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Don't forget previously noted opportunities closing this week.
ReThink Media is actively recruiting for:
- Associate Director for Voting Rights, Democracy Communications Collaborative – apply by Jan. 15
- Media Associate. Peace & Security Communications Collaborative – apply by Jan. 23
The Chamber of Digital Commerce has just passed along a call for Interns throughout 2017 (including the Jan.-Apr. term) – preference is given to applicants pursuing computer science, economics and finance degrees and/or an interest in public policy.
Aetna (search DC location or use req # for keyword)
- Senior Government Relations Specialist, NAIC (37690)
- Senior Government Relations Specialist, Medicare (37691)
- Senior Government Relations Specialist, Medicaid (37692)
Fair Trade USA, a social enterprise that promotes sustainable development & corporate social responsibility, seeks Vice President, Marketing & Communications to join their Oakland, CA-based team – search managed by Waldron
Honda Motor Co. – Manager, Federal Affairs – seeking candidates with 10-12 years of experience in federal government relations or on Capitol Hill
Palestine/Israel human rights & non-violence advocate Just Vision seeks Manager of Strategic Relationships with 3-5 years of experience in individual gifts, external relations, communications, and database management – DC or New York
Hager Sharp (scroll to find)
Public Policy & Advocacy Advisor (scroll to find) – minimum of 7 years' experience with in international relief & development, PR/communications desired – Capitol Hill background also sought – see also main career page
Urban Libraries Council – Communications Director – seeking candidates with 7-10 years of experience in communications for an international association, non-profit, public library system, or city/county government.
NAIOP/Commercial Real Estate Development Association – Director of Federal Affairs – 5 years' relevant experience working on federal legislative and/or regulatory issues required; Capitol Hill experience a plus
Milken Institute
- Director of Development, Melanoma Research Alliance
- Editor – DC or Santa Monica, CA
- Intern supporting Faster Cures
Sister Cities International – Director of Conferences & Events (scroll to find) – 3+ years of relevant logistics & event management experience required.
Literacy Council of Northern Virginia – Communications Coordinator – 2+ years' experience in marketing, communications, design, or related field required.
Visa – Director, Government Relations – 8-10 years of substantive experience in government relations, legislative affairs and advocacy required; background in the payments, technology, judiciary or intellectual property sectors strongly preferred.
State Dept. – Public Affairs Specialist – $66k-$103k – apply by Jan. 12
George Washington University
Community Program Organizer, School of Public Health – casual/on-call position – Baltimore, MD
… and finally: Royal Caribbean UK & Ireland seeks Intern to take a three-week tour of paid Instagram inspiration this Summer – apply with your best travel pics by Jan. 31


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Get Your Tiger Gear!
The DWU Campus Bookstore has more than just books! In addition to buying and renting textbooks, you'll also find official DWU clothes, gear and gifts, school supplies and more. And when you're done with your textbooks, you have the option to trade them back in.
We're located in the McGovern Library and are open Monday through Friday from 8 a.m. until 5 p.m.
Check out the DWU bookstore online.
If you have any questions, contact us or give us a call at (605) 995-2805.

