gibson Rd
7609 140Th Pl NE
Mr. Rooter of Fairbanks
875 Old Richardson Hwy
North Pole
North Pole
Paternity Express
P.O. Box 390182
3900 E Sprague
S.A.D. Fitness
Delta Junction
Senior Health Direct
719 E. Grand River Ave.
Simpson Sales Academy
5650 Colleyville Blvd
Stables, Dr. Janice L.
1867 Airport Way
Tanana Valley Clinic
1001 Noble St
Test Me DNA Fairbanks
1606 23rd Ave
The Calorie Clinic LLC
309 Range Rd
The Standing Company
5848 Dixie Hwy
The WalkEZstore.com
2309 West Smith Valley Road
Trinity Medical Pharmacy
9332 St Rd 54 Ste 203
United Health Care
PO Box 30555
Valued Relationship Inc
1400 Commerce Ctr Dr
Wal-Mart Automotive
537 Johansen Expy
Walmart Supercenter
537 Johansen Expy
Whiter Image Dental
670 Endeavor Circle
Join Angie's List to get the best local reviews in Delta Junction.
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text: Home Recipes Course Desserts Pie Recipes Banana Pie Recipes Share: Banana Pie Recipes Looking for recipes for banana pie? Taste of Home has the best banana pie recipes from real cooks like you, featuring reviews, ratings, how-to videos and tips. Recipe Spotlight Chocolate & Peanut Butter Pudding Pie with Bananas I created this pie in tribute to Elvis, who was my favorite entertainer, and to Hershey, Pennsylvania, the town where I was born. —Penny Hawkins, Mebane, North Carolina View Chocolate & Peanut Butter Pudding Pie with Bananas Recipe More Banana Pie Recipes Sort: Highest Rated Alphabetical Newest < prev | 1 | 2 | 3 | 4 White Chocolate Banana Pie White Chocolate Banana Pie I developed this recipe based on a popular pie served at an Atlanta restaurant. Everyone looks forward to this dessert at the annual Christmas party we host on our farm. Chocolate curls as a garnish are a nice contrast to the creamy white pie. View Recipe Banana Berry Tarts Banana Berry Tarts If you need a no-fuss dessert that will please kids but is elegant enough for a grown-up gathering, try this dessert. Marshmallows, banana and raspberries are stirred into the creamy treats for a fun dinner finale.—Barbara Nowakowski, North Tonawanda, New York View Recipe Banana Chocolate Cream Pie Banana Chocolate Cream Pie English toffee bits add a buttery crunch to this cool and creamy layered pie created by our Test Kitchen. View Recipe Advertise with us ADVERTISEMENT Bananas Foster Pie Bananas Foster Pie I love peanut butter with banana, so it only made sense to me to create a pie with that combination. I also used the flavors of banana's foster to create this delectable pie.—Emily Hobbs, Ozark, Missouri View Recipe Cherry Banana Cream Pie Cherry Banana Cream Pie From Hanover, Ontario, Denise Elder shares this dreamy dessert. "The crunchy crust is spread with a rich butter layer, then topped with a fluffy filling flavored with banana, cherries and chocolate," she says. "Guests always tell me the pie reminds them of a banana split...and then ask for seconds. View Recipe Banana Cream Meringue Pie Banana Cream Meringue Pie I grew up on a farm in Alberta, and I still remember my mom's pies, fresh from the oven for supper. This creamy banana variety is so good, no store-bought version can compare! View Recipe Banana Fudge Pie Banana Fudge Pie This dessert, which is like a banana sundae, is both light and good. I make it often. View Recipe Banana Split Pie Banana Split Pie How to Thaw Cool-Whip Whipped Topping: The best way to thaw frozen COOL WHIP Topping is in its container in the refrigerator. Depending on the size of the tub you have, the thawing time varies. Recommended thawing times are: 8-oz. tub-4 hours; 12-oz. tub-5 hours; 16-oz. tub-6 hours. We DO NOT recommend thawing COOL WHIP Topping in the microwave. View Recipe Mini Banana Cream Pies Mini Banana Cream Pies Mini pies are all the rage, and this cool and luscious banana cream pie is no exception. Whether it's just the two of you or a party of five, savor this classic dessert without the fear of wasteful leftovers. —Carol Maertz, Spruce Grove, Alberta View Recipe Quick Creamy Banana Pie Quick Creamy Banana Pie "I tried a lot of different banana pie recipes before I found one that could be modified into a low-fat version with great results," writes Martha Domeny of San Diego, California. View Recipe < prev | 1 | 2 | 3 | 4 Advertise with us ADVERTISEMENT Advertise with us ADVERTISEMENT


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Original recipe yields 12 servings
Amount per serving ({{servings}} total)
Based on a 2,000 calorie diet
Ready In
This was a very good recipe. The only thing I recommend is to first cook the mushrooms and onions in the butter for a while. Then add the rest of the ingredients. We found we liked it this wa...
I cannot see at all why this recipe is rated so highly. It turned out to be a flavorless flour-based gravy instead of a mushroom sauce. It was way too thick--I ended up using 50% more stock than...
Excellent topper for grilled pork chops!! I too sauteed the onions in the butter first, then added the shrooms for a minute or so, then added the other ingredients. MMMM-MMM GOOD. Thanks Sara!


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Reducing Prison Suicides
An examination of prison staff experience informed the Harris Review of self-inflicted deaths amongst young adult prisoners.
RAND Europe's policy research focuses on eight specific areas. By establishing new angles of enquiry and, in some cases, new methodologies for research, we bring fresh approaches and add valuable contributions to traditional as well as new RAND research areas.


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Gay magazines tend to be packed with celebrity, hunks, and (maybe) some politics.
When we get to the theater, the ticket vendor fans herself, swooning at the sight of all the hunks.
Oh, I think there are plenty of hunks left on Game of Thrones.
1813, "large piece cut off," possibly from West Flemish hunke (used of bread and meat), which is perhaps related to Dutch homp "lump, hump." Meaning "attractive, sexually appealing man" is first attested 1945 in jive talk (in Australian slang, it is recorded from 1941).


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May We Suggest
This blog provides customized book recommendations to our patrons. To get your own, just fill out the May We Suggest form and you can expect results within 10 days. You can also like May We Suggest on facebook.
Bird Books for the Young at Heart
Emu by Claire Saxby ; illustrated by Graham Byrne
Nest by Jorey Hurley
Flamingos by Melissa Gish
Parrots over Puerto Rico by Susan L. Roth and Cindy Trumbore ; collages by Susan L. Roth
Do you really want a bird? by Bridget Heos ; illustrated by Katya Longhi
Have you heard the nesting bird? by words by Rita Gray ; pictures by Kenard Pak
Mama built a little nest by Jennifer Ward ; illustrated by Steve Jenkins
Parenting an Athlete
Parenting young athletes: developing champions in sports and life by Frank L. Smoll and Ronald E. Smith
Concussions and our kids: America's leading expert on how to protect young athletes and keep sports safe by Robert Cantu and Mark Hyman
Parenting an athlete by Annette Reiter
Playing to win: raising children in a competitive culture by Hilary Levey Friedman
Fun and games: 100 sport-related activities for ages 5- 16 by Anthony Dowson, Keith E.J. Morris
The most expensive game in town: the rising cost of youth sports and the toll on today's families by Mark Hyman
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Michigan Mysteries
Check out these great mysteries for kids, written by authors from right here in Michigan!
