text:
Models and Frameworks for Implementing Evidence-Based Practice: Linking Evidence to Action
Jo Rycroft-Malone, Tracey Bucknall
John Wiley & Sons, May 10, 2010 - Medical - 268 pages
The Evidence-Based Nursing Series is co-published with Sigma Theta Tau International (STTI). The series focuses on implementing evidence-based practice in nursing and mirrors the remit of Worldviews on Evidence-Based Nursing, encompassing clinical practice, administration, research and public policy.
Models and Frameworks for Implementing Evidence- Based Practice: Linking Evidence to Action, looks at ways of implementing evidence gained through research and factors that influence successful implementation. It acknowledges the gap that exists between obtaining evidence and the practicalities of putting it into practice and provides direction to help to close this gap. This, the first book in the series, helps the reader to make decisions about the appropriateness of using various models and frameworks. A selection of models and frameworks are examined in detail including examples of their use in practice. The book concludes with an analysis and synthesis of the included models and frameworks.
The Models and frameworks that have been included are based on a number of criteria: that they are internationally recognised, have undergone widespread evaluation and testing, are transferable across different settings, and can be used by different disciplines. Models and frameworks include:
KeyĀ Points
Other titles in the Evidence-Based Nursing Series:
What people are saying - Write a review
We haven't found any reviews in the usual places.
Selected pages
doing the right thing for patients
Intended audience
Future plans for framework modifications
Stetler model
Other editions - View all
Models and Frameworks for Implementing Evidence-Based Practice: Linking ...
Jo Rycroft-Malone,Tracey Bucknall
No preview available - 2013
Common terms and phrases
achieving evidence-informed adoption Advanced practice nurses available research evidence barrier to implementing best available research Ciliska Clinical nurses specialists clinical practice Confirmation phase decision makers Decision phase Diffusion of Innovations Dobbins et al Engage stakeholders evaluating strategies evidence in decision evidence into practice Evidence-Based Nursing evidence-informed decision-making process example facilitating evidence-informed decision graduate students health care health care professionals health policies health professionals hypothesized Identify barriers implementation evidence Implementation phase Implementation Science implementing our model individuals and organizations informed decision infrastructure and support Innovations Theory Intended users interventions knowledge broker knowledge mobilization Knowledge phase knowledge translation strategies KTE models KTE strategies literature models and frameworks Oxman Persuasion phase policy and practice policy settings process of evidence-informed promote evidence-informed decision Public Health qualitative research randomized controlled trial research utilization Science identified specific strategies to promote studies evaluating substance use issues support to promote systematic reviews translate research evidence women with substance


text:
A lawn desert garden with pool and porch
I recollect the first occasion when I saw and utilized a patio swimming pool. I was in review 2. I couldn't accept that some person could have their pool in a terrace.
I used around 8 hours in that pool. It was a day I'll always remember… you know those mysterious days as a youngster that make such an impression and were so fun and cheery that you revel in simply recalling those days.
From that day on, I fell head over heels in love for pools. In any case I adore them. I utilize one 3 to 5 times each week as a grown-up. Some of my best memories are used in pools … they're excellent, fun, unwinding and an extraordinary spot to hang out with family and companions.
I know they're a ton of work and exceptionally lavish. They're unquestionably not for everybody, except on the off chance that you love them to the extent that I do, the exertion and expense is worth the trou

subset: commoncrawl


text:
The sale process for Yahoo's core business is finally steaming into the first round per CNBC reports on Monday, and there are some familiar faces circling.
Microsoft, for one, is said to be willing to "lend significant financing" to buyout firms interested in bidding for Yahoo, according to Re/code. The outreach comes five years after the tech giant teamed up with Silver Lake, Andreessen Horowitz and Canada Pension Plan Investment Board to try to buy a minority stake in Yahoo, and eight years after Microsoft made an unsolicited bid for the company.
It shouldn't be a surprise to see Microsoft in the mix again when it comes to Yahoo. The pair are still part way through a 10-year search agreement that essentially turned over Yahoo's search service to Microsoft's Bing, while Yahoo ran search-related ad sales using Microsoft's technology. Though that agreement was tweaked last year, it remains of financial importance to Yahoo, which disclosed that the fees generated from that agreement represented 35 percent of its revenue for the past two years. It's also arguably worked out OK for Microsoft:
"Bing exceeded 20% U.S. market share as we focused our advertising business on search" - Microsoft (2015 Annual Report)
Back in 2011, the Microsoft consortium was competing against other private equity firms including TPG. In the end, no deal happened with any party, likely in part because of strong opposition from Yahoo's then-activist, Dan Loeb's Third Point. (Loeb said any transaction would be a "sweetheart" deal that would effectively transfer control of the company without the payment of a premium and entrench then-CEO and co-founder Jerry Yang, who the firm was angling to oust.)
Third Point's involvement in Yahoo eventually influenced the hiring of Marissa Mayer as chief executive as well as the sale of part of Yahoo's Alibaba stake back to the company. Those moves ended up reaping roughly $1 billion in profit for Loeb's fund by the time it dumped its stake.
Yahoo's current activist in residence, Starboard Value, might not be quite as fortunate even if a Microsoft-backed private equity team or other interested bidders like Verizon line up to strike a deal.
Starboard first disclosed its stake in November 2014 and now has 1.7 percent of the company in its grips. Even if the core business is sold for anything close to analyst estimates (anywhere from $3.5 billion to $8 billion) and proceeds are distributed entirely to shareholders, the fund will be hard-pressed to lock in gains of more than $150 million.
It's also notable that Starboard is pushing to unseat a CEO installed by a fellow activist, which is fairly uncommon.
Given the revolving door (and growing presence) of such shareholders, many of whom have pushed for new management across corporate America, it's unlikely to be the last time that one firm locks horns with a target intending to undo the efforts of a rival.
This column does not necessarily reflect the opinion of Bloomberg LP and its owners.
To contact the author of this story:
Gillian Tan in New York at firstname.lastname@example.com
To contact the editor responsible for this story:
Beth Williams at email@example.com


text: Sniper Rile Proficiency Skill at handling sniper riles. Structure Volume: 0.01 m3 Miscellaneous Training Time Multiplier: 5x Base Price: 567,000 ISK sniper rifle damage bonus: +3% per level Required Skills Sniper Rifle Operation - level 5 Retrieved from "http://dust514.wikia.com/wiki/Skill:Sniper_Rifle_Proficiency?oldid=6582" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Skills Add category Cancel Save


text: Wonderful news! I hope this is a proper forum for posting feedback on the pre-release. Installed it from the Ubuntu Jaunty PPA in Jaunty 64 bit. On first run, I was able to drag an AVI (XVID MPEG-4, 640x480 at 30fps with Mono ADPCM audio at 44.1KHz) into the clip library, but got an error: GStreamer encountered a general stream error. gstavidemux.c(4163): gst_avi_demux_loop (): /GstPipeline:pipeline0/GstBin:bin0/GnlComposition:gnlcomposition0/GnlSource:gnlsource5/pitivi+elements+singledecodebin+SingleDecodeBin:pitivi+elements+singledecodebin+singledecodebin5/GstAviDemux:avidemux5: streaming stopped, reason not-linked This resulted in no video playing back from the timeline. However, when right-clicking a clip in the library, I was able to play it from there. Since doing this once to one clip, I'm now able to drag all my files of this type to the timeline with no errors, and playback so far is flawless. It seemed as if playing a clip from the library caused playback in the timeline to work, ignoring or fixing the previous error. If this is not the right place for this sort of comment, please accept my apologies, reply with a link to the proper forum, and I'll post it there. Thank you! -Mark Edward Hervey wrote: > > Hi all, > > We got a bit delayed, but it's happened at last. The PiTiVi team is > proud to announce the first 0.13 pre-release. > > Tarball available here: > http://ftp.gnome.org/pub/GNOME/sources/pitivi/0.13/pitivi-0.13.0.2.tar.bz2 > > For those using Jaunty, we've also made pitivi (and all required > dependencies) available on the gstreamer-developers PPA: > https://launchpad.net/~gstreamer-developers/+archive/ppa > > Testing, feedbacks, bug reports are welcome as usual on bugzilla: > http://bugzilla.gnome.org/enter_bug.cgi?product=pitivi > > Unless blockers popup, expect the 0.13.1 release next monday (25th > May). > > Edward > > > ------------------------------------------------------------------------------ > Register Now for Creativity and Technology (CaT), June 3rd, NYC. CaT > is a gathering of tech-side developers & brand creativity professionals. > Meet > the minds behind Google Creative Lab, Visual Complexity, Processing, & > iPhoneDevCamp asthey present alongside digital heavyweights like Barbarian > Group, R/GA, & Big Spaceship. http://www.creativitycat.com > _______________________________________________ > Pitivi-pitivi mailing list > Pitivi-pitivi@... > https://lists.sourceforge.net/lists/listinfo/pitivi-pitivi > > -- View this message in context: http://www.nabble.com/PiTiVi-0.13.0.2-pre-release-tp23651154p23672867.html Sent from the pitivi-pitivi mailing list archive at Nabble.com.