subset: commoncrawl


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Rave Reviews
"Jesi B did an absolute amazing job on capturing my kids personalities and beauty. I had been looking forward to working with her for a long time and waiting patiently for the perfect time of year with the perfect location. Boy did she deliver. We were fortunate to get the amazing barn location and Jessica spent endless hours finding that perfect spot....now that is customer service! I have had shoots before with my kids and the preparing and anticipation of it always sent me crazy, but Jessica was so great in giving me tips for clothing and when we arrived on location, she had such patience and ease that I was immediately calmed. My kids warmed up to her and really showed off their personalities that she was able to capture so well. I was beyond thrilled with the proofs and my order of a huge collage up my stairs is on order and I am so anxious to see the final pieces of her art on my wall. Thank you Jessica for creating such beautiful memories that I will forever cherish!!" T. Van Houten
Working with photographer Jessica Brosamle gifted me with much more than I'd planned for. During our photo session, she provided me not only with a full complement of amazing headshots for my business but also an experience that helped me to see my personality and uniqueness shine through the lens of her camera. Her professionalism and charm made me comfortable from the start and her commitment to ensure that my experience – and my photos – were only the best and has made me an immediate fan. I am confident that my photos will captivate clients and readers throughout my business presence and it is due to the unique talent of JesiB. Dawn Mena, Captivating Copywww.getcaptivatingcopy.com
Jessica-Thank you so much for taking such beautiful maternity and announcement pictures for our family. You created a very comfortable and relaxed environment for my kids. Thanks to you, they actually cooperated AND had fun! You did an amazing job capturing the moment. Thanks again,Tessa H. and Family
I'm so overwhelmed with emotion right now! Those are the most beautiful pictures we've ever had, Jessica. You have captured the very essence of our family and I am so grateful! From now on, you will be our family photographer for all of our special memories. You have such an amazing gift! Words fail to express my gratitude. Kelley F.
Oh my gosh, these bring tears to my eyes! She looks so beautiful! I cannot believe that this is what you got from our crazy session - you have a real talent, Jessica. Emma wasn't exactly in the mood for pictures and I was really worried that we wouldn't get any good shots of her. Between the peeing, pooping, and crying - you managed to get some really amazing shots of her. I love them all and choosing is going to be so hard. I am not too busy to look at these gorgeous pictures! Thank you again for being SO patient and kind with Emma - I can't wait for you to take more pictures of her! Cheryl Y.
Hi Jessica,I have a super busy day at work today, but wanted to take a minute to say THANK YOU again for doing such an awesome job with our family pictures. I cannot even begin to tell you how many compliments I have gotten on how great they are. As I'm sure you noticed, I posted a few on fb last night but also shared them with a few family members and friends that are not on fb. We are very happy with the way they all turned out and will definitely be using your services in the future and recommending you to others! Cindy S.
" Jesi has an artful eye for capturing the true essence of each member of my family. She definitely brings out the "WOW" factor in every frame! Her engaging and creative nature makes for a very comfortable, fun experience in front of the lens. Your search for the best ends here!" ~ K Fox
" I just can't get enough of Jesi B! The way she handled my newborn so gentle and got the BEST pictures of him in the poses she put him in. She also did my maternity pictures...never thought someone could make me look so good and feel sexy at a time like that! Well she did it! They were beautiful, look no further you have found your girl here!" Amber S
"Jesi B took our family pictures and we loved them all so much we had a difficult time choosing just one to use for our Christmas card. She was so easy to work with and took every detail into consideration. We have received so many compliments and will be using her services again for newborn pictures when our 3rd daughter arrives early next year." C. Smith
Jesi captured pictures of my family that no portrait studio has ever done. She is patient with kids and has a great eye for locating amazing scenery! I love the fact that I was able to always communicate with her and get her opinion on the clothes I picked out for the photo shoot...... I cant wait to book again !!! C Keeler
" Jesi B did an amazing job on our family photos for 2011!! Just got more pictures today and we ABSOLUTELY love them. She is always so friendly and we will definetly use her again for our next set of pictures! Can't wait! " Sydney R
" Jesi B Photography captured the most amazing memories for my family. Jessica has a natural way when working with children that made my girls feel so comfortable. They were having so much fun they forget they were having their pictures taken! The photos she took that day are all over my house and every time a new person sees them they rave about how beautiful they are. I will be using Jesi B the next time and will continue to highly recommend her!!!! " M. Steinberg


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Inspired in the simplistic lines of early Asian design, the Tokyo family of coffee tables brings back minimalistic elegance that brings balance to the surrounding elements. The low profile combined with the geometric lines of the Tokyo family are the perfect complement to enhance the mood higher and taller objects brings to living spaces. Dimensions: H14 x W59 x D24 in (H35 x B150 x T62 cm)
Note: Please feel free to contact us for large, high quality images.
© 2007-2011 evincodesign.com. All rights reserved.