Mike's mystery by created by Gertrude Chandler Warner ; illustrated by Dirk Gringhuis
The baseball mystery by [Carole Marsh]
The vengeance of the witch-finder by John Bellairs ; completed by Brad Strickland ; frontispiece by Edward Gorey
Blue Bay mystery by created by Gertrude Chandler Warner ; illustrated by Dirk Gringhuis
Mystery explosion! by Johnnie Tuitel and Sharon Lamson
People of Literature
A home for Mr. Emerson by Barbara Kerley ; illustrated by Edwin Fotheringham
Leaving China: an artist paints his World War II childhood by James McMullan
The Brontë sisters: the brief lives of Charlotte, Emily, and Anne by Catherine Reef
The trouble begins at 8: a life of Mark Twain in the wild, wild West by Sid Fleischman
Reporter in disguise: the intrepid Vic Steinberg by Christine Welldon
Kids it's Time for an Adventure!
Up [videodisc] by Walt Disney Pictures presents a Pixar Animation Studios film
Rio [videodisc] by Twentieth Century Fox Animation presents a Blue Sky Studios production
Madagascar [videodisc] by produced by Teresa Cheng, Mireille Soria ; directed by Eric Darnell, Tom McGrath ; written by Mark Burton, Billy Frolick, Eric Darnell, Tom McGrath
Ratatouille [videodisc] by Walt Disney Pictures presents a Pixar Animation Studios film
Fresh Recipes
The kitchen gardener's handbook by Jennifer R. Bartley
Fresh grilling: 200 delicious good-for-you seasonal recipes by Better Homes and Gardens ; project writer, Lisa Kingsley
Fresh: recipes for enjoying ingredients at their peak by [editor, Jan Miller]
Fresh from the farmers' market [electronic resource]: year-round recipes for the pick of the crop by Janet Fletcher ; photographs by Victoria Pearson ; introduction by Alice Waters
Everyday food: fresh flavor fast: 250 easy, delicious recipes for any time of day by from the kitchens of Martha Stewart Living
Detective Manga
Death note. Vol. 1, Boredom by story by Tsugumi Ohba ; art by Takeshi Obata ; [translation & adaptation, Pookie Rolf ; touch-up art & lettering, Gia Cam Luc]
Case closed. Vol. 1 by Gosho Aoyama
Arisa. Vol. 1 by Natsumi Ando ; translated and adapted by Andria Cheng ; lettered by North Market Street Graphics
Young Miss Holmes. Casebook 1-2 by Kaoru Shintani
Lizzie Newton: Victorian mysteries, Vol. 1 by story, Hey-jin Jeon ; art, Ki-ha Lee ; [translation, Lauren Na ; adaptation, Janet Houck ; lettering & design, Nicky Lim]
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Beginning Chapter Book Series to Love
Cam Jansen: the mystery of the U.F.O. by David A. Adler ; illustrated by Susanna Natti
Clementine by Sara Pennypacker ; with illustrations by Marla Frazee
Mercy Watson to the rescue by Kate DiCamillo ; illustrated by Chris van Dusen
Junie B. Jones and the stupid smelly bus by Barbara Park ; illustrated by Denise Brunkus
Bad Kitty gets a bath by Nick Bruel
Horrid Henry by Francesca Simon ; illustrated by Tony Ross
Teach Your Cat Tricks
The cat behavior answer book: practical insights & proven solutions for your feline questions by Arden Moore ; illustrated by Matt Ambre ; foreword by Nancy Peterson
The cat whisperer: why cats do what they do— and how to get them to do what you want by Mieshelle Nagelschneider
I just got a kitten. What do I do?: how to buy, train, understand, and enjoy your new kitten by Mordecai Siegal ; photographs by Ginger S. Buck
The cat owner's problem solver: how to manage common behavior problems by thinking like your cat by Margaret H. Bonham
Getting started: clicker training for cats by Karen Pryor
You can train your cat: secrets of a master cat trainer by Gregory Popovich
Slimming Down
The diet fix: why diets fail and how to make yours work by Yoni Freedhoff, MD, CCFP, Dip. ABOM
The 20/20 diet: turn your weight loss vision into reality: 20 key foods to help you succeed where other diets fail by Phil McGraw
The biggest loser boot camp: the 8-week get-real, get- results weight-loss program by the experts and trainers of the Biggest Loser
The ultimate diet revolution: your metabolism makeover by Jim Karas
The beauty detox power: nourish your mind and body for weight loss and discover true joy by Kimberly Snyder, C.N.
The diet trap solution: train your brain to lose weight and keep it off for good by Judith S. Beck, Ph.D., and Deborah Beck Busis, LCSW


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3/14/15 Lo Tide Run- Team LIB- We are
a top Sponsor!! Carolina Beach
Blue Ribbbon Run Autumn Hall
Low TIde Pub- A Night Under the Stars Low Tide Pub
6/13/15 2nd Annual Susan S. Allen Memorial Golf Tournament
Clayton NC
Will post more information soon about these events.
You can register for the Lo Tide Run at http://shop.lotiderun.org/. Don't forget to select team Love is Bald!!!


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Sporting Gifts
eSportsonline has great sports gifts and ideas for men, women and children. Give her the gift of fitness and fun with indoor and outdoor games and sporting equipment. For kids we have jumbo beach balls & more to provide them with hours of excitement and activity. Shop today for speedy next day shipping!
Razor E100 Electric Scooter Sweet Peaeach
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Razor E300 Electric Scooter Greyeach


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The Only EKG Book You'll Ever Need
Malcolm S Thaler, "The Only EKG Book You'll Ever Need"
Lippincott Williams Wilkins | 2009 | ISBN: 1605471402 | 336 pages | File type: CHM | 14,4 mb
Now in its Sixth Edition, this popular, practical text presents all the information clinicians need to use the EKG in everyday practice and interpret hypertrophy and enlargement, arrhythmias, conduction blocks, pre-excitation syndromes, and myocardial infarction. It is an ideal reference for medical students in ICM courses, house officers, or anyone directly involved in patient care, whether student, teacher, or practitioner.
Summary: Good for beginners or wanting to learn more
Rating: 5
I am a paramedic and love this book only because it helped with school and still has some good information for reference. This book isn't your solve all problems book but it is a great book to teach you and get you started on understanding ECG rhythms and will help you in the future of your medical career. This book helped our class out a lot only because our paramedic books only went over the minimal requirements and most of us wanted to learn more and advance our knowledge and this book did in fact help. Again if you are learning ECG and already know it and want to learn more into it than this book is for you. I have this book sitting on my shelf in case I even forget something or see something new and want to know why it happened or what it means. Good reference material.
Summary: The only EKG Book You'll ever need
Rating: 5
The title speaks for the book! Definitely a great buy! If you are a nurse you need this book!!
[Fast Download] The Only EKG Book You'll Ever Need
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1884 1C One Cent, Judd-1721, Pollock-1929, R.5, PR65 NGC....