text:
Jason Chong thought five, maybe six, schools in the district would join.
Instead, 16 of 20, and nearby Buena Park Junior High, signed on.
A longtime math and science teacher in the Fullerton School District, Chong last year was introduced to LEGO Robotics by a colleague at Parks Junior High.
The school's robotics team later qualified for the FIRST LEGO League Challenge, an international competition held sectionally, and then regionally, that pits student-made robots against others.
Piggybacking off of that idea, and off of Cal Poly Pomona's Robotics Education through Active Training program, Chong conceived a similar showcase.
On Saturday, the Fullerton School District hosted more than 300 students at its first Robot Nation Competition.
"We wanted to start something these kids would be proud of, something we could own," said Chong, a district teacher on special assignment and the event's coordinator. "I'm very amazed at how quickly this came together."
Last year, Chong visited elementary and junior high schools within the district. He found that most had robotics clubs.
For 90 minutes once, sometimes twice, a week, up to 50 kids met with teachers to learn programming basics. Sometimes students from Troy High and Sunny Hills High attended meetings.
His idea for a district competition green-lighted, Chong and local robotics teachers sought area businesses to sponsor school teams.
Those that did often brought pizza, ice cream and other treats to club meetings. The Anaheim Ducks S.C.O.R.E. program hopped on to sponsor the event.
"What we really wanted to do was emphasize partnerships," Chong said. "We wanted local businesses, local technology companies, to pair with us so there's a bond, a connection to our local industries."
Laguna Road Elementary first grade teacher Mary Nguyen said rather than walk kids through steps in the 10 weeks leading to Saturday's competition, she and other adult volunteers encouraged trial and error.
"The kids love the challenge, and seeing that immediacy of success or of failure back," Nguyen said. "You give them a goal and they have this competitiveness, this intrinsic motivation, inside them."
Through a local connection, Code Tech – a global consulting and technology services company – became an event partner. Employees helped students with coding and engineering.
Parents with comparable knowledge also lent their talents, Nguyen said.
"We wanted to build a kindergarten-to-career pathway for kids," Chong said. "This competition does the work in promoting and encouraging student engagement."
Said Nguyen: "We see kids who aren't really engaged in class … take off in this robotics club."
Contact the writer: 714-796-7724 or firstname.lastname@example.com


text:
The ULTIMATE $2000 Intel gaming PC build guide has landed. Enjoy! It uses the latest "Haswell" 4th Generation Core series processor family and some other really cool stuff that makes it incredibly efficient and silent without compromising on performance.
This video build guide was sponsored by Intel.
Sponsor link:
Parts List:
Intel Core i7 4770K "Haswell" CPU
Intel 520 Series 240GB SSD
ASUS Sabertooth Z87 motherboard
Fractal Design Define R4
GeForce GTX 780 video card
WD Green 3TB hard drive
Corsair Vengeance 16GB DDR3 RAM
Seasonic G Series 550W power supply
bequiet! Dark Rock 2 CPU cooler
Beyerdynamic Custom One Pro headphones
Razer Deathadder 2013 gaming mouse
Filco Majestouch Cherry MX Brown 104 key Stealth printed mech. keyboard
ASUS Pro Art 248Q 24" monitor
Join our community forum:
Music by Adhesive Wombat used with permission. Check out his work here:


text:
Turtle Island, Fiji
Last stop
I just landed in Fiji I have just noticed that this is my last destination till I go back home to Canada, I am very sad that my journey is ending but I am happy to go see my family back home. Fiji is one of the most beautiful places in the world that most people cant go to. In my 3 day stay here in Fiji I plan to go scuba diving, fishing, and relax on the beach and enjoy this warm weather.
The resort that I am staying at is called the Turtle Island resort, its a 5 star resort that get great reviews online, it cost about 755$ per night so for two nights this will cost me 1510$ in total.
Day One
Today I am going scuba diving, Fiji is known to be one of the best places to go scuba diving because of the different types of under water species they have. First they got trained me how to scuba dive and they show me all the safety precautions, then after all the training was done we went on a boat and traveled 30 mins out to sea, this is where we went scuba diving.
Day Two
After that eventful day yesterday I have decided to take it is and go fishing for the day, fishing is also really in popular in Fiji because of the vast population of aquatic animals. I have rented a boat for the day and it cost me 50$.
It is now 7 pm and I am heading back to the hotel room, today I caught 13 fishes and the largest one was 15 inches long, I was very impressed with my luck today because i usually go the whole say without catching anything.
Day Three
The final day of my dream trip I have decided to do what I love most which is swim on the beach. All of the beaches in Fiji are amazing and the water is really warm and has a beautiful blue colour to it.
Now I am on my way to end my trip of a life time and head home, I am really sad to end this journey but I got to see a lot of really cool places that most people will not see in their life time. I have a 10 hour flight to auckland then another 14 hour flight to Toronto, this will cost me 1400$.
The End of my dream trip...
Posted by noahlinhares 07:15 Comments (0)


text:
This property has been removed by the agent. It may be let or temporarily removed from the market
Property Description
Letting information:
|Added on Rightmove:
|11 November 2016 (284 days ago)
Key features
- Town center location
- 2 Large double bedrooms
- EPC Rating - E51
- Yard to rear
- Council tax band B
- Post Code LS29 8EA
Full description
Fully redecorated and carpeted 2 bed terrace in an ideal town centre location, close to the station with its excellent commuter links. Briefly the property comprises, living room, fully fitted kitchen, stable style door to yard, cellar head, first floor with large double bedroom, bathroom with 'p' shaped bath and shower over, second floor with very large bedroom with long reaching views. UNFURNISHED, available NOW.
Map & Street View
View in fullscreenView in fullscreen
Street View is unavailable in this location
Disclaimer - Administration fees may apply when renting a property in England, Wales or Northern Ireland. For more details, please contact the agent.
Property reference IK000683. The information displayed about this property comprises a property advertisement. Rightmove.co.uk makes no warranty as to the accuracy or completeness of the advertisement or any linked or associated information, and Rightmove has no control over the content. This property advertisement does not constitute property particulars. The information is provided and maintained by Angus Roberts, Ilkley.
Please refer to our Renting Guides for further information on how to safely rent a property.
Map data ©OpenStreetMap contributors.


text:
UK's new Oil and Gas Authority appoints seven senior executives as Prime Minister David Cameron appoints new Energy Secretary.
Monday saw the appointment of several people to key oil and gas regulatory positions in the UK.
As well as the selection of Conservative politician Amber Rudd MP as the country's UK Secretary of State for Energy and Climate Change, after last week's General Election, the newly-formed Oil and Gas Authority (OGA) announced the appointment of seven men and women to management roles within its organization.
New directors at the OGA include: Gunther Newcombe as director of exploration and production; Dr Angela Seeney as director of technology and projects; Hedvig Ljungerud as director of policy, performance and economics; and John Ogden as the OGA's chief financial officer.
Newcombe joins OGA from Indonesia, where he was president and CEO of VICO – a joint venture between BP and ENI. He has 35 years' experience in the oil and gas industry.
Seeney has spent most of her career with Royal Dutch Shell, where she acquired extensive global experience in global oil, gas and alternative energy technologies. Ljungerud is an expert in policy and economics, having spent nine years at HM Treasury. Ogden has held a variety of leadership roles both in the public sector in a private business, working at Sony, Hutchison 3G and Ericsson.
As well as appointing its directors, the OGA has also completed three senior managers: Brenda Wyllie as area manager, northern North Sea; Scott Robertson as area manager, central North Sea; and Mick Richardson as exploration and new ventures manager.
The selection of a candidate for a further position – area manager, southern North Sea and Morecambe Bay – will be announced in the coming weeks, the OGA said.
View Full Article
Click on the button below to add a comment.
Generated by readers, the comments included herein do not reflect the views and opinions of Rigzone. All comments are subject to editorial review. Off-topic, inappropriate or insulting comments will be removed.
Related Companies
More from this Author
Most Popular Articles
Regional News
From the Career Center
Jobs that may interest you


text:
WASHINGTON – Political uncertainty more than health treatment could spell doom for tens of millions of Us citizens collaborating in Obamacare marketplace exchanges.
And Iowa could be the initially point out to working experience a blackout by insurance companies who produce guidelines to persons who do not get protection from an employer and are also not eligible for Medicare or Medicaid.
Two of Iowa's insurers — Aetna and Wellmark Blue Cross & Blue Defend — declared past thirty day period they are pulling out of the point out following year thanks to uncertainty in Washington and money losses.
Medica, the lone remaining Iowa marketplace insurance company, noted past week it may well likely observe match for the very same factors, leaving extra than 70,000 Iowans without having health insurance.
Tens of millions of lower and middle-revenue persons residing in 1-3rd of the nation's counties, significantly in rural spots, could similarly uncover them selves without having sponsored insurance. Oklahoma and various other states are also down to 1 insurance company.
"Still no selection on 2018," reported Kristen Cunningham, spokeswoman for Blue Cross & Blue Defend of Oklahoma, the only insurance company however collaborating in that state's marketplace exchange.
Oklahoma's point out insurance commissioner, John Doak, reported in March 140,000 persons in the Faster State depend on sponsored insurance by means of the exchanges. He explained the possible blackout in Oklahoma as a "dire predicament."
The Trump administration could aid address the disquiet by continuing to give subsidies to the marketplace insurance companies. The subsidies are employed by the insurers to hold deductibles and co-payments cost-effective to persons in the exchanges.
There is little likelihood Congress will go promptly on the contentious Republican health treatment monthly bill narrowly passed by the Household past Thursday. It is now right before the Senate and at minimum 1 moderate Republican, Sen. Susan Collins of Maine, reported she believes the Household edition is so unacceptable a rewrite from scratch is in buy.
The dilemma is insurance companies have to determine by following thirty day period no matter if they will give options in the marketplace exchanges following year. Proper now they are in limbo and nervous about generating commitments.
13 Senate Democrats — which include Pennsylvania's Bob Casey, Massachusetts' Elizabeth Warren and Ed Markey, and Michigan's Debbie Stabenow — wrote President Donald Trump's spending plan director Monday, criticizing the administration for not performing adequate to inspire companies to proceed promoting insurance in the exchanges.
The market team, America's Well being Insurance Designs, reported a vital element identifying no matter if insurers stay or depart states is no matter if the administration proceeds to pay out insurers $seven billion for every year in subsidies to lower deductibles and co-payments less than the present regulation.
The Trump administration has but to determine no matter if it will cease generating the payments further than this thirty day period.
Medica Vice President Geoff Bartsh sent shock waves by means of Iowa past week with his announcement Medica is very seriously contemplating departing the point out "without swift motion by the point out or Congress to give stability to Iowa's particular person insurance marketplace."
The firm demands "a stable marketplace, established procedures of the highway and a distinct knowledge of the market's risk" to stay, he reported in a statement.
Medica's announcement set off a blame recreation.
Household Speaker Paul Ryan and other Republican leaders pointed to Iowa as an instance of why Obamacare is failing and demands to be replaced.
Democrats' countered insurers are contemplating fleeing the marketplace exchanges for the reason that the Republican-managed Congress has been not able to pass a substitute monthly bill, making uncertainty more than subsidies needed to make guidelines cost-effective to persons in the exchanges.
"The Republicans' phone for repeal without having a distinct system has made needless uncertainty for vendors, insurers and Iowans alike," reported Rep. Dave Loebsack, D-Iowa, in a statement Monday. "It is extensive previous time that Republicans realize that health treatment is not a soccer to be thrown all over for political gain. We are speaking about true people's lives here."
The Senate Democrats letter to White Household Spending budget Director Mick Mulvaney pointed to the subsidies as essential to lifting the ambiguity struggling with insurers.
"The uncertainty getting caused ty the administration's rhetoric is threatening the foreseeable future of the ACA," the letter reported. "Holding the health treatment of tens of millions of people today hostage as a bargaining chip is unacceptable."
Contact CNHI Washington reporter Kery Murakami at email@example.com.