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As of now, Goodreads has links on book pages to numerous retailers. For authors, this means that when a new reader checks out a book on Goodreads, she has the option to be directed to the retailer of her choice to make a purchase. A comment on the announcement blog post, left by a Goodreads employee, states, "... We have no plans to change how the links on our book pages work. ..." (emphasis mine) Maybe I'm a cynic, but I can't see the links staying as they are. (More likely, this is Goodreads' way of saying, "We won't change the way links work, but once Amazon is in control, all bets are off.") I can't imagine Amazon would ever, on a site it owns, link directly to its competition. Just like on IMDB, another Amazon-owned site, all links will point back to the mother ship.
Reviews, the backbone of the Goodreads community...with Amazon playing Big Brother, will the Goodreads review policy be changed? A statement, again found in the comments thread of their announcement post, added by a Goodreads employee, says, "We don't expect to make changes to our review policy. Our reviews are obviously a strength of our site, and we don't plan to change how we handle them." (emphasis mine) Again, the clever wording leads me to believe that they are leaving things open to change under the reign of King Amazon.
Amazon's review policy, while seemingly innocuous, is actually a huge problem for reviewers, many of whom have stopped reviewing on Amazon and now review strictly on personal blogs and Goodreads, because:
- Amazon claims ownership on reviews, meaning all the time and effort you put into crafting your review? ... that now belongs to Amazon, and they can use your words however they see fit.
- Authors are not allowed to review books on Amazon. You're an author? And a reader? Preposterous! Not too long ago, Amazon quietly (read: without warning) began deleting any reviews written by authors of books in the same genre. So I'm a YA author. And, not so coincidentally, I LOVE to read YA. But review it? Not allowed, according to Amazon.
- Amazon has very strict rules about the language reviewers use, ostensibly because they now own the words and don't want to be held responsible for anything inflammatory or offensive. While I personally prefer to read reviews that are honest, but kind (yes, even a negative review can be kind), many reviewers like to be snarky and even use - gasp! - some foul language. That's a no-go on Amazon.
Goodreads is a place where reviewers can say what they like, how the like, without fear of having their review removed without warning, or fear of their words being used to benefit a company (when they themselves are no longer even allowed to use the review on their own website, technically). Like I said above, the statement as of today is that there is no expected change for the review policy, but given Amazon's track record, reviewers are nervous, and rightly so.
While, on the author side of things, changes to the Buy Links and review policy may be the most worrisome aspects of this acquisition, readers may have a bigger issue with:
The loss of a neutral place to meet up and talk about books we love.
For years, that's exactly what Goodreads was. We could go and talk with fellow book nerds about the books and authors we adore, and there was no pressure to buy, because Goodreads had no vested interest in book sales. With Amazon owning the company, you better believe there's a vested interest; every member is now a potential customer. How long before our Goodreads pages look like Amazon sales copy?
So what is a reader to do?There is very little out there by way of social network sites geared toward readers. Goodreads was basically it. Sure, Shelfari had a brief run where it looked like it might become popular. Then Amazon bought the site, failed to support it, and let it all but die. (Will this be the future for Goodreads? I doubt it.)
Another option is Library Thing. On the surface, it looks great: perhaps not as fully featured as Amazon, it has potential. After one adds 200 books, it is no longer free, but the pay options are very reasonable: $10 a year, or $25 for life. Under the surface, however, you will find that Library Thing is owned 40% by Amazon. (A note on that link: AbeBooks is now owned by Amazon, which is how ownership of Library Thing was acquired.)
So, yeah. If you want social networking revolving around books? It's owned by Amazon. That's your option, sorry. This Goodreads purchase just blasted open a HUGE niche in the social network market. Geek Husband and I are already scheming. :)
Your turn! What do you think about Amazon buying Goodreads? Are you concerned at all? Leave it in the comments.


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Naperville Law Firms
About Randall J Yorke
Randall J Yorke
1155 S Washington St
Naperville, IL, 60540
This listing is under a verification process.
If you're the owner please contact us.
* Disclaimer: While every care is taken to ensure the accuracy of the data, This site does not represent or guarantee the accuracy, reliability, completeness or suitability of its content for any particular purpose. Please contact us if you discover an error.
Randall J Yorke map