2007 Milwaukee, WI (ANA) Signature Coin Auction #444
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Flashy 1884 Annular Cent, Judd-1721, PR651884 1C One Cent, Judd-1721, Pollock-1929, R.5, PR65 NGC. The first of three examples of this unusual annular pattern for the cent, struck in nickel. Apparently these patterns were well-preserved as the two major services have together certified 13 in PR65 with 23 finer (7/07). The fields on this piece are highly polished. There is a slightly depressed area surrounding the hole, most likely caused by the pressure of the punch that removed the center. Mostly brilliant, there are a couple of spots of color on the obverse (date side). (PCGS# 62150)
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text: community Meet Patti Hogan, featured in Soapbox about her efforts to improve her East Price Hill neighborhood By Mark Heyne • Apr 9, 2015 Provided, Soapbox Media, by Scott Beseler Listen Listening... / 18:55 One of Cincinnati's oldest neighborhoods, Price Hill, was once praised as the city's most popular and distinctive suburb. Commonly divided into East Price Hill, West Price Hill, and Lower Price Hill, the area covers over 6 square miles. Like many neighborhoods, Price Hill has experienced its ups and downs, but its residents are proactive in targeting problems and working together to make their community a better place to live. The new People's Liberty wants to know how you'd use $100k to make our city better By Mark Heyne • Aug 11, 2014 Listen Listening... / 24:25 New Life in Over the Rhine By Mark Perzel • Aug 24, 2012 Mark Perzel shares a few minutes with Ensemble Theatre of Cincinnati's Artistic Director D. Lynn Meyers talking not just about their upcoming season, but the current renaissance in the theater's Over the Rhine neighborhood.


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Amazon has just issued a firmware update (5.3.3) for the Kindle Paperwhite. Leaving your device's wireless enabled will eventually install it automatically. If you can't wait, go here
The Ultimate HCG Diet Cookbook is available free at the moment right here.
Amazon has also rolled out a new 'rental' service. A few books are available for a month's rental fee of about half the retail price. Keep it more than a month and you are charged more until after six months when you owe them full price. I don't think I will be taking advantage of that option.
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Some broker implementations require creating a copy the message forwarding it over to the backend. The broker also might slightly modify things like addressing headers etc. on the message for proper message routing within the DMZ. The problem is that we see a very high CPU cost in creating this copy message and this also results in lower throughput. Note: Streamed transfer mode is not in scope for this article.
For all performance issues we need to measure and profile and to investigate this issue we initially try to simulate the pattern of the broker by just copying over the message and then forwarding it to a backend dummy service. We then take profiles of this to understand how much the actual cost of copying is.
An 8% cost for copying seems to be acceptable considering the value of making the copy and able to do other things if required. But then again this was not what is being observed. In the profiles from the actual broker we notice about 40% cost for creating a copy. This means that almost half the time is spent in creating a message copy. So effectively your throughput would almost drop to half when the broker is configured to create a copy of the message. This is excluding costs like logging etc. Evidently our simulation is not accurate so we need to isolate this further. We take in more functionality from the broker so that we hit this expensive path. One of the key observations was that the message is copied just before it is being forwarded. This also means that there are a bunch of manipulations that was done on the message and in our simulation we didn't perform any manipulation. So to get this closer we need to probably change some things on the message. To keep it simple we did something like removing some header and adding another header to the message since most brokers modify headers before forwarding it over.
int headerIndex = input.Headers.FindHeader(header.Name, header.Namespace);
if (headerIndex >= 0)
Eureka!! We observed our throughput went down and this was in line with what we were seeing in our broker. So we can see that CreateMessage and CreateBufferedCopy have increased in cost quite a bit.
So this was performance data we collected.
Copy and Forward
Copy forwarding with new header
98.6 %
98.7 %
Now that we have identified the root cause we also need to identify the solution so that the broker can achieve the functionality without taking up so much CPU.
The solution is actually quite simple "Modify your message after you create the buffered copy". I wanted give the solution before the analysis since most of you would probably not be interested in the analysis but if you are then the rest would be interesting.
The most common way to create a copy your message is using Message.CreateBufferedCopy(int).
If headers have been modified then CreateBufferedMessage takes an alternative path using the DefaultMessageBuffer. The reason is that a fully copy of the message has to be created if any buffered header has been modified. An internal property called headers.ContainsOnlyBufferedMessageHeaders is used to distinguish if the faster BufferedMessageBuffer can be used to create the buffered copy or not. If there are any modified headers then this means we need to assure that the message is fully marshaled over and the buffer itself cannot be copied(e.g. the user can add a reference type to the header) and so we fall back to a path that would fully reparse the message and create a fully deserialized copy of the modified message. The main point here is a copy should always be a deep copy and any kind of modification should not result in a message with shallow copied message parts. When you copy and create a message from the original then your message objects get its own copy of headers that it can play around with without affecting the original incoming message. Message copy by itself is a fast operation as you can see from the above profile and copying a modified message can be very CPU intensive when using buffered transfer mode.


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Shrouded in Silence - eBook
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Wired, Tribulation Survival Series #1Robert L. WiseHachette Books / 2004 / Trade Paperback$23.99Availability: Usually ships in 24-48 hours.CBD Stock No: WW6691631
Publisher's Description
Author Bio
Father Robert (Robert L. Wise, PhD) is both a priest and an archbishop in the Communion of Evangelical Episcopal Churches. He is the author of thirty-two published books and the ghost author of six published novels, including The Death of Innocence, the parents story of the death of JonBenét Ramsey. Father Roberts books have been translated into Spanish, Dutch, Chinese, Japanese, and German. He has made numerous television appearances discussing his adventures in researching his stories.
Product Reviews
Frances StoneAge: Over 65Gender: female4 Stars Out Of 5February 17, 2012Frances StoneAge: Over 65Gender: femaleQuality: 4Value: 4Meets Expectations: 3I AM ENJOYING THIS EBOOK BECAUSE IT HAS A CHRISTIAN THEME AND IS AN EXCITING STORY.
LAFrederickAge: 25-34Gender: female2 Stars Out Of 5December 26, 2011LAFrederickAge: 25-34Gender: femaleQuality: 2Value: 2Meets Expectations: 2I found the book to be sort of over the top which made it extremely predictable, unbelievable and very, very dull! I'm very disappointed as it started off so well.
RShafferNorwalk, OhioAge: 45-54Gender: male5 Stars Out Of 5Fantastic storyNovember 17, 2011RShafferNorwalk, OhioAge: 45-54Gender: maleQuality: 5Value: 5Meets Expectations: 5This book was amazing!!! Twists and turns. Great characters and a brilliant story!!!
livinginozAge: 45-54Gender: female4 Stars Out Of 5October 7, 2011livinginozAge: 45-54Gender: femaleQuality: 4Value: 5Meets Expectations: 4Great book, I really enjoyed the read. Would love to see more books like this on the free ebook download page!
SkyPilotCanadaAge: 55-65Gender: male4 Stars Out Of 5Good Writing Sabotaged by Bad EditingMay 27, 2011SkyPilotCanadaAge: 55-65Gender: maleQuality: 3Value: 5Meets Expectations: 4This story is gripping and will hold your attention until the end. If you are a fan of Mr. Wise, you will remain one after reading this story. If this is your first meeting with the author, you will be tempted to read more of his works.
Good as it is though, this book suffers from a major failing on the part of the editorial staff responsible for bringing it to its published form.
The repetition of the same word in successive sentences is frowned upon in fiction publishing circles and is something that good editing is intended to remedy. Ex.: "Bandages covered most of his face with red seepage along the side of his chin. She only saw one eye still closed, the other was covered by bandages. A thick bandage covered the top of his chest with with plastic tubes running down the side to bags on the rail. A bag of glucose hung from a rack..." (Note that the words "bandage" and "bag" are repeated in close proximity in a span of three sentences.)
There also seems to be a misunderstanding of the use of the words 'past' and 'passed' throughout and other places, in the Ebook, at least, where words are missing.