text:
Check out @tileexcellence's Tweet: https://twitter.com/tileexcellence/status/836095165148315648?s=09Read More
Tile Installation DIY Consumer Help Blog Says Bye to Bloggerin: affordable answer questions. expert contractors arrogant chris lawson consumer help blog help with tile blog need guest bloggers no longer associated tile excellence unethical
Chris Lawson No Longer Associated With Consumer Help
Blog | Ask Tile Excellence
Effective last month, but officially finishing up this week, Chris Lawson, chief tile blogger here on Tile Installation| Consumer Help Blog is no longer a member of this tile focused DIY and Consumer Help Community.
He plans on semi-retiring to Florida sometime in the near future, where he'll find Pool Around Tile Installations and Gemstone Tiles and Tile Art, three businesses/websites I created for Chris over the past 4+ years waiting for him. Plus of course E-Bay.
He plans on semi-retiring to Florida sometime in the near future, where he'll find Pool Around Tile Installations and Gemstone Tiles and Tile Art, three businesses/websites I created for Chris over the past 4+ years waiting for him. Plus of course E-Bay.
I'm also pleased I could be instrumental in making a difference in Chris's life, and help him build up his tile business which has been thriving and lucrative for years, well known throughout North Boston and Cape Ann, Mass. This has allowed him many future opportunities he hadn't planned on.
We are looking for someone to step up and take his place. Guest tile bloggers are welcome! We'll soon introduce our "Tile Excellence Tile Installers" in the North Shore area who provide tile excellence in their services!
We are looking for someone to step up and take his place. Guest tile bloggers are welcome! We'll soon introduce our "Tile Excellence Tile Installers" in the North Shore area who provide tile excellence in their services!
Good luck and Best Wishes.
Recent Custom Installation Slate, Round Glass Tile Floor, Natural Stone Fireplacein: ask chris bathrooms chris lawson ethics.tile installers floor tile shower walls tile installation
Sorry not a lot of time lately to write here on the blog, but I haven't gotten any questions here either! Come on, guys! Thanks for the emails and calls though, keep them up, but if you write here, I get a notice from my site and it helps remind me to write!! I've just been so busy, I could use the break. I'll be posting some shower pics I did on a handicap accessible shower a couple of weeks ago. Thanks.
Pictures from tile installation jobs of Chris Lawson, Tile Excellence August 2012
Natural Slate Tile Installation
Two Bathroom Showesr, Floors & Wall and Customized Tile Installation including custom made soap and wall inserts
Natural Marble Bathroom Floor Tile
Round glass mosaic tile installed on bathroom floor
Tile Installation- Time Vs. Pricein: ask chris backsplash bathroom remodeling chris lawson cost do it yourself estimates fair glass tile help help blog kitchen marble questions scam tile excellence tile installation time
First of all, let me apologize. I'm new to all this technology stuff, but I guess my "Welcome to Our Blog" post was supposed to come before I shared stories. However, I thought the following information might be helpful, and save some people some money, so it took priority. I'll try and post the first entry, but we'll see. Hang in there with me folks!
So, onto the information I wanted to share. I wanted to share some insight with people out there that need tile work done. Unfortunately, many people within the industry are out to get as much as they can for installation jobs. One of the most common ways to increase the charges for the installation is to drag the job out. Here's a good example I recently came across.
I recently installed a kitchen backsplash where the customer had gotten several estimates and done his research. Quotes were anywhere from $500-$750.00. Time frames varied from 2-4 days. As soon as I talked to him on the phone, I knew this was an installation that would take me a matter of hours. So, I priced the installation accordingly, which happened to be considerably lower than the above mentioned estimates because I knew through my 30+ years experience, that the job would take only half a day-and it would be flawless.
After our phone conversation, the potential customer checked out pictures of my work and references on my website, http://www.tileexcellence.com/ and quickly scheduled the job. The bottom line here is, there are installers out there who will drag out the job to justify the price or are too inexperienced to do the job in a reasonable time frame, thus charging more. To save yourself from this situation, be sure to get three or more quotes for any installation and get references. Check out their work if you can.
Tile Excellence, Inc. did the job in about 1/2 a day, and it costs the customer only $350.00 in labor. To see this project, before and after, and/or to see what Scott from Wakefield had to say about his new backsplash and the quality of the work, click here to visit our customer testimonial page.
Next blog post, I'll talk about hardyboard vs. durock. Stay tuned, and send me your questions or share your experience. Thanks.
Owner, Operator
Tile Excellence, Inc.
Gloucester, MA
(978) 471-9127
Owner, Operator
Tile Excellence, Inc.
Gloucester, MA
(978) 471-9127
Tile Installation Guest Bloggers
It is so nice hearing about other tile professional's experiences and seeing materials and how-to instruction; very enlightening. That's what this blog is all about; sharing experiences, ideas and advise for consumers and DIY project enthusiasts. Thanks!
Guest Bloggers
Tile Installation Excellence Consumer Help Blog
Trugard Direct
Contact: Jake KriserEmail: firstname.lastname@example.com
Website: trugarddirect.com
Contact: Tile Gal
Phone Number: 1-866-695-8992
Website: MyTileBacksplash
Social Bookmarking
(C) 2010-2016 Diana Smith, SEO Powered Websites All Rights Reserved. Powered by Blogger.


text:
Architectural Signs & Letters Installed In All Major US Cities and Shipped Nationwide Free !
Precision Architectural Signs, Letters & Logos.
Made In The USA Since 1976!
Is The National Full Service Architectural Sign & Architectural Letter
Company. We
install & ship nationwide. You will find our superb quality letters, logos, graphics and signs
enhancing business image,
identifying corporations, & communicating messages all over the entire USA.
Experience, knowledge, expertise and really good old fashion craftsmanship combined with the latest cnc technology enables us to create the finest architectural signage in the industry.
We cut out, fabricate and polish finish all metals
including stainless steel, brass, bronze, muntz metal, aluminum, copper,
gold, silver, nickel, pewter and metal laminates into the form of logo signs.
When your business sign / letter / logo project requires good old fashion high
quality, we welcome the opportunity to provide the quality you expect
and beyond as we have done for thousands of very satisfied, happy clients
since 1976.
Water Jet Cut Out, Laser Cut Out, Router Cut Out Fabricated, LED Lighted, Cast Metal, Injection Molded
Stainless Steel,
Aluminum, Brass, Bronze,
|Architectural Signage Alloys & Finishes Information:
Architectural Signage / Letters / Logos are made from 6061 aircraft quality metal and
yields superb chrome like mirror polishing. 5052 H32 is used for satin
brushed and paint finishes. Aluminum is used when anodize finish is
required. Anodized finish letters are available in polished &
satin clear, polished & satin gold,
polished & satin black, dark, medium & light duranodic brown. Aluminum
is also used for baked enamel, powder coated, and pantone color match painted
metal letters.
Brass Architectural Signage / Letters / Logos are made from non lead metal C46400 60% copper, 39.25% zinc, .75% tin, 0% lead. We also offer yellow navel brass if needed. Both yield quality mirror polish & satin brush, oxidizing and patina finishes. Brass is used for quality nickel plating, chrome plate and gold plated lettering.
Muntz Architectural Signage / Letters / Logos are made from non leaded metal C280 61% copper, 39% zinc 0% lead. Muntz metal has the same characteristics as brass for mirror polish, satin brush & oxidize & patina finishing. Muntz also chrome plates well.
Bronze Architectural Signage / Letters / Logos are made from non lead C22000 90% copper, 10% zinc, 0% lead. Quality bronze that mirror polishes up beautifully & satin finishes well. This metal is ideal for oil rubbed, antique, green patina & oxidize finishes.
Copper Architectural Signage / Letters / Logos are made from C11000 pure solid copper metal, and takes mirror polishing & satin brushed finishing, oil rubbed, antique, blackened & oxidize finishes with outstanding quality results.
Stainless Steel Architectural Signage / Letters / Logos are made from 304 alloy as standard which has good corrosion resistance and we also make metal letters with 316 alloy marine grade stainless that is practically tarnish and corrosion proof. 316 alloy marine steel is for all applications and especially used on yachts, boats and by salt water. Both mirror polish excellent and chrome well, both satin brush #4 finish and other grain finishes well and both take titanium gold impregnation excellent for a polished gold and satin brush gold that last in exterior applications. 304 is best for black gun metal finish. 316 alloy stainless steel / titanium gold is best for harsher environments. 304 & 316 alloy stainless steel nickel chrome plate and real gold plate superb.
Gold, Silver, Nickel, Pewter, Architectural Metal Signs / Letters / Logos are made from pure solid metals and we can do just about any finish you can imagine. We can craft any metal into any letter, just ask, we can do it.
Metal Faced Architectural Signage / Letters / Logos are made from exactly that, thin gauge metal face laminated to a substrate material which creates dimension. We use the best material, Chemmetal, for the faces of our Metallam letters. Chem Metal offers dozens of rich looking metal finishes. Chem Metal Laminated to various substrate depths / thicknesses combined with smooth laser polished edges, painted finish edges in contrasting colors, a matched color or metal simulated colors, metalam letters & logos are an attractive and a less expensive solution to achieve high quality 3D / dimensional simulated solid metal looking lettering and logos. We can also make metallam lettering with any mica / metal mica face.
Acrylic Architectural Signage / Letters / Logos are cut out from extruded acrylic in 32 standard colors. Our acrylic cut with 1000 watts of power yields superb smooth polished edges for standard colors. Acrylic letters are durable and precise and can be painted to match any pms pantone color. Ideal for interior or exterior signage.
Plastic Architectural Signage / Letters / Logos are precision formed by injection mold process using cellulose acetate butyrate BTE plastic. Highly impact resistant and yet economical, plastic letters are an excellent for building lettering. Used mostly for exterior applications, plastic letters can be made in any font or logo. Available in 31 standard colors and can be paint finished to match any pantone color.
Architectural Architectural Signage / Letters / Logos Finishes Summary: Mirror polish, satin brush, hand rubbed, gun metal, swirl, non directional grained, duranodic, antique, oxidize, color anodize, turquoise verde patina, turquoise patina, nouveau patina, nickel and chrome plated, titanium gold, sand blast, glass bead, etched, powder coating, baked enamel, pantone PMS color match painting, polyurethane, Nickolas clear silver lacquer, metal flake, metallic spray paint, air brush, custom textured & all Chemetal real metal & metal mica laminate finishes.
SIGNMASTERS is innovative, if there is another material or finish you want to incorporate into your architectural sign / lettering project, let us know, we can do anything architectural & designer both Interior & exterior.
The Choice For Architectural Signs & Letters!
Installed in the USA Since 1976.
Hi-Tech Architectural Signage In All Materials, All Fonts, All Sizes, All Thicknesses, All Mountings, All Finishes
Signmasters.com Architectural Signs Copyright and Trademark Notice