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Proverbs 15:19 "The way of the sluggard is blocked with thorns, but the path of the upright is a highway." (NIV) Today's Thought Have you seen the Zootopia movie? It came out from Disney last year. In one scene, police officer Judy Hopps (a bunny) and Nick Wilde (a fox) go into the Department of Motor … Continue reading The pablum of the weak
Month: March 2017
A verse we read with one eye closed
I don't know about you, but when I read the Bible there's a silent conversation happening in my head. With a lot of stories and verses I'm saying things like 'Yeah, that's awesome, that's good too, oh yup…" But then I run into passages like this one and yup turns into gulp: "If anyone wants … Continue reading A verse we read with one eye closed
Inflated with purpose
Hebrews 13:1 "Let mutual love continue." (NRSV) Today's Thought These days I'm thinking a lot about purpose. I just think that knowing your purpose has the potential to inflate your days with meaning, energy, zest and faith. And if you don't ever think about how your daily purpose connects up to God's bigger plans, I … Continue reading Inflated with purpose
In Christian teaching over the past 70 or 80 years there has been an emphasis on heaven and life after death. In a sense, I get that. It's huge! But I sometimes worry it has overshadowed (to an unhealthy degree) what we're summoned to do in this life BEFORE we die. One of the thinkers … Continue reading Co-Renovators
Your Magic Toeshoes
Alongside literary giants like Shakespeare and Tolstoy are the Berenstain Bears. (Maybe that's a stretch, but just go with me on this one.) In one of their timeless classics we learn about The Magic Toeshoes. Basically, a young bear wants to be a ballerina. But her toes are wobbly. So an old ballet master takes pity … Continue reading Your Magic Toeshoes
What's the point?
Micah 6:8 "And what does the LORD require of you?" (NIV) Today's Thought What's the point? Have you ever asked yourself that question? I have. It's a question we ask when we're feeling overwhelmed or alone or frustrated. But it's also a question we ask as life goes on and we haven't figured out how … Continue reading What's the point?
When conformity and self-discovery fall flat
In the movie Witness, a young Amish woman named Rachel falls in love with a police officer who is clearly not Amish. Her father Eli tells her it's forbidden and that the community elders could have her punished for her actions which, he says, are childish. "I'll be the judge of that!" she says. "No, … Continue reading When conformity and self-discovery fall flat
Hey! I'm one of the important ones!
Proverbs 3:34 "…but to the humble he gives favor." (ESV) Today's Thought Recently I was at the hospital and saw a group of doctors having coffee together. All of them were wearing stethoscopes around their necks. I wonder why. Maybe they just didn't have time to put them away. It's hard to be sure. And I … Continue reading Hey! I'm one of the important ones!
The more seriously you take your death…
Genesis 3:19 "...for you are dust, and to dust you shall return." (ESV) Today's Thought There are certain things we just don't like to think about. Things like taxes. Things like flooded basements. Things like that embarrassing picture of you in grade 11 going up on Facebook! And things like… Death. But do you know … Continue reading The more seriously you take your death…
Staring at a loaded gun she still said Yes
Is your belief in God rooted in how good God is to you, or in the goodness of God? One of the most inspiring biblical stories is of Shadrach, Meshach and Abednego. Because of their unswerving loyalty to God they refuse to worship the false gods of King Nebuchadnezzar. So they're threatened. Either bow down … Continue reading Staring at a loaded gun she still said Yes


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Steffen Rayburn-Maarup: Speech Bubbles and Lettering
Part of this assignment was to make two pages out of written text with no visual references.
The first two pages are inked by myself and the smaller two pages were done by one of my classmates, Tina Burholt.
I'm aware there's a severe lack of speech-bubbles and text, but it was un-needed in this case, however, it was a solid exercise and very good lecture about placement and planning for text and speech-bubbles.


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Previous File Entry | Next File Entry
- Wed, 17:43: Should we worry about "RomneyCare" & his global warming? One would think the Republican-lead US House would keep any GOP president in check.
- Wed, 19:13: An Iowa couple married for 72 years died one hour apart last week in the hospital as they held hands. KCCI Des Moines: http://t.co/HeOeVKHe
- Wed, 20:07: 2011 takes the title as wettest year in Cleveland weather history dating back to 1870s | http://t.co/oZiUjqe4 http://t.co/wiPXs5o8
- Wed, 21:51: Texas Rangers said that if they lose the World Series they'll do what Democrats have done - just blame Busch.
- Wed, 23:12: #OccupyCleveland intends to break the law, remain on Public Square. Permits expire midnight Friday. #HurryWinter http://t.co/NhcpzzQU
- Wed, 23:27: Sorry, I should have tweeted #OccupyCleveland plans to block Christmas decorations from Public Square. #MyApologies http://t.co/NhcpzzQU
- Thu, 12:09: Qaddafi got killed. I feel real bad another dictator bit the dust - NOT.
- Thu, 13:44: You can go ahead and clap. Go ahead, nothing wrong with it.