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Although relative quantification of real-time RT-PCR data can provide valuable information, one limitation remains the selection of an appropriate reference gene. No one gene has emerged as a universal reference gene and much debate surrounds some of the more commonly used reference genes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). At this time, no gene encoding for a plasma membrane protein serves as a reference gene, and relative quantification of plasma membrane proteins is performed with genes encoding soluble proteins, which differ greatly in quantity and in targeting and trafficking from plasma membrane proteins. In this work, our aim was to identify a housekeeping gene, ideally one that codes for a plasma membrane protein, whose expression remains the same regardless of drug treatment and across a wide range of tissues to be used for relative quantification of real-time RT-PCR data for ATP binding cassette (ABC) plasma membrane transporters.
In studies evaluating the expression levels of two commonly used reference genes coding for soluble proteins and two genes coding for membrane proteins, one plasma membrane protein, plasma membrane calcium-ATPase 4 (PMCA4), was comparable to the two reference genes already in use. In addition, PMCA4 expression shows little variation across eight drug-treated cell lines and was found to be superior to GAPDH and HPRT1, commonly used reference genes. Finally, we show PMCA4 used as a reference gene for normalizing ABC transporter expression in a drug-resistant lung carcinoma cell line.
We have found that PMCA4 is a good housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters and receptors.
Relative quantification for real-time RT-PCR requires the use of a reference gene for normalization [1]. An "ideal" reference gene should be expressed at similar levels in different cell types and under various treatment conditions. Many commonly used reference genes or "housekeeping" genes do not always possess these two necessary attributes. Several commonly used housekeeping genes were originally used as references for more qualitative assays such as Northern blots and conventional RT-PCR, and their use in quantitative RT-PCR was not originally re-evaluated[2]. Therefore, the selection of the appropriate reference gene for performing relative quantification has been a topic of much discussion and evaluation [1,3,4]. This becomes more critical when examining the mRNA expression of membrane proteins, since many of the frequently used reference genes are soluble proteins, which differ significantly in total amount and in targeting and trafficking from membrane proteins.
Membrane proteins are a major component of the human genome, as they comprise nearly 30% of the total human genome [5]. These proteins are also involved in approximately 85% of cell signalling pathways. The biogenesis of membrane proteins and cytosolic proteins varies greatly. At transcription, specific mRNA-binding proteins are added to nascent mRNA to ensure RNA integrity and to oversee RNA export from the nucleus, subcellular localization, translation and stability [6]. Membrane localization of mRNA has been reported in membrane proteins, and this localization may facilitate cotranslational import of some membrane transporters as is seen in Atm1, a yeast ABC transporter of the inner mitochondrial membrane, and Ist2, a yeast plasma membrane putative ion channel [7]. Investigators have also shown that mRNAs that encode for secreted or membrane proteins are preferentially found in membrane-bound polysomes [8]. The translation of secretory, integral membrane proteins as well as that of luminal and membrane proteins of the endoplasmic reticulum (ER) take place on membrane-bound ribosomes of the ER [9], whereas the synthesis of cytosolic proteins occurs on the ribosomes within the cytoplasm. Polytopic membrane proteins have multiple membrane spanning domains, and they are believed to cotranslationally incorporate into the membrane of the ER with the assistance of the translocon [10].
An important superfamily of membrane proteins is the ATP-binding cassette (ABC) transporter family. Several ABC transporters are linked to disease conditions in humans including cystic fibrosis, Stargardt disease, Dubin-Johnson syndrome, Pseudoxanthoma elasticum and adrenoleukodystrophy [11]. In addition, the overexpression of ABC transporters is the predominant cause of multidrug resistance in cancer [12], and understanding the triggers for overexpression of these proteins upon exposure to various drug regimens would help researchers find ways to circumvent multidrug resistance. Therefore, a reliable reference gene that does not change following drug treatment is vital for the relative quantification of ABC transporter gene expression. In this work, our goal was to identify a housekeeping gene, ideally one that codes for a plasma membrane protein, whose expression remains the same regardless of drug treatment and across a wide range of tissues for normalization of real-time RT-PCR data for ABC transporters.
We hypothesized that plasma membrane proteins could function as reference genes and that a plasma membrane reference gene would be most appropriate for relative quantification of membrane proteins consisting of 4–12 membrane spanning helices otherwise known as polytopic membrane proteins. As mentioned previously, membrane protein biogenesis, in particular for polytopic membrane proteins, involves several steps that are specific to membrane proteins; therefore, a reference gene encoding for a protein which undergoes similar process of biogenesis would be best to use as a reference measurement for gene expression. First of all, to evaluate this hypothesis, two commonly used soluble protein reference genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyl transferase 1 (HPRT1), and two commonly expressed plasma membrane proteins, plasma membrane calcium-ATPase 4 (PMCA4) and the alpha subunit of the Na+ /K+-ATPase (ATP1A1), were evaluated using real-time RT-PCR with a variety of human tissue RNA samples. PMCA4 and ATP1A1 were chosen because both are ubiquitously expressed polytopic plasma membrane proteins. We determined that the consistency of PMCA4's expression profile was comparable to that of the commonly used reference genes across a panel of tissues. We then compared these four genes in eight drug-treated cell lines, and PMCA4 was found to be superior to GAPDH and HPRT1. Finally, we used PMCA4 to normalize ABC transporter expression from real-time RT-PCR for drug-resistant lung carcinoma cells.
Results and Discussion
PMCA4 shows comparable expression to GAPDH and HPRT1 in various tissues
To determine the optimal concentration of total RNA to use within our studies, the efficiency of the RT-PCR reaction was evaluated over a concentration range. The standard curve for two primers for the detection of HPRT1 and PMCA4 indicates that the efficiency is nearly 2 (Fig. 1). Two hundred nanograms of total RNA was found to be within the linear range of the reaction, and this quantity was used to evaluate the primers for this and all subsequent studies comparing the four genes.
Figure 1. Standard curve to evaluate efficiency of RT-PCR reaction. The HPRT1 (blue circle) and PMCA4 (red diamond) primers were evaluated over a range of total RNA concentrations (30 ng-300 ng total RNA). The average crossing point values are given for the n = 4, and the error bars represent ± SD.
To evaluate the use of ATP1A1 or PMCA4 as reference genes, their expression patterns for a variety of human tissue RNA samples were compared to those of two commonly used reference genes, GAPDH and HPRT1. The average crossing point values determined by real-time RT-PCR for the 4 housekeeping genes across different tissue samples with standard deviation are shown in Fig. 2. The selected tissue types are known to express ABC transporters at moderate to high levels. GAPDH and HPRT1 showed the least amount of variation for the panel of tissues tested (Mean = 22.24 and variance = 1.26; Mean = 24.75 and variance = 1.64, respectively). However, similar to GAPDH and HPRT1, PMCA4, which encodes a plasma membrane calcium pump that removes bivalent calcium ions from cells against very large concentration gradients [13-16], is consistently expressed in this varying panel of tissues (Mean = 21.17 and variance = 1.87). ATP1A1, the alpha subunit of the plasma membrane Na+ /K+ ATPase [17], shows a greater variance in expression compared to either PMCA4 or the commonly used reference genes (Mean = 20.84 and variance = 2.09). This suggests that unlike PMCA4, ATP1A1 is not the best choice for tissue samples as a reference gene. Further statistical analysis using a one-sided Fisher variance-ratio, comparing the variance for the PMCA4 samples to the variance for each other gene samples separately, indicates that there is no statistically significant difference (p > 0.05) between PMCA4 expression and that of the other genes (ATP1A1 p = 0.383; GAPDH p = 0.187; and HPRT1 p = 0.356). The variances for the ATP1A1 samples were also compared to the variances for the GAPDH and HPRT1 samples; the p values were 0.118 and 0.252, respectively. Additional studies with cell culture lines from other tissue types generated similar results (Table 1). The F-test for the cell culture lines did indicate that the variance of the ATP1A1 expression profile is statistically different than that of PMCA4 (p = 0.041), whereas there is no statistically significant difference between the variances of the PMCA4 and the GAPDH samples (p = 0.093) or the variances of the PMCA4 and the HPRT1 samples (p = 0.074). There was also a statistically significant difference between the variance of ATP1A1 and those of GAPDH and HPRT1 (both p values = 0.001). This suggests that PMCA4 is comparable to two established reference genes, GAPDH and HPRT1, while ATP1A1 is not.