text:
59328Re: [existlist] Re: Existentialist Concepts?
Expand Messages
- Feb 9, 2013[a consummation in a deployment of the spirit's self knowledge and
self-power of its divine being and consciousness is the culmination]
One could very well propose that there is an evolution of humanity [assuming
we will last much longer to actually evolve further], since we already know
that is what is happening.
But your previous statement was that there is a meaning in the cosmos. So,
what is that meaning??
If the meaning is that humans should arise and somehow reach a level of
divine being ... why?? Did the cosmos in its origin of the Big Bang also at
the time have a focus upon a humanity which would not appear for another 12
billion years or so??
-----Original Message-----
From: devindersingh
Sent: Friday, February 08, 2013 9:37 PM
To: email@example.com
Subject: [existlist] Re: Existentialist Concepts?
There are, according to Sri Aurobindo, three stages that cover the entire
process of manifestation that is relevant to evolution. An involution of the
spirit in the inconscience is the beginning;
evolution in the ignorance with its play of possibilities of a partial
developing knowledge is the middle; a consummation in a deployment of the
spirit's self knowledge and self-power of its divine being and consciousness
is the culmination. It may be argued that the two stages that have already
occurred seem at first sight to deny the possibility of the later
consummating state of the cycle, but it is stressed that logically they
imply its emergence. For, it is argued, if the inconscience has evolved
consciousness, the partial consciousness already reached must surely evolve
into complete consciousness, considering that just as the impulse towards
Mind ranges from the more sensitive reactions of Life in the metal and the
plant up to its full organization in man, so in man himself there is the
same ascending series, the preparation, if nothing more, of a higher and
divine life. It is, therefore, contended that it is a supramentalised,
perfected and divinized life for which the earth-nature is secretly seeking,
and that a progressive manifestation of this kind can only have for its
secret or significance, the evolution of a Being in a perfect Becoming.
According to Sri Aurobindo, the present age of human development has a
possibility of becoming a gate for the arrival of a Spiritual Age in which
the ideal of brotherhood can come to be practised; it is only in that
condition that a new form of human unity can be forged in which the
individual and the collectivity, even on a global scale of organization, can
come to be harmonized. In the meantime, however, it is to be realized that
the advent of the Spiritual Age and even a transition to that advent is
bound to be difficult, and it is, in fact, riddled with a series of crisis.
And there are deeper reasons for this crisis.
Sri Aurobindo considers the present stage of crisis as an
evolutionary crisis in which the human will is called upon to make a free
choice. This is the crisis where the human reason has evolved up to a point
where it is obliged to play the role of lifting up humanity to create a life
of unity, mutuality and harmony. On the other hand, there is a force that is
striving to assert stark barbarism which has still survived in the
civilized man. There is thus an acute conflict between the ideals of Reason
and the gravitational pull of barbarism supported by Unreason. Within the
larger framework of this conflict, there are three alternative
possibilities, which are also in conflict with each other. For it is
possible that the humankind may be able to utilize the present scientific
and technical knowledge to such an extent that an order of existence can be
created in which physical and vital wants of the human being can greatly, if
not fully, be satisfied, and it may also be conceived that this order of
existence can be maintained by mechanical devices and application of the
power of machines. This possibility may push humanity to the creation of a
framework that can be sustained only by the imprisonment of the human
spirit. There is also a second alternative in which human reason can
continue to spin out larger or narrower circles propounding great dreams but
find itself unable to fulfill them. But there is also a third alternative in
which the human beings might consent to rise to the higher levels than those
of the Reason and consent to be spiritualized.
--- In email@example.com, eduardathome wrote:
> [The purpose of the creation of the cosmos is your own individual
> evolution]
> In brief, what you are saying is that humanity is evolving. We already
> know
> that ... even if one were to accept that it is an evolution towards a
> cosmic
> consciousness.
> That doesn't answer the question of what is the "meaning". Am I here for
> some particular purpose or reason?? What is there in all this for the
> individual??
> If the end point is to be able to link our consciousness with a cosmic
> consciousness, why is this important? If for some reason we do not reach
> that point ... so what? And who or what is monitoring our progress?
> eduard
> -----Original Message-----
> From: devindersingh
> Sent: Friday, February 08, 2013 10:49 AM
> To: email@example.com
> Subject: [existlist] Re: Existentialist Concepts?
> The meaning for you Eduard is this:Each plane that emerges in the
> evolution contains the seed of the following planes. So the plane of
> matter which emerges first contains the seed of the planes of life and
> mind. Each planes also has involved in it even higher planes, including
> the planes of the spirit. The spirit is involved in all the planes that
> emerge. One of the goals of life is for us to enable the spirit to
> emerge in matter, life, and mind. That is one of our roles of destiny;
> to release the spirit in all things.
> Life is involved in Matter; Mind is involved in Life; and Supermind in
> Mind. (Actually Mind is involved in Matter and Life, and Supermind in
> all three.)There is a process of unfolding of one plane from the
> previous plane. One plane emerges from the previous because it is
> already involved there as potential; and under the right circumstances
> emerges to the next highest plane. However, what really compels the
> emergence of the next plane is the underlying spirit that is embedded in
> each plane, pushing that plane's potential to emerge from its current
> plane into the next plane. Since spirit is infinitely creative, what
> emerges in the next plane is dynamically new and creative; what could
> not be conceived. http://www.gurusoftware.com/GuruNet/Creation.htm
> What then is the next step of your evolution Eduard?The ordinary human
> consciousness is confined to one's own individual consciousness.
> That is, we are directly aware of only our own thoughts, feelings and
> sensations. What lie outside our personal consciousnessâ€"the
> consciousness of other beings and things in the worldâ€"are
> apprehended by us only indirectly, through external sense contacts,
> emotional empathy or mental inference. However, the personal
> consciousness of our separate individual self is part of and one with
> the consciousness of the Cosmic Self. Our individual physical, vital and
> mental consciousness is derived from and is a part of a universal
> physical, vital and mental consciousness. But there is a wall of
> Ignorance between the individual consciousness and the universal
> consciousness. Consequently, the individual self feels itself to be
> separate from the universe. By means of a spiritual discipline our
> ordinary consciousness, which is more or less gross and dense, becomes
> more subtle and is able to perceive what lies outside our individual
> consciousness more directly through an inner perception. It becomes a
> direct perception of consciousness all around us through our own
> consciousness. Further, by a widening of the individual consciousness,
> it extends itself progressively so as finally to be identified with the
> consciousness of the universe, seeing oneself in everything in the
> cosmos and everything in the cosmos in oneself. One is then said to have
> cosmic consciousness. Thus, "When one has the cosmic consciousness,
> one can feel the cosmic Self as one's own self, one can feel one with
> other beings in the cosmos, one can feel all the forces of Nature as
> moving in oneself, all selves as one's own self" (1972d, p.
> 1071).http://www.ipi.org.in/texts/ipyc/ipyc-full/asdalal.php
> The purpose of the creation of the cosmos is your own individual
> evolution:http://www.gurusoftware.com/GuruNet/AurobindoMother/TheLifeDiv\
> ine/HTML/LifeDivineIntro.htm --- In email@example.com,
> eduardathome wrote:
> >
> > [Is the cosmos meaningless, as expressed by various thinkers called
> > "Existentialists". I think not. ]
> >
> > If the cosmos has meaning, then what is this meaning?? And for this
> the
> > meaning should be a meaning for the individual. What does all this
> mean to
> > me. That is the issue. It is pointless to say that the cosmos has
> meaning
> > if this is not a meaning for the individual.
> >
> > So Debashish if you think the cosmos has meaning, please tell what is
> the
> > meaning.
> >
> > eduard
> >
> > -----Original Message-----
> > From: devindersingh
> > Sent: Friday, February 08, 2013 4:35 AM
> > To: firstname.lastname@example.com
> > Subject: [existlist] Re: Existentialist Concepts?
> >
> >
> >
> > would I agree with Camus that life is absurd? Life has a purpose.
> > Is the cosmos meaningless, as expressed by various thinkers called
> > "Existentialists". I think not.
> > > Dick: I exist so what is it all about and what does life offer in
> the way
> > > of experience.
> > This talk considers Sri Aurobindo's spiritual experiences.
> > The presentation is partly based on Dr. Debashish Banerji's recently
> > published book "Seven Quartets of Becoming: A Transformative Yoga
> Psychology
> > based on the Diaries of Sri Aurobindo".
> >
> > Dr. Debashish Banerji is a Dean of academic affairs at the University
> of
> > philosophical research in Los Angeles:
> > http://www.youtube.com/watch?NR=1&v=Xhzui09n8Z0&feature=endscreen
> >
> > --- In email@example.com, "William" wrote:
> >
> > > --- In firstname.lastname@example.com, "Dick." wrote:
> > > >
> > > > Existentialist Concepts?
> > > >
> > > > [ Eduard, Agreed. We don't know how a zealot or sadist makes
> decisions.
> > > > Existentialist context is for existentialists, and there don't
> seem to
> > > > be many left anymore. It's pointless to argue whether anyone else
> is
> > > > acting authentically. I don't think anyone is inherently anything,
> and
> > > > I'm not accommodating terrorists by suggesting they're bound to an
> > > > ideology. Are there any existentialists concepts you agree with?
> Mary ]
> > > >
> > > > Well he is certainly right on that at least, they are brainwashed
> by an
> > > > ideology. However, do a numbered list of what you see as being THE
> > > > Existentialists Concepts and we can go though them one at a time.
> I read
> > > > them all many years ago and was most amazingly unimpressed and
> downright
> > > > bloody bored stiff. My own existentialist outlook is only one
> concept â€"
> > > > I exists so what is it all about and what does life offer in the
> way of
> > > > experience. I was asking that question, in a less sophisticated
> way,
> > > > when I was three. So, what does it have to offer us? What have you
> > > > found? I have told what I found (six books and a hundred poems and
> > > > thousands of letters and emails). Now you have a go. Or are you
> like the
> > > > gnostics and who say that they will not tell?
> > > >
> > > > Dick Richardson
> > > >Dick, read the existentail primer. It is not too long and is very
> concise
> > > >and direct. CSW has continually updated it . You need not task us
> on our
> > > >own site when the whole philosophy is well outlined by the Owner.
> Bill
Please support the Existential Primer... dedicated to explaining nothing!
Home Page: http://www.tameri.com/csw/existYahoo! Groups Links
- << Previous post in topic Next post in topic >>