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I have written over 80 books for K-YA. Many are titles in my middle grade series: American Diaries, Survival, and Hoofbeats. The Unicorn's Secret and The Faeries' Promise series are for young readers. ((excerpts are up on http://www.kathleenduey.com)) Dark, atypical fantasy for teens and adults is my new love. My first YA novel,Skin Hunger, was a 2007 National Book Award finalist. Sacred Scars was released in 2009. The third book in the trilogy is finished and in the production process now.
- kathleen duey
- http://www.kathleenduey.com / Twitter: @kdueykduey FB:www.facebook.com/kathleen.dueyhttp critique information here: http://kathleenduey.blogspot.com/2011/08/critiques-short-answer-is-yes-if-my-own.html ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ FABULOUS world-wide (free shipping!) bookstore:: http://www.bookdepository.com/
Monday, November 24, 2014
Limori : Book Three Post # 23
I made this basket years ago...long before writing took over my life. I noticed it this morning high on a shelf, and started thinking about Sadima, her childhood, her weaving, her easy connections with animals and her love for everything alive. Then Franklin had found her that long ago day when he was looking for children Somiss wanted to observe. I wish I could go back there now and when she started down the dirt road to go to Limori, I wish could catch her hand and talk her out of it. But I can't. Not now.
2 comments:
- MollyMom103 said...
I need her to take this terrible journey. I'm not the only one.
- kathleen duey said...
Yes. I know. You are right and I am afraid, too....but I know she has to do this...
Thank you ....for saying it aloud.
Subscribe to: Post Comments (Atom)


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And a big 'hola' to everyone that has posted a dare so far. We are big fans and I recently tried tanning in just a thong which I have never worn before in public. We went to the beach for the day and I ended up a little red in some areas that are not used to seeing the sun (i.e. butt cheeks). I'm sure you'll be able to see what I'm talking about in the pictures. We'd love to get your feedback and to save some of you some typing, yes, I know to wear sunscreen next time hehe.
gabyl55 @ yahoo.com
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Custom Made Flags
Tudor House is very proficient at producing custom-made flags for
- Businesses
- Companies
- Local Governmant offices like the Towns, Cities and Shires
- Sporting Clubs like Bowling Clubs, Yacht Clubs, Football Clubs, Basketball Clubs, Netball Clubs
- Associations
- Schools
- Religious groups
Artwork is required in a high resolution file, otherwise simply send us a copy of the logo you would like us to print on the flag and our highly specialized team will provide quality workmanship . Click here to view the artwork requirements.
Custom made flags can be made in several shapes and sizes to suit the occasion. Flags in the size of 1800 mm x 900 mm are recommended for 6 m Flag Poles.
Although printed flags are more the norm these days and also more economical, Tudor House can also create Appliqued flags for that special, authentic look.


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Friend's Email:
Subject: I have found a DVD that I think you would enjoy
|Nature San Diego Zoo
Actors: F. Murray Abraham, Paul Christie, Howard McGillin, Peter Coyote, Jay O. Sanders
Director: Nigel Cole
Genres: Kids & Family, Educational, Documentary
NR 2004 1hr 0min
Larger Image
Movie Details
Similar Movies
Similarly Requested DVDs
Movie Reviews
Fine episode of Nature
Amateur Stargazer | 04/07/2007
(5 out of 5 stars)
"This episode is a tour of the San Diego Zoo and it shows how the animals are cared for. It is a very enjoyable program for any animal lover or fan of the Nature series. This is one of the few older episodes available with narration by George Page. The animals featured include pandas, cheetahs, rhinos, gorillas, giraffes, and many more. There are also two short videos on the DVD made by the staff of the zoo to educate the public. One is about the polar bears and the other discusses marsupials-- mainly the koala. Polar bears and koalas are among the zoo's diverse exhibits of animals. The videos are good bonus features for an entertaining, appealing DVD."


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Cute Cats and The Arab Spring
Filed under: Podcasts | |
I saw this mentioned on BoingBoing last week: it is a discussion from the 2011 Vancouver Human Rights Lecture series discussion the importance of mainstream internet venues in democratic movements: unlike small networks (whose shutdown may be invisible to the mainstream), use of popular networks like Flickr, Facebook and Youtube means that heavy-handed shut-downs will likely make more people aware that something is going on. A very Freakonomics bit of analysis!
CBC.ca | Cute Cats and The Arab Spring. Via BoingBoing
photo credit: Dr. Hemmert