Table 1. Comparison of Housekeeping Gene Expression in Various Cell Linesa
Figure 2. Quantification of commonly expressed genes encoding soluble or membrane proteins in various human tissue samples using real time RT-PCR. The expression levels of two polytopic membrane protein genes (panel A and B) and two soluble protein genes (panel C and D) in eight human tissue samples known to express ABC transporters were evaluated using RT-PCR. A) ATP1A1; B) PMCA4; C) GAPDH; and D) HPRT1. The error bars represent standard deviation (n = 4).
PMCA4 expression is unchanged under a variety of treatment conditions
Reference genes must be expressed at similar levels in different cell types and more importantly must remain consistent under various treatment conditions. Our previous results address tissue expression of PMCA4. In an attempt to further examine the utility of PMCA4 as a reference gene; its expression was evaluated in 8 drug-treated cell lines. RNA was isolated from four drug-treated sublines and from the drug-sensitive parental cells, MCF-7 and KB-3-1. The MCF-7 (human breast adenocarcinoma) sublines were the following: MCF-7/ADR [18], MCF-7/VP16 [19], MCF-7FL-1000 [20], and MCF-7-ADRVP [21]. The KB-3-1 (human cervical squamous cell carcinoma) sublines were selected with either doxorubicin (KB-A1), vinblastine (KB-V1) or colchicine (KB-C1 and KB-8.5-11) [22]. These drug-selected cell lines are known to overexpress ABC transporters at the mRNA level. We also re-evaluated ABC transporter expression levels within these lines using RT-PCR to confirm that overexpression was present (data not shown). The average crossing point values are given for the different cell lines in Fig. 3. Interestingly, in this analysis, GAPDH, a commonly used reference gene, performed poorly under these conditions (Mean = 17.24 and variance = 1.21), and PMCA4 expression was the most consistent across the various high dose drug treatments (Mean = 21.4 and variance = 0.29). HPRT1 showed slightly more variation than PMCA4 (Mean = 20.77 and variance = 0.41). Again, the results for ATP1A1 (Mean = 19.49 and variance = 0.55) show that the variance is nearly two-fold larger than that for PMCA4. This taken together with the results from the tissue and cell line expression studies suggests that ATP1A1 is not suitable as a reference gene.
Figure 3. Comparison of reference gene expression in drug-resistant cell lines. The two parental cell lines, MCF-7 and KB-3-1, and several of their drug-resistant subclones were evaluated using RT-PCR. The drug-resistant MCF-7 cell lines were as follows: MCF-7/ADR (doxorubicin-treated)[18]; MCF-7FL-1000 (flavopiridol-treated)[20]; MCF-7/VP16 (etoposide-treated) [19]; and MCF-7-ADRVP (adriamycin- and verapamil-treated)[21]. The KB cell lines were KB-A1 (doxorubicin-treated); KB-V1 (vinblastine-selected) and KB-C1 and KB-8.5-11 (colchicine-selected)[22]. A) ATP1A1; B) PMCA4; C) GAPDH; and D) HPRT1. The error bars represent standard deviation (n = 4).
PMCA4, on the other hand, was found to be superior to all other reference genes tested in this study. An F-test was used to individually compare the variance of the PMCA4 samples to the variance of the samples for the other genes. The variances for ATP1A1 and GAPDH, p = 0.027 and 0.00001, respectively, were statistically different than the variance for PMCA4. No statistical difference was found between PMCA4 and HPRT1, p = 0.146. From these results, we first identified a reference gene which was foremost, a membrane protein and which would also be consistent for all cell lines examined, regardless of drug selection conditions. PMCA4 was chosen as the reference gene for our subsequent work because of the convincing data from these studies. To our knowledge, this is the first report of the use of a gene encoding a membrane protein for normalization of real time RT-PCR data for polytopic membrane transport proteins.
PMCA4 is a good reference gene for polytopic plasma membrane proteins
We have shown that PMCA4 expression is consistent and that it should be used as a reference or housekeeping gene for membrane proteins. To demonstrate the use of PMCA4 for normalizing the ABC transporter expression profile for multidrug resistant cells, we evaluated parental COR-L23P and drug-resistant COR-L23R cells. COR-L23R was the only doxorubicin-resistant cell line reported to overexpress the ABC transporter, ABCC1, by step-wise selection using high doses of doxorubicin. Further characterization of multidrug resistance-linked ABC transporter gene expression has not been reported to date for the COR-L23R cells. Crossing point values for each transporter were normalized to the respective crossing point values for PMCA4. Data are presented as a fold change in gene expression for the drug-resistant cell line compared to the parental cells using the delta delta Ct method. Surprisingly, our results show that only this ABC transporter is highly overexpressed in the resistant cells. COR-L23R overexpresses ABCC1 (> 90-fold) with doxorubicin-selection compared to parental COR-L23P cells (Fig. 4), and no differences were found between COR-L23P and COR-L23R expression of PMCA4 (Cp = 21) at 300 ng of total RNA per reaction. However, if GAPDH was used as the reference gene, this fold change would be greatly inflated, since the crossing point values between COR-L23P (Cp = 18) and COR-L23R (Cp = 20) vary by 2 crossing point values. Alternatively, the mean expression value for HPRT1 for the COR-L23P cells was 19.8 while that of the COR-L23R cells was 20.1. Although these differences in Cp values appear to be minimal, the calculated fold change would be highly amplified when either GAPDH or HPRT1 was used with the delta-delta Ct method (Table 2).
Figure 4. Evaluation of selected ABC transporter expression in lung carcinoma COR-L23 (parental and resistant) cell lines. A) The fold change in expression in COR-L23R cells compared to the parental cell line is depicted for the gene expression of ABC transporters. Two individual determinations of crossing point values are given. B) Western blotting with MRPr1 antibody (1:1000) shows that only COR-L23R cell lysates express ABCC1[27]. Lane 1, 20 μg of COR-L23P; lane 2, 10 μg of COR-L23R; and lane 3, 20 μg of COR-L23R. C) C219 (1:1000) was used to detect ABCB1[28]. Lane 1, COR-L23P (100,000 cells); lane 2, COR-L23R (100,000 cells); and lane 3, positive control, KB-A1 (100,000 cells). D) ABCG2 was detected with BXP-21 (1:1000)[29]. Lane 1, COR-L23P (100,000 cells); lane 2, COR-L23R (100,000 cells); and lane 3, positive control, MCF-7-FL1000 (100,000 cells). E) Functional ABCC1 is expressed in COR-L23R cells as determined by calcein-AM efflux assays. Brown solid line histogram, COR-L23P; blue solid line histogram, COR-L23P with 20 μM MK-571; red solid line histogram, COR-L23R control; and green solid line histogram, COR-L23R with 20 μM MK-571. The results from a typical experiment are shown in the panel. Similar results were obtained on two additional experiments.