text:
Tamako Market specials focused on Choi-chan
EyeonAnime.com is the best place to watch Tamako Market: Absent-Choinded episodes online free. To begin watching Tamako Market: Absent-Choinded, choose a episode below. Have fun watching!
Alternative Name: -
Category: Anime
Date Added: 7 months ago
Released In: -
Status: Completed
Rank: 3497 (46 views)
Subscribe: RSS


text:
|Home | About | Journals | Submit | Contact Us | Français
Brain metastasis of breast cancer profoundly affects the cognitive and sensory functions as well as morbidity of patients, and the 1 year survival rate among these patients remains less than 20%. However, the pathological mechanism of brain metastasis is as yet poorly understood. In this report, we found that metastatic breast tumour cells in the brain highly expressed IL-1β which then 'activated' surrounding astrocytes. This activation significantly augmented the expression of JAG1 in the astrocytes, and the direct interaction of the reactivated astrocytes and cancer stem-like cells (CSCs) significantly stimulated Notch signalling in CSCs. We also found that the activated Notch signalling in CSCs up-regulated HES5 followed by promoting self-renewal of CSCs. Furthermore, we have shown that the blood-brain barrier permeable Notch inhibitor, Compound E, can significantly suppress the brain metastasis in vivo. These results represent a novel paradigm for the understanding of how metastatic breast CSCs re-establish their niche for their self-renewal in a totally different microenvironment, which opens a new avenue to identify a novel and specific target for the brain metastatic disease.
Breast cancer is a second leading cause of cancer death among women and more than 90% of deaths are still attributed to metastatic diseases (Jemal et al, 2010; Weigelt et al, 2005). At the late stage, most patients develop metastatic lesions which is always fatal and the brain is one of the major sites (Palmieri et al, 2007). Because of the location of metastatic lesions, a surgical approach is limited and most chemotherapeutic drugs are ineffective due to the blood-brain barrier (BBB; Steeg et al, 2011). Despite this clinical importance, the molecular basis of breast tumour metastasis to the brain is poorly understood.
According to the cancer stem cell theory, cancer stem-like cells (CSCs) are capable of initiating tumourigenesis and are also responsible for metastatic growth (Li et al, 2007; Pang et al, 2010; Visvader & Lindeman, 2008). They have an ability to drive self-renewal replication at a niche which provides specific growth factors and activates distinct signalling such as Notch, Hedgehog and Wnt (Malanchi & Huelsken, 2009; Moore & Lemischka, 2006). These signalling pathways are evolutionarily conserved and play critical roles in embryonic stem cells; however, aberrant expression of these pathways is often observed in various types of cancers (Reya et al, 2001; Takebe et al, 2010). When CSCs metastasize to a distant organ, they need to re-model the microenvironment to generate a suitable niche and reactivate these signalling pathways (Li & Neaves, 2006; Scadden, 2006). It is virtually unknown how the metastasized CSCs adapt themselves to the brain which has a totally different microenvironment and how the CSCs communicate with specific brain cells and re-establish a niche for their own growth.
Astrocytes are the most abundant glial cells in the brain and they are activated under certain pathological conditions which can be identified by a high level of glial fibrillary acidic protein (GFAP) expression (Gomi et al, 2010; Li et al, 2008). Interestingly, they are also frequently observed around the brain metastasis lesions, suggesting a potential role of activated astrocytes in promoting brain metastasis (Marchetti et al, 2000; Papadopoulos et al, 2004). In this report, we found that CSCs generate their niche in the brain by communicating with astrocytes through reciprocal interaction, which in turn activates Notch signalling in CSCs followed by promoting self-renewal of CSCs. We have also shown that blocking Notch signalling by a BBB-permeable drug significantly suppressed brain metastasis in an animal model.
To understand how metastasized breast cancer cells thrive in the brain by remolding the local microenvironment, we first examined the effect of conditioned medium (CM) acquired from brain-metastatic breast cancer cell lines on primary rat astrocytes. 231BrM and CN34BrM cells that can specifically metastasize to the brain were originally isolated from MDA-MB231 (MDA231) and CN34, respectively, through in vivo selection (Bos et al, 2009). As shown in Fig 1A and B, both mRNA and protein levels of Notch ligand, JAG1 were significantly increased by the CM from 231BrM and CN34BrM but not by the CM from their parental cells, indicating that the CM of 231BrM and CN34BrM contain soluble factor(s) which can up-regulate the JAG1 expression in astrocytes. It should be noted that up-regulation of Notch ligand by CM was specific to JAG1, and none of the other Notch ligands including JAG2, DLL1, DLL3, and DLL4 were responsive to CM (Supporting Information Fig S1A). The up-regulation of JAG1 was also observed in immortalized human astrocytes that were treated with CM of 231BrM (Fig 1C). Moreover, the result of our immunocytochemical analysis indicates that the expression of both JAG1 and GFAP, a marker of reactive astrocytes, were strongly augmented by the CM from 231BrM cells (Fig 1D). We have also examined the tissue-specificity of JAG1 activation by culturing primary human microglial cells, another major component of brain cells, with CM of MB231 and 231BrM cells. We found that JAG1 was almost undetectable in microglial cells by immunocytochemical staining and that the level of JAG1 was unchanged by the treatment of CM (Supporting Information Fig S1B).
To identify the secretory factor(s) which stimulated JAG1 expression in the CM of brain metastatic cells, we performed a cytokine antibody array analysis and found that IL-1β, which is known to promote tumour growth, angiogenesis and invasion, was the most significantly enriched cytokine in the CM of 231BrM cells (Fig 2A; Supporting Information Fig S2A). In addition, we analysed the existing GEO data base (GSE12237) which contains comprehensive gene expression profile of MB231 and 231BrM cells and found that IL-1 β was indeed significantly over-expressed in 231BrM cells compared to other cytokines or chemokines (Supporting Information Fig S1B). The up-regulation of IL-1β in 231BrM cells (Fig 2B and C) and CN34BrM cells (Fig 2D) compared to their parental cells was also confirmed by qRT-PCR, Western blot and ELISA. To investigate the clinical relevance of IL-1β in brain metastasis, we analysed a series of clinical microarray cohort data (GSE12276, GSE2034, GSE2603, GSE5327, and GSE14020) that contain the brain relapse information of a total of 710 patients. We found that the high level of IL-1β but not IL1-α was significantly correlated with a poor brain metastasis-free survival of breast cancer patients (Fig 2E). Furthermore, the results of our IHC analysis also indicate that primary tumours from patients who eventually developed brain metastasis (n = 6) expressed significantly higher IL-1β compared to the tumours from overall metastasis-free patients with the similar clinical grades (n = 11; Fig 2F and Supporting Information Fig S2C). Therefore, it is plausible that IL-1β secreted from brain metastatic cells plays critical roles in metastatic growth by up-regulating the Notch ligand in astrocytes.
To directly examine whether IL-1β up-regulates the Notch ligand, we tested the effect of recombinant IL-1β on JAG1expression in primary rat and human astrocytes. We found that IL-1β was indeed capable of up-regulating JAG1 in primary human and rat astrocytes (Fig 3A and B) as well as in immortalized human and rat astrocytes cell lines (Supporting Information Fig S3A) in both dose and time dependent manners. It should be noted that IL-1α which has been found to be highly expressed in 231BrM cells was also able to up-regulate JAG1 in astrocytes (Supporting Information Fig S3B). However, the expression of this cytokine was not significantly correlated to the status of brain metastasis (Fig 2E). On the other hand, the rest of the soluble factors that were found to be enriched in the CM of 231BrM cells failed to activate JAG1 expression in astrocytes (Supporting Information Fig S3C), suggesting that JAG1 activation in astrocytes is specific to IL-1. Moreover, IL-1β was shown to strongly activate JAG1 and GFAP in rat astrocytes by our immunocytochemical analysis and Western blot (Fig 3C and Supporting Information Fig S3D). To further investigate whether IL-1β in CM of 231BrM cells is indeed the factor which activates JAG1 in astrocytes, we examined JAG1 expression in rat astrocytes that were treated with CM of 231BrM in the presence or absence of IL-1 receptor antagonist (IL-1RA) or IL-1β antibody. As shown in Fig 3D and E, the expression of JAG1 in rat astrocytes was significantly decreased in the IL1RA or IL-1β antibody treated cells but not by the treatment with the anti-IL1α antibody (Supporting Information Fig S3E). Furthermore, we examined the mRNA level of other Notch ligands in rat astrocytes after IL-1β treatment and found that only JAG1 was significantly up-regulated by IL-1β (Supporting Information Fig S3F). We also found that the NF-κB inhibitors, PDTC or RO 106-9920, significantly abrogated the IL1β-mediated JAG1 expression in astrocytes, indicating that IL-1β up-regulates the JAG1 expression through the NF-κB pathway (Fig 3F and Supporting Information Fig S3G). Taken together, our results indicate that IL-1β secreted from brain-metastatic cells specifically activates JAG1 in reactive astrocytes.
In order to test whether the activation of JAG1 in astrocytes indeed triggers the Notch signalling in tumour cells through cell–cell interaction which is generally required for Notch activation, we first cultured astrocytes in monolayer followed by infecting lentivirus carrying sh-JAG1 or sh-scramble, and the knockdown of JAG1 was confirmed by Western blot after 48 h (Supporting Information Fig S4A). In parallel, GFP-labelled 231BrM cells were seeded on top of the astrocyte monolayer and they were co-cultured for 2 days followed by examining the activated Notch signalling in 231BrM cells by immunocytochemistry using anti-NICD antibody (Fig 4A). We found that the Notch signalling in the cancer cells was strongly activated when cells were co-cultured with rat astrocytes and this activation was almost completely abolished by the knockdown of JAG1 expression in astrocytes and the treatment of the cells with γ-secretase inhibitor, DAPT. The Notch pathway has been reported to play a critical role in the self-renewal of various types of stem cells (Bouras et al, 2008; Pannuti et al, 2010). To further examine the role of the reactive astrocytes in promoting self-renewal of CSCs, we co-cultured 231BrM cells with rat primary astrocytes and found that the CSCs population in 231BrM cells was significantly increased after the co-culture in a time dependent manner, indicating that interaction with astrocytes indeed promotes the self-renewal ability of CSCs (Fig 4B; Supporting Information Fig S4B). In addition, we treated astrocytes with recombinant IL-1β and co-cultured with the parental cell, MDA231. We found that IL-1β significantly increased the CSCs population (Supporting Information Fig S4C). This result strongly supports our idea that IL-1β enhances the self-renewal of CSCs by activating astrocytes. We also treated MDA231BrM cells with anti-IL1α or anti-IL1β antibodies and co-cultured with rat astrocyte for 72 h. We found that inhibition of IL-1β significantly decreased the CSCs population, while anti-IL1α antibody failed to decrease the JAG1 expression in astrocytes and did not affect the CSCs population of 231BrM cells in this assay (Supporting Information Fig S4D and S3E). These data strongly suggest that IL-1β but not IL-1α is the major regulator of JAG1 activation and CSCs population. Furthermore, we isolated CSCs (CD24−, CD44+, ESA+) from 231BrM cells by Magnetic-activated cell sorting (MACS; Supporting Information Fig S4E) and they were co-cultured with rat primary astrocytes, NIH3T3 or mouse brain endothelial cells followed by FACS analysis for CSC markers. As shown in Fig 4C and D, the population of CSCs was significantly increased when these cells were co-cultured with astrocytes but not with other types of cells and this effect was drastically abrogated by the DAPT treatment. On the other hand, the population of differentiated cells which express high level of CK18 (cytokeratin 18) was significantly increased (Supporting Information Fig S4F). Taken together, these results strongly support our notion that IL-1β secreted from metastatic cells activates astrocytes which in turn stimulate the self-renewal of CSCs by activating Notch signalling. To further investigate the role of Notch signalling in the self-renewal of CSCs, we constructed a stable 231BrM cell line which carries the tetracycline-inducible NICD gene. The expression of Notch down-stream targets, HES1, HEY1 and HES5, were all up-regulated upon tetracycline induction in this cell line, confirming the inducible Notch signalling (Supporting Information Fig S4G). Importantly, the population of CSCs was significantly increased after the induction of the NICD expression in this cell line, suggesting that Notch signalling plays a key role in maintaining the stemness of the CSCs. (Fig 4E).