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Today's volatile marketplace demands that both corporates and financial institutions have best-in-class technology, people and process to remain competitive and profitable. Decision-makers in every industry are being asked to increase efficiencies, maximise existing resources and enable transparency across the financial supply chain to manage liquidity and increase profitability. From banks and financial institutions to insurance providers and card processors, GXS has a solution to support your B2B transaction needs.
The GXS Financial Integration Suite has been designed to provide the ideal combination of services and software to allow financial institutions and corporations to connect and exchange critical financial data in a secure, reliable and scalable environment. With GXS, you have access to the solutions you need to integrate existing ERP systems, leverage legacy platforms and successfully reallocate resources to increase straight-through-processing and visibility of financial transactions.
Learn how the GXS Financial Integration Suite can support your various product lines:
- Cash Management—Through our Managed Services platform GXS can manage the connectivity between your corporate clients' ERP systems and your bank's cash management applications. GXS enables straight-through processing of transactions such as payment instructions and account statements using a variety of messaging standards including SWIFT, ISO 20022 XML, SAP IDoc and EDI. By leveraging a Managed Services approach, GXS clients have been able to demonstrate measurable improvements in time to on-board new clients and flexibility to support new business.
- Supply Chain & Trade Finance—Through our Active Orders and Active Logistics products, you can gain visibility into the supply chains of your corporate clients. Numerous banks have leveraged the visibility to Open Account transactions gained through GXS services to build new Enhanced Open Account and Supply Chain Finance products. Time-to-market can be accelerated by leveraging GXS expertise in on-boarding of suppliers in emerging markets and mapping of documents such as purchase orders, bills of lading and commercial invoices.
- Commercial Cards—Through our Managed Services GXS can manage the connectivity and integration with your partners—whether they are merchants, issuers, processors or corporate clients. Our secure, hosted platform for message and file exchange complies with the PCI (Payment Card Industry) Data Security Standards. GXS Managed Services can support a wide variety of use case scenarios. In the commercial cards segment, GXS facilitates the integration of Level 3 card detail data with corporate accounting applications.
- Insurance Group Benefits—Through our Managed Services GXS can manage the connectivity with the human resource systems of your corporate clients and the processing applications of your Third-Party Administrators (TPAs). GXS can enable the automated exchange of employee census, reconciliation and billing files to support Group Disability, Group Life, Employee Absence Management and other group benefits products. By leveraging a Managed Services approach, GXS clients have been able to demonstrate measurable improvements in time to introduce new products and flexibility to bid on new sales opportunities.
- Securities—Through our Managed Services platform securities, GXS can manage the connectivity between broker/dealers, investment managers, global custodians and centralised depositories. GXS enables straight-through processing of equity, fixed income, mutual fund, foreign exchange and derivatives trading and enables asset servicing activities such as corporate actions and account reconciliation. GXS has experience with ISO 15022, ISO 20022 XML, FIX and other popular securities standards. GXS clients have been able to satisfy customer-specific integration needs, reduce time-to-revenue and enhance international connectivity offerings.
GXS actively participates in these financial services industry standards initiatives: Association for Financial Professionals (AFP), Common Global Implementation (CGI), International Securities Association for Institutional Trade Communication (ISITC), MACHA, NACHA and Society for Worldwide Interbank Financial Telecommunication (SWIFT).


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We all have the nebulous and sometimes daunting idea that the road to business growth lies overseas. Organizations spend millions of dollars to establish a presence in emerging markets. They hire employees from around the world. But do we really have an idea of how well this is working? What is the return on investment?
Last year, we commissioned a survey of 500 executives of companies with more than 500 employees in offices all over the world. The results have been made available in a handy infographic entitled Business Value in Language. We sought to break down the language needs of organizations as detailed as possible, including how employees with language skills have added value to their businesses.
Improved relations with customers
79 percent of our respondents believed that improved language skills among their employees led to improved customer relations. Linguistic lines are blurring, not only overseas but within the US. For example, in a time where Latinos have become the majority-minority in the country, Spanish language skills can make a significant difference in the bottom line.
Increased sales opportunities
72 percent responded that improved language skills have led to increased sales opportunities. For most, the goal in increasing their company's international footprint is to gain new customers. Obviously, without the appropriate language skills, those new customers will have a difficult time communicating their needs, and you will have a tougher time communicating your value proposition.
Increased productivity
68 percent of respondents stated that language skills have led to increased productivity. That's not surprising. In businesses large and small, due to the growth of communications technologies, teams can be made up of employees from all over the world. It's important for these team members to be able to communicate effectively. When they do, the job gets done more efficiently.
To see more results of the survey, click here to check out the infographic Business Value in Language.
Find more posts about: Business, company growth, international strategy, language learning