Table 2. Evaluation of ABCC1 Fold Change Using Different Housekeeping Genesa
No other ABC transporter in our panel (consisting of the 12 ABC transporters linked to multidrug resistance) was overexpressed, and only the transporter which was previously reported remained the ABC transporter conferring resistance as seen by Western blotting (Fig. 4C and 4D). ABCC1 was not detectable in the parental COR-L23P cells, as seen in this Western Blot. Calcein-AM efflux assays using MK-571, the typical ABCC1 inhibitor, further verified that COR-L23R cells overexpress functional ABCC1 (Fig. 4E). These results are consistent with previous reports on the drug resistance profiles of these cells [23].
In this work, we identified a consistent housekeeping gene, PMCA4, for plasma membrane proteins which was comparable to other established reference genes when examined in a diverse panel of tissues and cell lines. Our work investigated the effects of drug treatment and tissue type on the selection of a housekeeping gene; additional work evaluating other conditions is currently under way. PMCA4 was also found to be superior to these reference genes, since it showed less variation regardless of drug treatment for the KB-3-1 and MCF-7 cells. We also thoroughly characterized the ABC transporter gene expression profile for a multi-step doxorubicin-selected cell line using PMCA4 as the reference gene. Overexpression of ABCC1 was verified in the COR-L23R cells at both the mRNA and protein levels. Although we have found that PMCA4 works quite well for the described conditions, as for all known housekeeping genes, there are conditions where alternative housekeeping genes may work best. For PMCA4, other investigators reported that increased Pi levels in cattle resulted in increased PMCA4 levels; neuronal development was also linked to increased PMCA4 levels [24,25]. PMCA4 appears to be a suitable housekeeping gene for normalization of gene expression for polytopic membrane proteins including transporters, ATPases and receptors.
Chemicals and other reagents
Acetoxy-methyl ester of calcein (calcein-AM) was purchased from Molecular Probes (Eugene, OR). Doxorubicin was purchased from Calbiochem (San Diego, CA). RT-PCR reagents were purchased from Roche Applied Sciences (Indianapolis, IN). RNA from various tissue types examined in Fig. 2 was purchased from Clontech (Mountain View, CA).
Cell Lines
The human large-cell lung tumor line COR-L23P and its doxorubicin-selected MRP1-overexpressing multidrug-resistant variant COR-L23R were a generous gift from Margery A. Barrand (Department of Pharmacology, University of Cambridge, Cambridge, UK) [23]. Both COR-L23P and COR-L23R cells were cultured in RPMI 1640 medium with 10% (v/v) fetal calf serum supplemented with 100 units of penicillin/streptomycin/ml (Invitrogen) at 37°C in 5% CO2 humidified air. The COR-L23R cells were also maintained in the presence of 0.2 μg/ml doxorubicin.
The cervical epidermal carcinoma cell line KB-3-1 and its drug resistant subline, KB-A1, were generous gifts of Michael M. Gottesman (Laboratory of Cell Biology, National Cancer Institute, NIH, Bethesda, MD) [22], and the MCF-7 breast cancer cell line and the subline MCF-7/ADR were a generous gift of Kapil Mehta (MD Anderson Cancer Center, Houston, TX) [18]. These cell lines were cultured in DMEM with 10% (v/v) fetal calf serum supplemented with 2 mM glutamine and 100 units of penicillin/streptomycin/ml (Invitrogen) at 37°C in 5% CO2 humidified air. The drug resistant subline KB-A1 was kept under constant selection in 1 μg/ml doxorubicin while 0.5 μg/ml doxorubicin was added to the MCF-7/ADR cells every other passage.
RNA Isolation
RNA was isolated from cells grown in 6-well plates to characterize reference gene expression in all cell lines and to characterize ABC transporter expression in the parental COR-L23P and drug-resistant COR-L23R cell lines. In these studies, 6-well plates were seeded at 200,000 cells per well for each cell type, and RNA was isolated following 72 hr of growth. Drug-resistant cell lines were plated with the appropriate concentration of drug during the 72 hr period. The medium and any detached cells were removed from the wells. RNA isolation was performed on the cells that remained attached using the Qiagen RNeasy kit (Valencia, CA) as per the manufacturer's protocol with a 15 minute on-column DNAse incubation step. RNA samples were isolated in triplicate. The pure RNA was quantified using the Nanodrop ND-1000 Spectrophotometer (Wilmington, DE). The integrity of the RNA was verified using the Agilent 2100 Bioanalyzer (Palo Alto, CA) with the Eukaryote Total RNA Nano assay. The RNA samples were stored at -80°C until needed.
Quantitative RT-PCR
Real-time quantitative RT-PCR was used to measure the mRNA expression levels of the four reference gene candidates and the selected ABC transporters. The LightCycler RNA Master SYBR Green Kit and LightCycler II or LightCycler 480 instrument (Roche Biochemicals, Indianapolis, IN) were utilized in these studies. Primers for these genes were designed using the Roche Probe Design 2 software. All primer sets were tested prior to use in this work to ensure that only a single product of the correct size was amplified for all primer sets. The primer set for PMCA4 was designed to a region which is common to each of the splice variants and does not differentiate between them. (The list of primers is given in Table 3).
Table 3. List of Primers used for RT-PCR
Two hundred nanograms of total RNA with 250 nM primers were used to evaluate the primers for the studies comparing the four reference genes. The LightCycler RNA Master SYBR Green Kit and LightCycler 480 instrument (Roche Biochemicals, Indianapolis, IN) were utilized in these studies to determine which of the four reference genes would be the most consistent across tissue types and treatment conditions. The average crossing point values for 4 housekeeping genes across different tissue samples with standard deviation were determined, and the RT-PCR conditions were as described in the manufacturer's protocol for the RNA Master SYBR Green kit. Negative controls consisting of no-template (water) reaction mixtures were run with all reactions.
The RT-PCR reaction for ABC transporters was performed on 300 ng total RNA with 250 nM specific primers under the following conditions in the LightCycler II: reverse transcription (20 min at 61°C), one cycle of denaturation at 95°C for 30 seconds, and PCR reaction of 45 cycles with denaturation (15s at 95°C), annealing (12s at 58°C), and elongation (30s at 72°C with a single fluorescence measurement). The PCR reaction was followed by a melting curve program (65–95°C with a heating rate of 0.1°C per second and a continuous fluorescence measurement) and then a cooling program at 40°C. Negative controls consisting of no-template (water) reaction mixtures were run with all reactions. PCR products were also run on agarose gels to confirm the formation of a single product at the desired size. Crossing points for each transcript were determined using the 2nd derivative maximum analysis with the arithmetic baseline adjustment. Crossing point values for each transporter were normalized to the respective crossing point values for reference gene plasma membrane calcium ATPase 4. Data are presented as a fold change in gene expression for the drug resistant cell lines compared to the parental cells using the delta delta Ct method.
Western Blotting
For the Western blotting assays, cells were harvested and the cell lysates were prepared from drug-resistant and parental cells using a lysis buffer containing 10 mM Tris, pH 8.0, 1% Triton X 100, 2 mM DTT, 1% aprotinin, 1 mM AEBSF, and DNase. Following brief sonication and the addition of SDS, equivalent numbers of cells were loaded and separated by 7% NuPAGE gels. The electrophoressed proteins from the gel were transferred to a nitrocellulose membrane and probed with the appropriate primary antibodies specific for the ABC protein of interest. Immunoblotting was performed using the C219 (1:2000) antibody for ABCB1, MRPr1 (1:1000) (Alexis) for ABCC1, Anti-BCRP (1:1000) for ABCG2, and the anti-mouse IgG-horseradish peroxidase (HRP) conjugated (1:10000) secondary antibody. HRP-dependent luminescence was developed using Western lighting chemiluminescence reagent plus (PerkinElmer, Wellesley, MA). The blot was exposed to Hyperfilm within a Hypercassette for various times. The intensity of bands on the blot was quantitated using a scanner and ImageQuant software (Piscataway, NJ).