To further clarify the downstream targets of Notch signalling which promotes the self-renewal of CSCs after interacting with astrocytes, we first co-cultured 231BrM-GFP or CN34BrM-GFP cells on top of the rat astrocyte with or without knockdown of JAG1 for 48 h. GFP+ cells were then sorted out by FACS and the expression level of HES5 was examined by qRT-PCR. We found that HES5 was significantly up-regulated in 231BrM-GFP (Fig 5A) as well as in CN34BrM-GFP (Supporting Information Fig 5A) after co-culturing these cells with rat astrocyte and that knockdown of JAG1 in rat astrocyte significantly abolished this effect. Interestingly, when we analysed existing clinical breast cancer cohort data, we found that the high expression level of HES5, but not HES1 or HEY1 was significantly correlated with a poor brain metastasis-free survival of breast cancer patients (Fig 5B). Furthermore, we examined the expression of HES5 in paraffin embedded primary and brain metastatic tumours by Taqman PCR and found that HES5 was indeed significantly over-expressed in metastatic tumours in the brain (n = 8) compared to the primary tumours (n = 5; Fig 5C). To verify the role of HES5 in self-renewal of CSCs, we knocked-down the HES5 gene in 231BrM Tet/NICD cells by infecting lenti virus expressing shRNA with or without an induction of NICD followed by examining the CSCs by FACS. We found that the induction of NICD significantly increased CSCs population; however, the knock-down of HES5 significantly abrogates the enrichment of CSCs and mammosphere forming abilities that were induced by NICD (Fig 5D and E and Supporting Information Fig 5B). Interestingly, knock-down of HES1 and HEY1 which are another two important downstream targets of Notch pathway failed to suppress the CSCs population in 231BrM cells (Supporting Information Fig 5C). We then ectopically expressed HES5 in 231BrM cells by infecting cells with lenti virus carrying HES5 expression plasmid followed by FACS analysis. As shown in Fig 5F, the ectopic expression of HES5 significantly increased CSCs population after 72 h of viral infection. To further validate our result in clinical samples, we obtained primary tumour from advanced breast cancer patients, and the tissue was passaged only once in NOD/SCID mouse without in vitro culture. The tumour cells were dissociated and the cells were infected with pSin-puro, pSin-HES5 or PLKO-shHES5 lenti virus and they were cultured in an ultra-low attachment plate. We then measured CSCs population by FACS after 72 h and their mammosphere forming ability by counting the number of spheres after 10 days (Supporting Information Fig S5D). As shown in Fig 5G and H, we again found that HES5 significantly enriched the CSCs population and mammosphere forming ability in the primary breast cancer cells. Whereas, the knock-down of HES5 significantly decreased the mammosphere forming ability and blocked the enrichment of CSCs. These results strongly suggest that the activated Notch signalling promotes self-renewal of CSCs through up-regulation of HES5.
To examine the role of Notch signalling in promoting brain metastasis in vivo, we first isolated CSCs from 231BrM cells which express the luciferase gene followed by the limiting dilution analysis of CSCs in nude mice to confirm that they are the population with higher metastatic and tumour initiating ability (Fig 6A; Supporting Information Fig S6A). In addition, we performed microarray analysis using the Affymetrix expression array for CSCs that were isolated from MB231, 231BrM and 231BoM which preferentially metastasizes to bone. As shown in Supporting Information Fig S6B and C, IL-1β was exclusively expressed in CSCs from 231BrM cells among these tested CSCs. The isolated CSCs from 231BrM cells were then transplanted into nude mice by intracardiac injection followed by monitoring tumour growth by bioluminescent imaging for 4 weeks. The mice were sacrificed and their brains were removed and subjected to ex vivo luciferase assay (Supporting Information Fig S6D). We then performed immunohistochemical analysis on the brain sections with JAG1 and GFAP antibodies and found strong co-localized signals of GFAP and JAG1 in the reactive astrocytes that surrounded the metastatic lesion (Fig 6B). Interestingly, a group of CD44+ESA+ cells were also found to be significantly enriched in the invasion front of metastatic tumour, suggesting that these CSCs may be responsible for the growth of the tumour cells in the brain (Fig 6C, Supporting Information Fig S6E). To further investigate the role of Notch signalling and IL-1β in promoting brain metastasis, we inoculated CSCs that were isolated from 231BrM, 231BrM-DNMAML or 231BrM-shIL1β, through intracardiac injection into nude mice. We found that suppressing Notch signalling by DNMAML or knocking down IL-1β expression by shRNA in CSCs of 231BrM significantly attenuated their metastatic growth in the brain compared to that of 231BrM parental cells (Fig 6D). Next, we ectopically expressed IL-1β in MDA231 cells by using the lentivirus expression system and transplanted the established cell line into mice by intracardiac injection. As shown in Fig 6E, we found a significant increase in the incidence of brain metastasis in the group which received MDA231 with IL-1β over expression compared to the control group. In addition, we performed the in vitro BBB transwell assay by first culturing mouse brain endothelial cells on the transwell membrane until confluent followed by seeding GFP-labelled cancer cells on top of the transwell. We found that the invasive ability of MDA231 was significantly enhanced by IL-1β (Supporting Information Fig S6F). Taken together, our data suggest that IL-1β contributes to both the invasive ability of cancer cells and to the activation of Notch signalling through astrocytes.
Our in vitro results also suggest that a Notch inhibitor may serve as an effective therapeutic drug for the treatment of brain metastasis of breast cancer. As a first step towards this goal, we tested the efficacy of a γ-secretase inhibitor in our model system. Nude mice which have been inoculated with CSCs were treated with Compound E, a potent BBB permeable γ-secretase inhibitor (Grimwood et al, 2005; Sonoshita et al, 2011), by i.p. injection followed by monitoring brain metastasis by BLI (bioluminescence imaging). We found that this drug significantly suppressed the incidence of metastasis as well as growth of CSCs of 231BrM in the brain by blocking HES5 expression (Fig 6F; Supporting Information Fig S6G and H). Collectively, these data strongly suggest that when breast cancer cells are metastasized to the brain, they secrete IL-1β which activates JAG1 expression in astrocytes and that the direct interaction of the activated astocytes with CSCs turns on the Notch pathway followed by promoting self-renewal of CSCs (Fig 6G).
Organotropism is one of the most distinct properties of cancer metastasis which indicates that metastatic cancer cells can only thrive in permissive microenvironment (Hu et al, 2009; Lu & Kang, 2007). Glial cells constitute a major part of the brain cells and astrocytes are the most abundant glial cells (Grosche et al, 1999; Ventura & Harris, 1999). Fitzgerald et al previously found that a large amount of glial cells were trapped within the inner-tumour mass in surgically resected brain samples and demonstrated that reactive glial cells can be recruited by cancer cells to promote tumour growth in the brain (Fitzgerald et al, 2008). Furthermore, reactive astrocytes are known to protect cancer cells from chemotherapy by activating signalling pathway related to cell survival (Langley et al, 2009; Lin et al, 2010). It is also noteworthy that brain-metastatic lung cancer cells were shown to stimulate the production of pro-inflammatory cytokines in astrocytes, which significantly promoted the growth of cancer cells (Seike et al, 2011). In our study, we have shown that reactive astrocytes appeared abundantly around the brain metastatic regions and that the activated astrocytes were indeed able to promote self-renewal of CSCs by direct interaction. We have also shown that brain-metastatic cancer cells secrete excessive amounts of IL-1β and activate astrocytes which in turn promote Notch signalling in CSCs. Therefore, our results indicate that CSCs establish their niche in the brain through reciprocal interaction with astrocytes, which plays a pivotal role in pathogenesis of brain-specific metastasis of breast cancer.
Interleukin-1 (IL-1) is one of the most well studied cytokines that play key roles in cancer progression, and two forms of IL-1 have been identified, namely IL-1 α and IL-1 β (Elaraj et al, 2006; Voronov et al, 2003). IL-1β is processed by interleukin-1β-converting enzyme (ICE) before it becomes functional as a secreted cytokine, while IL-1α can localize in the cytosol and mediate intracellular signalling (Aotsuka et al, 1991; Debets et al, 1995; Miller et al, 1993). The secreted IL-1β induces inflammatory response and alters tumour microenvironment; however, it was also shown to enhance the growth and invasion abilities of cancer cells in an autocrine fashion (Aotsuka et al, 1991; Kawakami et al, 1997). IL-1β is also known to promote cancer progression by upregulating pro-metastatic genes such as matrix metalloproteinases and stimulate adjacent cells to produce angiogenic proteins or growth factors including VEGF, IL-8, IL-6, TNF-α and TGF-β (Lewis et al, 2006). Many solid tumours are known to express a high level of IL-1β which is shown to correlate with patient survival (Elaraj et al, 2006; Lee et al, 2006; Liu et al, 2006). Notably, we have shown that the expression levels of IL-1β in the primary tumours of breast cancer patients were significantly associated with their brain metastatic statuses, suggesting that IL-1β may serve as a potential prognostic marker and a therapeutic target for brain metastasis. Interestingly, treatment with IL-1RA, a potent IL-1 inhibitor, was shown to significantly decrease the growth and metastases of colon and lung cancer cells in mouse models (Lewis et al, 2006). However, BBB permeability of IL-1RA is still unknown and it has a relatively short half-life (4–6 h), therefore, developing a more effective small molecule mimicking IL-1RA is needed.
Metastatic growth is believed to be initiated by CSCs at the distant organs that constitute totally different microenvironment from the primary tumour sites. Similar to embryonic stem cells, CSCs also require specific niche which provides factors to activate various pathways for the maintenance of stemness of CSCs through direct cell–cell interaction or by secreting growth factors. In this context, it is noteworthy that Karnoub et al reported that bone mesenchymal stem cells (BMSC) generate a 'pre-metastatic niche' at the distant organs even before metastatic cells arrive at the site (Karnoub et al, 2007). Interestingly, Li et al recently found that prostaglandin E2 (PGE2) was secreted by BMSCs in response to cancer cell-derived IL-1 and that the BMSC-derived PGE2 significantly enhanced the CSCs population via Akt/GSK-3/β-catenin signalling axis (Li et al, 2012). However, the 'pre-metastatic niche' hypothesis may not be applicable to brain metastasis because the brain is a highly specialized organ and also due to the brain-blood barrier, it is unlikely that BMSC reach the brain before metastasis, although this possibility cannot be totally excluded.
Increasing lines of evidence suggest that the Notch pathway plays a crucial role in maintaining the stemness of CSCs in a particular microenvironment (Charles et al, 2010; McGowan et al, 2011). A hallmark of Notch signalling is the requirement of the ligand–receptor interaction through direct cell–cell contact, which may occur between tumour cells or tumour cell–stroma interactions (Sethi et al, 2011; Xing et al, 2011). Butler et al have recently shown that bone marrow endothelial cells which express Notch ligands were indeed required for the self-renewal of haematopoietic stem cells in a Notch dependent manner (Butler et al, 2010). We have shown that direct interaction of CSCs and activated astrocytes is essential for up-regulating Notch signalling and the following self-renewal of CSCs in the brain. Our data also indicate that this activated Notch signalling up-regulated the HES5, which significantly augmented self-renewal of CSCs. It has been reported that HES5-expressing telencephalic cells are maintained as neural stem cells during embryogenesis, indicating a possible role of HES5 in maintaining self-renewal of CSCs (Ohtsuka et al, 2001). In this report, we have discovered a novel pathological mechanism by which breast CSCs establish a niche in the metastasized brain through interaction with activated astrocytes. Our results have revealed a vicious paracrine loop of IL-1β and Notch signalling through direct interaction of CSCs and astrocytes, which in turn promotes the growth of metastasized CSCs in the brain. Importantly, we have also shown that a BBB-permeable Notch inhibitor can serve as an effective therapeutic drug to suppress metastatic growth of breast cancer in the brain. These discoveries open a window of opportunity to identify a novel therapeutic target for brain metastasis.