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"If you're unlucky enough to be hit by a car, pray it wasn't made by Audi, BMW or Vauxhall. It's a disgrace that makers are ignoring pedestrian safety when, for the sake of spending a bit more, they could potentially help save hundreds of lives each year," says Malcolm Coles, editor of Which? magazine published by the U.K.-based consumer advocacy group of the same name.
"In recent EuroNCAP tests the Audi TT, Skoda Superb and Suzuki Grand Vitara were all found to be potential death threats to pedestrians despite the fact it's cheap and easy for manufacturers to make cars more pedestrian-friendly. The cars all scored zero points and show no sign of thought given to preventing pedestrian injury in a lower speed collision," according to a press statement from Which?. "Simple design improvements - costing an average of just £53 for each new car - could save 255 lives and 6,300 serious injuries in the UK each year. Which? is also naming and shaming other popular cars that did badly in the tests, putting pedestrians at too high a risk; they include the BMW 1-series, 5-series and X5, Jaguar X-Type, Range Rover and Vauxhall Astra. "
Tests gave relatively high marks to models from Citroen, Honda, Seat and Volkswagen.


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Category: Hip-Hop music
If you like fast dance songs, the music of Hip-Hop in mp3 format will suit best. On our site you will find many albums by individual performers, as well as the great collections in the collections that you can listen even in the car, so as not to miss. Download Hip-Hop torrent is very easy, it is the easiest and most reliable way to obtain the desired music of good quality and free of viruses. We have a hip-hop can be downloaded free of charge, which is much nicer than on other portals that offer to pay first, and then have to wait for an SMS message with the code.
Recall that Hip-Hop as a music genre was born in 1979 in the African-American areas of New York. This subculture has evolved both musically and in the dance tracks. The origins of hip-hop are such genres like funk, soul, reggae, disco and others. Many DJs started to use the so-called new style of music, using the technique of scratching and recitative. The texts of this genre is raised as a socially significant, political themes and memories of bygone times. In the 90s hip – hop became especially popular, there are a large number of sub-areas: crunk, snap, R & B, reggaeton, hip – house and so on.