Assay of transport of fluorescent substrates by flow cytometry
A FACSort flow cytometer equipped with Cell Quest software (Becton-Dickinson, Franklin Lakes, NJ) was used for FACS analysis. Briefly, 300,000–500,000 cells were harvested after trypsinization by centrifugation at 500 × g and then resuspended in IMDM supplemented with 5% Fetal Bovine Serum. 0.25 μM calcein-AM was added to the cells in 4 ml of IMDM in the presence or absence of a specific inhibitor for the ABC transporter evaluated. For example, 20 μM MK571 was used to specifically inhibit ABCC1. The cells were incubated in a water bath at 37°C in the dark for 10 minutes for a calcein-AM efflux assay prior to being pelleted by centrifugation at 500 × g. The cell pellet was then resuspended in 300 μl of PBS containing 0.1 % BSA and then analyzed immediately using the flow cytometer as described previously [26].
Comparisons between the expression profiles for PMCA4 and each of the other genes was performed using a one-tailed F-test two sample for variances, with a confidence level of 0.05.
ABC, ATP binding cassette; calcein-AM, calcein acetoxy-methyl ester; MRP, multidrug resistance protein; MK-571, (3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-(dimethyl amino-3-oxo propyl)thio)methyl)thio)propanoic acid; PAGE, polyacrylamide gel electrophoresis; Pgp, P-glycoprotein; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; HPRT, Hypoxanthine phosphoribosyl transferase 1; PMCA4, Plasma Membrane Calcium- ATPase 4; ATP1A1, Na+ /K+-ATPase
Authors' contributions
AMC performed the RT-PCR studies and drafted the manuscript. KJC assisted AMC with these studies. CPW evaluated the COR-L23 cells by Western blotting and flow cytometry. SVA supervised the studies and finalized the manuscript. All authors read and approved the final manuscript.
We thank Dr. Michael M. Gottesman for his encouragement and helpful discussions. We also thank Dr. Margery Barrand (University of Cambridge, UK) for the gift of COR-L23 cell lines, Dr. Susan Bates (NCI, NIH, Bethesda, MD) for providing the MCF-7 cell lines and Dr. Kapil Mehta (MD Anderson Cancer Center, Houston, TX) for the MCF-7 and MCF-7/ADR cell lines. We thank Dr. Zuben Sauna for his helpful suggestions and George Leiman for his editorial assistance. This research was supported by the Intramural Research Program of the National Cancer Institute, NIH, Center For Cancer Research. AMC was supported by the NIGMS Pharmacology Research Associate (PRAT) Program.
Bustin SA: Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems.
J Mol Endocrinol 2002, 29:23-39. PubMed Abstract | Publisher Full Text
Huggett J, Dheda K, Bustin S, Zumla A: Real-time RT-PCR normalisation; strategies and considerations.
Genes Immun 2005, 6:279-284. PubMed Abstract | Publisher Full Text
Dheda K, Huggett JF, Chang JS, Kim LU, Bustin SA, Johnson MA, Rook GAW, Zumla A: The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization.
Anal Biochem 2005, 344:141-143. PubMed Abstract | Publisher Full Text
Wong ML, Medrano JF: Real-time PCR for mRNA quantitation.
Biotechniques 2005, 39:75-85. PubMed Abstract
Stevens TJ, Arkin IT: Do more complex organisms have a greater proportion of membrane proteins in their genomes?
Proteins: Structure, Function, and Genetics 2000, 39:417-420. Publisher Full Text
Aguilera A: Cotranscriptional mRNP assembly: from the DNA to the nuclear pore.
Curr Opin Cell Biol 2005, 17:242-250. PubMed Abstract | Publisher Full Text
Jansen RP: mRNA localization: message on the move.
Nat Rev Mol Cell Biol 2001, 2:247-256. PubMed Abstract | Publisher Full Text
Diehn M, Eisen MB, Botstein D, Brown PO: Large-scale identification of secreted and membrane-associated gene products using DNA microarrays.
Nat Genet 2000, 25:58-62. PubMed Abstract | Publisher Full Text
Nicchitta CV: A platform for compartmentalized protein synthesis: protein translation and translocation in the ER.
Curr Opin Cell Biol 2002, 14:412-416. PubMed Abstract | Publisher Full Text
Turner RJ: Understanding the biogenesis of polytopic integral membrane proteins.
J Membr Biol 2003, 192:149-157. PubMed Abstract | Publisher Full Text
Gottesman MM, Ambudkar SV: Overview: ABC transporters and human disease.
J Bioenerg Biomembr 2001, 33:453-458. PubMed Abstract | Publisher Full Text
Gottesman MM, Fojo T, Bates SE: Multidrug resistance in cancer: role of ATP-dependent transporters.
Nat Rev Cancer 2002, 2:48-58. PubMed Abstract | Publisher Full Text
Stauffer TP, Guerini D, Carafoli E: Tissue distribution of the four gene products of the plasma membrane Ca2+ pump. A study using specific antibodies.
J Biol Chem 1995, 270:12184-12190. PubMed Abstract | Publisher Full Text
Guerini D, Garcia-Martin E, Zecca A, Guidi F, Carafoli E: The calcium pump of the plasma membrane: membrane targeting, calcium binding sites, tissue-specific isoform expression.
Acta Physiol Scand Suppl 1998, 643:265-273. PubMed Abstract
Brini M, Coletto L, Pierobon N, Kraev N, Guerini D, Carafoli E: A comparative functional analysis of plasma membrane Ca2+ pump isoforms in intact cells.
J Biol Chem 2003, 278:24500-24508. PubMed Abstract | Publisher Full Text
Oceandy D, Buch MH, Cartwright EJ, Neyses L: The emergence of plasma membrane calcium pump as a novel therapeutic target for heart disease.
Mini Rev Med Chem 2006, 6:583-588. PubMed Abstract | Publisher Full Text
Kuhlbrandt W: Biology, structure and mechanism of P-type ATPases.
Nat Rev Mol Cell Biol 2004, 5:282-295. PubMed Abstract | Publisher Full Text
Mehta K: High levels of transglutaminase expression in doxorubicin-resistant human breast carcinoma cells.
Int J Cancer 1994, 58:400-406. PubMed Abstract
Schneider E, Horton JK, Yang CH, Nakagawa M, Cowan KH: Multidrug resistance-associated protein gene overexpression and reduced drug sensitivity of topoisomerase II in a human breast carcinoma MCF7 cell line selected for etoposide resistance.
Cancer Res 1994, 54:152-158. PubMed Abstract
Robey RW, Medina-Perez WY, Nishiyama K, Lahusen T, Miyake K, Litman T, Senderowicz AM, Ross DD, Bates SE: Overexpression of the ATP-binding Cassette Half-Transporter, ABCG2 (MXR/BCRP/ABCP1), in Flavopiridol-resistant Human Breast Cancer Cells.
Clin Cancer Res 2001, 7:145-152. PubMed Abstract | Publisher Full Text
Lee JS, Scala S, Matsumoto Y, Dickstein B, Robey RW, Zhan Z, Altenberg G, Bates SE: Reduced drug accumulation and multidrug resistance in human breast cancer cells without associated P-glycoprotein or MRP overexpression.