Human breast carcinoma cell line, MDA-MB231 (MDA231), was purchased from American Type Culture Collection. MDA-MB231BrM (231BrM), CN34 and CN34BrM were kind gifts from Dr. Massague (Memorial Sloan-Kettering Cancer Center). 231BrM and CN34BrM are derivatives of MB231 and CD34, respectively, and they are highly metastatic to brain (Bos et al, 2009). Cells were maintained in RPMI 1640 supplemented with 10% FBS, streptomycin (100 mg/ml), penicillin (100 units/ml) and grown at 37°C in a 5% CO2 atmosphere. Primary rat astrocytes were purchased from BrainBits LLC and maintained in Neuro basal medium (Invitrogen) with 10% horse serum and 3 mM glutamine (Invitrogen). Normal Human primary astrocytes were purchased from Lonza and maintained in AGM medium supplemented with BulletKit (Lonza). SV40 immortalized neonatal rat astrocyte (NRA) was kindly provided by Dr Stanimirovic (NRC-Institute for Biological Sciences) and E6/E7/hTERT immortalized human astrocyte (UC1) was a kind gift from Dr Russell Piper (University of California-San Francisco). Primary human breast tumour cells which maintained in xenograft tumour of NOD/SCID mouse were obtained from Conversant Biologics, Inc. shRNA-expressing lentiviral plasmids for IL-1β and HES5 were obtained from OpenBiosystems. Recombinant IL-1β, 1-Pyrrolidinecarbodithioic acid ammonium salt (PDTC) and -[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) were purchased from Sigma Co, and IL-1 RA and IL-1β antibody were obtained from R&D. Compound E was purchased from Enzo life sciences.
The expression plasmid of NICD cDNA with a Myc-tag was provided by Dr. Bresnick (University of Wisconsin Medical School, Madison, Wisconsin). MSCV-Mam (12–74)-EGFP was a kind gift from Dr. Pear (University of Pennsylvania). The tetracycline-inducible system T-Rex (Invitrogen) was used to create a cell line with inducible NICD expression. First, the Myc-NICD cDNA was amplified by PCR and cloned into the BamHI/SalI site of pcDNA5/TO (Invitrogen). The human breast cancer cell line 231BrM was transfected with pcDNA6/TR encoding the Tet repressor, and a stable cell line (231BrM/Tet) was generated. Then, the pcDNA5/TO/Myc-NICD expression plasmid was stably transfected into the 231BrM/Tet cell line, and the resultant clones were designated as 231BrM/Tet-NICD.
Western blot analysis was performed as described previously using antibodies against JAG1 (1/500; Cell Signaling), IL-1β (1/500; R&D), GFAP (1/500; Cell Signaling Technology), HES5(1/500; Millipore), P50(1/1000; Thermo) and α-tubulin (1/1000; Cell Signaling Technology; Bandyopadhyay et al, 2006).
Total RNA was isolated from the cells and reverse transcribed as described previously (Bandyopadhyay et al, 2006). The cDNA was then amplified with a pair of forward and reverse primers for the following genes: rat JAG1 (5′-GGTGGACAGCTCTGTGACAA-3′ and 5′-CAGCCTGGAGAACACTCACA-3′), ratJAG2 (5′-CTCCTCATTCGGGGTGGTAT-3′ and 5′-GTCGTCATCCCCTTCCAGT-3′), hJAG1(5′-GATCATGCCCGAGTGAGAA-3′ and 5′-ATCGTGCTGCCTTTCAGTTT-3′) ratDLL1 (5′-CAGGGTTGCACATTTCTCC-3′ and 5′-GCACGGACCTCAAGTACTCC-3′), ratDLL3 (5′-CCTGCGCGCTGAATGTC-3′ and 5′-CATCGAAACCTGGAGAGAGG-3′), ratDLL4 (5′-CACACACTGGACTATAATCTGG-3′and 5′-ACACATTCGTTCCTCTCTTCTG-3′), HES1 (5′-CTATTATGGAGAAAAGACGAAGA-3′ and 5′-CCTCTTCTCTCCCAGTATTC-3′), HES2 (5′-AGAACTCCAACTGCTCGAAGCT-3′ and 5′-CGGTCATTTCCAGGACGTCT-3′), HES5 (5′-TCCTCTCGCCTGTAGGGAAG-3′ and 5′-GCGAGCCCCGGCACTACAAAT-3′), HEY1 (5′-AGATAACGCGCAACTTCTGC-3′ and 5′-TGGATCACCTGAAAATGCTG-3′), and β-actin (5′-TGAGACCTTCAACACCCCAGCCATG-3′ and 5′-CGTAGATGGGCACAGTGTGGGTG-3′). For HES5 TaqMan PCR (5′-CTGATGCGCGCTCACAGT-3′), and (5′-CATGCACCCACCCAT ACAAA-3′); TaqMan probe TCTCCACGATGATCCTTAAAGGATT. PCR reactions were performed using DNA Engine Opticon 2 system (MJ Research) and the Maxima® SYBR Green qPCR Master Mix (Fermentas Life Science). The thermal cycling conditions composed of an initial denaturation step at 95°C for 5 min followed by 40 cycles of PCR using the following profile: 94°C, 30 s; 58°C, 30 s; and 72°C, 30 s.
Metastatic diseases are responsible for the majority of the deaths in breast cancer patients, and brain is one of the most common metastatic sites. The metastatic tumour in the brain profoundly affects the cognitive and sensory functions as well as morbidity of patients, and the 1 year survival rate among these patients remains less than 20%. However, little is known about the pathogenesis of brain metastasis, and therefore, it is of paramount importance to elucidate the molecular mechanism of metastatic process in order to define a specific therapeutic target.
In this report, we found that (i) metastatic breast tumour cells in the brain highly expressed IL-1β which can 'activate' astrocytes, (ii) this activation significantly up-regulated the expression of Notch ligand in the reactive astrocytes, which in turn activated Notch signalling pathway of CSCs upon direct interaction, (iii) the activated Notch signalling in CSC then up-regulated HES5 followed by promoting self-renewal of CSCs, and (iv) BBB-permeable notch inhibitor, Compound E, can significantly suppress the brain metastasis growth in our animal model. These results represent a novel paradigm for the understanding of how metastatic breast CSCs re-establish their niche for their self-renewal in a totally different microenvironment, which opens a new avenue to identify a novel and specific target for the brain metastatic disease.
This study has three major impacts. First, we have revealed a novel pathological mechanism by which breast CSCs establish a niche in the metastasized brain through interaction with activated astrocytes. Secondly, we have identified a vicious paracrine loop of IL-1β and Notch signalling through direct interaction of CSCs and astrocytes, which promotes the growth of metastasized CSCs. Therefore, these discoveries open a window of opportunity to identify a novel therapeutic target for brain metastasis. Finally, we found that a BBB-permeable Notch inhibitor can indeed serve as an effective therapeutic drug to suppress metastatic growth of breast cancer in the brain. We do believe that these findings are very timely contributions to the field of tumour microenvironment and cancer stem cell research and also provide a paradigm shift in our future development of targeted therapeutic drugs for the brain metastasis.
Human breast cancer specimens were obtained from surgical pathology archives of the Akita Red Cross Hospital, Iwate Medical School and Cooperative Human Tissue Network. Human breast cancer brain metastasis samples were obtained from CHTN. All of the tissue sections were obtained by surgical resection. Sections of 4 µm thickness were cut out from the formaldehyde-fixed and paraffin-embedded tissue specimens. The sections were deparaffinized and antigens were retrieved by heating the slides in 10 mM sodium citrate (pH 6.0) at 85°C for 30 min. The slides were treated with 3% H2O2 and then incubated overnight at 4°C with anti-IL-1β antibody (1/100; R&D Systems, Inc.) and anti-HES5 antibody (1/200; Millipore). The sections were then incubated with secondary antibodies and visualized using the Envision-plus kit (Dako Corp.). For frozen sections, 5 µm-thick sections were cut out from the OCT embedded tissue specimens. Slides were fixed with 95% ethanol followed by incubation with 3% H2O2. They were then incubated overnight at 4°C with anti-IL-1 β goat polyclonal antibody (1/200; R&D).
Cells were plated (1000 cells/ml) in ultra-low attachment plates (Corning, Acton, MA, USA) with DMEM/F12 supplemented with 2% B27 (GIBCO), 20 ng/ml EGF (Sigma), and 4 µg/ml Insulin (Sigma). Mammospheres with diameters over 100 µm were counted and data was represented as the means ± SEM.
Cells fixed with 70% ethanol were washed with PBS and blocked by 2%BSA for 1 h. After blocking, cells were washed again with PBS and incubated with anti-JAG1 rabbit polyclonal antibody (1/200; Cell Signaling Technology), anti-NICD (1/200, Cell Signaling Technology) and anti-GFAP rabbit polyclonal antibody (1/200; Cell Signaling Technology) overnight at 4°C. Cells were then incubated with anti-rabbit IgG Alexa Fluor (R) 555molecular probe (Cell Signaling Technology) for 1 h at room temperature. Fluorescence images were taken by a fluorescent microscope. For frozen section staining, slides fixed with methanol were washed with PBS and blocked by 2%BSA for 1 h. After blocking, the slides were washed again with PBS and incubated with anti-JAG1 rabbit polyclonal antibody (1/200; Cell Signaling Technology), anti-GFAP rabbit polyclonal antibody (FITC conjugated, 1/200; Cell Signaling), primary anti-CD44 conjugated with FITC (1/200; Cell Signaling Technology) and anti-ESA conjugated with Alexa 555(1/200; Cell Signaling Technology) for 1 h at room temperature. Fluorescence images were taken by a fluorescent microscope (Olympus IX71).
For cell surface marker analysis, cells were suspended in FACS buffer (PBS with 0.1% BSA and 0.1% Tirton X100) followed by incubation with FITC conjugated anti-CD24 (eBioscience, Inc.), APC conjugated anti-CD44 (BioLegend), PE conjugated anti-ESA (eBioscience, Inc.) and PE conjugated anti-CK18 (Santa Cruz Biotechnology, Inc.) for 15 mins at room temperature. Cells were then washed with PBS and re-suspended in PBS for FACS analysis using the C6 Flow cytometer (Accuri LTD).
Cancer stem-like cells (CSCs) were isolated by magnetic bead sorting using MACS Separator (Miltenyi Biotec). 231BrM cells were incubated with specific antibodies as follows: anti-CD24-Biotin (StemCell Technologies), anti-CD44-APC (BioLegend), and anti-ESA-Biotin (Miltenyi Biotec). CD24−/CD44+/ESA+ cells were then enriched by using a MACS magnet and MS columns (Miltenyi Biotec). All MACS procedures were performed according to the manufacturer's instructions.
RNA was extracted, labelled and hybridized to Human gene 1.0ST chip (Affymetrix) using the manufacturer's protocol. Normalization of the chip was performed using RMA algorism. These expression data were submitted to the NCBI Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE25976. For cancer cohort analysis, we compiled a microarray dataset of 710 patients from GEO (accession numbers: GSE12276, GSE2034, GSE2603, GSE5327 and GSE14020). These datasets were all normalized using MAS5.0 and each microarray was centred to the median of all probes. For each patient, brain-metastasis free survival is defined as the time interval between the surgery and the diagnosis of metastasis.
For i.c. injection, the mouse was anaesthetized by intraperitoneal injection of ketamine/xylazine. After wiping the injection site with Betadine, 28-gauge needle was inserted into the second intercostal space 3 mm to the left of the sternum. When the needle was inserted into the left ventricle of the heart properly, blood pumped into the syringe. Cell suspension in100 µl PBS was injected slowly over a 20–40-s period. A successful intracardiac injection was indicated on day 0 by systemic bioluminescence distributed throughout the animal. All protocols were approved by the University of Mississippi Medical Center Institutional Review Board.
FX and KW developed the hypotheses, designed the experiments and wrote the manuscript; FX performed most experiments and analysed data; AK and MI conducted experiments including Western blotting and qRT-PCR; PRP assisted with generation of lenti virus expression plasmids; HO helped in clinical cohort data analysis; MW and SS provided technical support in tissue culture; SKP, BEM and EP performed immunohistochemistry and provided pathological review; SH provided breast cancer patients' specimens and pathological review; AK, HO, WL, SS, YL, KW and KF helped in mouse work; YYM and AW provided key experimental reagents and experimental advice; KW directed this study.
This work was supported by NIH (R01CA124650, R01CA129000 to KW), the US Department of Defense (BC085590, BC085424 to KW, BC096982 to AK), and Susan Komen Foundation.
Supporting Information is available at EMBO Molecular Medicine Online.
The authors declared that they have no conflict of interest.
As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.
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:
Plants vs Zombies Heroes v 1.16.10 Mod Apk
Download Game Plants vs Zombies Heroes v 1.16.10 Mod Apk – Selamat pagi sob 😀 berhubung lagi liburan jadi gaaada salahnya saya update game ringan dulu yang mungkin bisa kalian mainkan selagi bosen hehe. Dari game yang sangat populer yakni Plants vs Zombies Heroes v 1.16.10 Mod Apk. Siapa sih yang gak kenal dengan game yang satu ini ? pasti pada pernah mainin kan hehe kini Plant vs Zombies ini sudah update ke versi terbarunya sob yang mau mainin versi modnya yuk langsung dicek aja
Fitur :
Collect and choose from 20 unique Plant and Zombie Heroes and unlock their lawn-inspiring super powers. Will you pick Plants and deploy the masked, sharp-shooting avenger, Green Shadow? Or will you choose a Zombie Hero and call on the superbly superficial Super Brainz, Zomboss's secret weapon with a powerful punch and fondness for hair gel? Each Hero's selection of super powers will help determine your team's winning strategy.
Every Hero needs a crew. Build yours and diversify your skills by collecting teammates – from the PvZ characters you know and love, to daring new plants and zombies. There are hundreds to discover! Devise distinct strategies for your Hero by selecting compatible teammates and experiment with devastating combos. Or, use Auto Team-Builder to quickly create your team, try out new strategies, and automatically upgrade your teams as you collect new characters.
Journey through the universe of PvZ Heroes as each action-packed skirmish takes you further along an ever-blooming map. Battle brain-thirsty or botanical foes – each side has a different path to follow. Encounter challenging bosses with tricky powers and abilities, and defeat them for legendary rewards!
Test your team when you take on other players in exciting real-time matches. Earn rewards for defeating your bite-iest adversaries, and build your bank when you complete Daily Missions from Crazy Dave. Who will become the ultimate horticultural hero? Harvest your courage – the battle is on!
What's New
What's In The MOD APK :
- High Sun
- Requires Android: 4.1 and Up
- Version: 1.16.10
ScreenShot :
Download Plants vs Zombies Heroes v 1.16.10 Mod Apk
- Plants vs Zombies Heroes v 1.16.10 Mod Apk | Via GoogleDrive
- pass : www.yogitutorial.co