J Cell Biochem 1997, 65:513-526. PubMed Abstract | Publisher Full Text
Shen DW, Cardarelli C, Hwang J, Cornwell M, Richert N, Ishii S, Pastan I, Gottesman MM: Multiple drug-resistant human KB carcinoma cells independently selected for high-level resistance to colchicine, adriamycin, or vinblastine show changes in expression of specific proteins.
J Biol Chem 1986, 261:7762-7770. PubMed Abstract | Publisher Full Text
Barrand MA, Heppell-Parton AC, Wright KA, Rabbitts PH, Twentyman PR: A 190-kilodalton protein overexpressed in non-P-glycoprotein-containing multidrug-resistant cells and its relationship to the MRP gene.
J Natl Cancer Inst 1994, 86:110-117. PubMed Abstract
Yamagishi N, Miyazaki M, Naito Y: The expression of genes for transepithelial calcium-transporting proteins in the bovine duodenum.
Vet J 2006, 171:363-366. PubMed Abstract | Publisher Full Text
Kip SN, Gray NW, Burette A, Canbay A, Weinberg RJ, Strehler EE: Changes in the expression of plasma membrane calcium extrusion systems during the maturation of hippocampal neurons.
Hippocampus 2006, 16:20-34. PubMed Abstract | Publisher Full Text
Hrycyna CA, Airan LE, Germann UA, Ambudkar SV, Pastan I, Gottesman MM: Structural Flexibility of the Linker Region of Human P-Glycoprotein Permits ATP Hydrolysis and Drug Transport.
Biochemistry 1998, 37:13660-13673. PubMed Abstract | Publisher Full Text
Muller M, Yong M, Peng XH, Petre B, Arora S, Ambudkar SV: Evidence for the role of glycosylation in accessibility of the extracellular domains of human MRP1 (ABCC1).
Biochemistry 2002, 41:10123-10132. PubMed Abstract | Publisher Full Text
Kartner N, Evernden-Porelle D, Bradley G, Ling V: Detection of P-glycoprotein in multidrug-resistant cell lines by monoclonal antibodies.
Nature 1985, 316:820-823. PubMed Abstract | Publisher Full Text
Maliepaard M, Scheffer GL, Faneyte IF, van Gastelen MA, Pijnenborg ACLM, Schinkel AH, van de Vijver MJ, Scheper RJ, Schellens JHM: Subcellular Localization and Distribution of the Breast Cancer Resistance Protein Transporter in Normal Human Tissues.
Cancer Res 2001, 61:3458-3464. PubMed Abstract | Publisher Full Text


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LDWF: Enforce Louisiana State Law & Ensure Tony The Tiger Is Released To A Reputable Sanctuary
by Tony's Friends · 49,571 supporters
Petition Update
Owner Taking Tony The Tiger Case To Lousiana Supreme Court
Tony's Friends
Sep 14, 2013 — July 2, 2013: Mr. Sandlin's lawyers filed an appeal to the Louisiana Supreme Court. ALDF Senior attorney Matthew Liebman said the Animal Legal Defense Fund will be filing its opposition to Morris' request. "The application is entirely without merit, yet another attempt by Mr. Sandlin to delay the inevitable and prolong Tony's inhumane captivity," he wrote in an email. http://freetonythetiger.wordpress.com/2013/07/05/truck-stop-tiger-case-headed-to-louisiana-supreme-court/
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747 commercial properties for sale found
£6,500,000 Guide Price
Guide Price £6,500,000
13,044 sq ft
Hotel for sale Queens Avenue, Muswell Hill, London
A substantial town house hotel consisting of 3 adjoining converted Victorian terraced houses totalling 19,561 sq ft GIA and 13,044 sq ft NIA. p> The property is arranged over 4 floors and comprises a large lobby area, 50 en-suite bedrooms and conference facilities for up to 180 peo... More details ›
Offers Invited
2.3 acres
Land for sale Southgate, Thaxted Road, Saffron Walden, Essex, CB10 2UP
RESIDENTIAL DEVELOPMENT SITE with Outline Planning Permission for a DEVELOPMENT OF UP TO 52 DWELLINGS. Saffron Walden is an HISTORIC AND THRIVING MARKET TOWN in north Essex in the district of Uttlesford, 15 miles north of Bishops Stortford, 18 miles south of Cambridge and 43 miles north of London. More details ›
£3,000,000 Offers in Excess of
Offers in Excess of £3,000,000
32,234 sq ft
Warehouse for sale 13 Waxlow Road, London, NW10 7NU
The property is situated on Waxlow Road which is accessed via Acton Lane. The 8, 794 sqft warhouse sits within this 0.74 acre site. The building is situated on a LARGE SECURED GATED SITE and benefits from dual gated access points. More details ›
£2,750,000 Offers in Excess of
Offers in Excess of £2,750,000
Mixed Use for sale The Rows, Harlow, CM20
FREEHOLD INVESTMENT AND DEVELOPMENT OPPORTUNITY IN TOWN CENTRE LOCATION More details ›
Offers Invited
52,272 sq ft
Residential Development for sale Long Acre Farm, Long Lane, Bovingdon, HP3 0NE
The property is located in a QUIET COUNTRY LANE approximately half a mile from the village of Bovingdon. The property currently comprises 5 commercial buildings and hardstanding, with FULL PLANNING PERMISSION for 4 detached houses. More details ›
18,000 sq ft
Warehouse for sale Unit 1 and 2, Kelvin Industrial Estate, Long Drive, Greenford, UB6 8WA
Almost 18,000 Sq Ft WAREHOUSE / OFFICE SPACES based in Greenford, West London. There are EXCELLENT TRANSPORT LINKS. 15 minutes to Central London and Heathrow Airport and a 10 minute walk to Greenford Tube Station. 15 CAR PARKING SPACES AVAILABLE. Fully refurbished within the last 10 years. More details ›
£1,925,000 Offers in Region of
Offers in Region of £1,925,000
38,433 sq ft
Light Industrial for sale Kenrich House, Elizabeth Way, Harlow, Essex, CM19
The property provides a detached 1960s LIGHT INDUSTRIAL WAREHOUSE / FACTORY and offices over ground, first and mezzanine level. The front of the building is over ground and first floors, and faces onto Elizabeth Way. It consists of offices, STORE ROOMS, training rooms and a LARGE FRONT RECEPTION. More details ›
£1,750,000 Guide Price
Guide Price £1,750,000
14,412 sq ft
Bar / Nightclub for sale The Rayners Village Way East Harrow, HA2 7HD
A4/A3/A2/A1 Or D1/B1. A prominent Art Deco period building located at the corner of Imperial Drive and Village Way making it highly visible. Built in 1937, the building retains many of its original features with a wealth of original panelling and fittings. Grade II Listed More details ›
16,000 sq ft
Light Industrial for sale Bearwalden Business Park, Royston Road, Wendens Ambo, CB11
This substantial range of manufacturing premises falls into two main buildings, being OFFICES and MANUFACTURING accommodation INCORPORATING WORKSHOP and LABORATORIES. PARKING is included. The property is ideally situated on this established business park. More details ›
Office for sale Freehold Offices, Lincolns Inn Office Village, Lincoln Road, Cressex Business Park, High Wycombe, HP12 3RE
This attractive office development comprises 12 modern terraced office buildings, arranged around a spacious, landscaped central courtyard. The offices benefit from a modern specification and are available to let or for freehold purchase. Lincolns Inn is able to offer an excellent parking allocat... More details ›
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