text:
The New New Bond Girl
Apparently, the producers of the James Bond movies are trying to make me look foolish despite my praise for their uncanny hot-girl-finding abilities. As I reported yesterday, British actress Gemma Arterton is slated to be in the new Bond outing. But today, the BBC is claiming that uber-hot Ukrainian model Olga Kurylenko will also be in the film, and will 'eclipse' Arterton's role. If you ask me, it's an embarrassment of riches for Daniel Craig.
Stand by for updates as the female component of the new James Bond movie continues to balloon.


text:
This year the location of the festival is Sofiensaal, situated in the central part of the city. The location with its own history! Built in 1826, completely burned down in 2001, but rebuilt and opened once again in 2013. At different times it was a swimming pool, recording venue, Russian banja (sauna), concert hall, where balls and operas were held. Johann Strauss I performed there regularly and conducted at the opening ball of the house in 1848. Many of the Strauss family's waltzes were first performed there. Now came the time of PRO-AM Competition to be held in this magic venue.
This year we prepared 2 different Packages for you. First package is for beloved by our participants GRAND HOTEL WIEN, which has been famous the world over for exquisite hospitality and refined luxury. Here you can choose between 2 room categories. The shuttle bus to the event location will be provided.
Learn more about Grand Hotel Wien.
The second package is for RUBY SOFIE HOTEL, located in the venue of the festival, what makes it so convenient. The philosophy of the hotel is lean luxury – "Pay less for the luxury, concentrating on what really matters". You can choose out of 5 room categories in this hotel. Attention! No twin rooms, all double rooms have 1 double/king-size bed.
Learn more about Ruby Sofie Hotel.
First time at "Amazing Vienna" - 2017 ADW International Congress "PRO-AM System". The congress will be held on 10th December from 10:00 to 15:00. Please, plan your flights in order not to miss the great opportunity to get the latest information from the world's best couches and dancers.
The Congress will run in 2 parts:
- Part I (10:00-12:30) - theory on business model, structure, financial, ethics etc. (for PRO/Teachers only)
- Part II (13:00-15:00) – Workshop in American Smooth & Rhythm (open for everybody)
- Nick Kosovych, USA
- Leonid Pletnev, Russia-Austria
- Nick Cheremukhin & Viktorija, USA
The number of participants is limited, please buy your tickets in advance.
Tickets for ADW International Dance Congress
The final stage of the European Open RRO-AM Grand Prix Series will be held at "2017 Amazing Vienna", where the Grand Prix Champion will be announced.
And for the third time, only in Vienna – Viennese Waltz World Open PRO-AM Championship! The champions of the National Subtitles will get a free of charge participation in this competition. Already among them are Veronika Tsareva - Andrey Motyl from Russia and Svetlana Sergeeva – Alexey Katushkov from Russia.
Grand Titles competitions are open for PRO-AM couples from all dance organizations and/or independent couples. Only for these competitions, we use a "couple vs. couple" judging system instead of standard "student vs. student" usually used in PRO-AM Competitions.
For your beauty and comfort we provide makeup and hairstyle directly at our festival. Please, make an appointment in advance. Information here.
We are delighted to announce world's best couples performing at the "AMAZING VIENNA" Dance Festival. With those great dance masters it will be a night to remember!