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- Research
- Open Access
The Wnt/β-catenin pathway regulates growth and maintenance of colonospheres
© Kanwar et al; licensee BioMed Central Ltd. 2010
- Received: 16 February 2010
- Accepted: 6 August 2010
- Published: 6 August 2010
Recent evidence suggests that epithelial cancers, including colorectal cancer are driven by a small sub-population of self-renewing, multi-potent cells termed cancer stem cells (CSCs) which are thought to be responsible for recurrence of cancer. One of the characteristics of CSCs is their ability to form floating spheroids under anchorage-independent conditions in a serum-free defined media. The current investigation was undertaken to examine the role of Wnt/β-catenin pathway in regulating the growth and maintenance of colonospheres. Human colon cancer cells HCT-116 (p53 wild type; K-ras mutant), HCT-116 (p53 null; K-ras mutant) and HT-29 (p53 mutant) were used.
Colonospheres formed in vitro exhibited higher expression of colon CSCs markers LGR5, CD44, CD166 and Musashi-1 along with putative CSC marker EpCAM, compared to the corresponding parental cancer cells and also exhibit the ability to form spheroids under extreme limiting dilution, indicating the predominance of CSCs in colonospheres. Colonospheres formed by HCT-116 cells show over 80% of the cells to be CD44 positive, compared to ≤ 1% in the corresponding parental cells. Additionally, colonospheres showed reduced membrane bound β-catenin but had increased levels of total β-catenin, cyclin-D1 and c-myc and down regulation of axin-1 and phosphorylated β-catenin. Increased expression of β-catenin was associated with a marked transcriptional activation of TCF/LEF. The latter was greatly decreased following down regulation of β-catenin by the corresponding siRNA, leading to a marked reduction in CD44 positive cells as well as colonospheres formation. In contrast, upregulation of c-myc, a down-stream effector of TCF/LEF greatly augmented the formation of colonospheres.
Our data suggest that colonospheres formed by colon cancer cell lines are highly enriched in CSCs and that Wnt/β-catenin pathway plays a critical role in growth and maintenance of colonospheres.
- Veteran Affair Medical
- Stem Cell Medium
- Colon Cancer Stem Cell
- Karmanos Cancer Institute
- Term Cancer Stem Cell
A growing body of evidence supports the contention that epithelial cancers including the colorectal cancer are diseases driven by a small set of self renewing cells, termed cancer stem cells (CSC) or cancer-initiating cells, that are distinct from the bulk of the cells in the tumor [1]. Initially identified in hematopoietic tumors, CSCs have now been identified and isolated in a variety of solid tumors that include breast, central nervous system, pancreas, skin, head and neck, colon and prostate [2–7]. CSCs share all the fundamental traits of stem cells-self renewal by asymmetric division, reduced proliferation and differentiation and resistance to apoptosis [8]. CSCs are identified by specific surface epitopes, which in the colon include CD44, CD133 and CD166 [9, 10]. To select putative colorectal CSCs, a promising combination of three markers- EpCAM, CD44 and CD166 was described by Dalerba et al. [11]. Although EpCAM previously being considered as pan-epithelial marker in the normal human colon, its frequent expression in CSCs in breast, colon, pancreas and prostate tumors suggests that this surface epitope could be a putative marker for CSCs, particularly in human colon cancer-derived cell lines [12]. More recently Lgr5, Musashi-1 and aldehyde-dehydrogenase 1 (ALDH-1) have been added to the list of stem cell markers for colon cancer [13–15].
One of the recently reported characteristics of tumor derived CSCs is that they can be grown to form spherical colonies in vitro, when plated in limited numbers under anchorage-independent conditions in a serum-free defined media supplemented with growth factors [9]. With the objectives to promoting in vitro expansions of CSCs, methods have been developed to grow and study them in sphere-forming assays as reported for neurospheres [16, 17], mammospheres [18, 19] and colonospheres [13, 20]. Using this approach, we and others have identified and/expanded colon CSCs by generating colonospheres from colon cancer cell lines [10, 21–24]. However, little is known about the signaling events that regulate the growth and maintenance of colonospheres.
Different signaling pathways such as Wnt, Hedgehog, Notch and Bmi have been implicated in various cellular processes during development that include differentiation, migration and proliferation [25–27]. Recent studies have reported the pivotal role of Wnt/β-catenin signaling pathway in the regulation of epithelial stem cell self renewal [28, 29]. In contrast, dysregulation of Wnt/β-catenin signaling has been implicated in colon carcinogenesis [30, 31]. However, the regulatory role of Wnt/β-catenin signaling in the maintenance and growth of colonospheres still remains elusive. The current investigation was, therefore, undertaken to study the in vitro expansion of colonospheres that display the characteristics of CSCs and to delineate the role of Wnt/β-catenin pathway in regulating the growth and maintenance of colonospheres using three different human colon cancer cells: HCT-116 (p53 wild-type; K-ras mutant), HCT-116 (p53 null; K-ras mutant) and HT-29 (p53 mutant, K-ras wild-type).
Generation and characterization of colonospheres
Extreme limiting dilution analysis of colonospheres forming frequency of HCT-116 cells, transfected with non-targeted (vector) or β-catenin siRNA and by colonospheres-derived cells
Number of cells/well
Number of wells plated
Number of wells showing colonospheres
Colonospheres-derived cells
Parental/Vector transfected
Parental/β-catenin siRNA transfected
Sphere forming frequency (95% CI)
1/2 (1/3-1/2)
1/11 (1/17-1/7)
1/171 (1/287-1/102)
P value
<< 0.0001
The Wnt/β-catenin pathway is activated in colonospheres
β-catenin is important for the generation of colonospheres
Recent studies have suggested that many epithelial cancers, including colon cancer arise from a small sub-population of CSCs through their oncogenic transformations [38–40]. There is a strong evidence that colon cancer-derived cell lines represent the tumors from which they are originally isolated [41] and can develop structures similar to those found in the original tissue [42], suggesting the presence of CSCs in colon cancer-derived cell lines. Much of the advance on CSCs comes from the assays that have used FACS technology to isolate and enrich for CSCs from primary tumors, and then testing their propagation in immune-deficient mice [20, 43]. Such assays are cumbersome and expensive, and do not readily enable either the characterization of CSCs or their functional properties. However, unlike the primary tumor, the cell lines represent a valuable resource that can be used repeatedly over time, and can be readily characterized by various CSCs assays. There are clearly many advantages to working with CSCs derived from cell lines, both for the possible development of novel targets for drug development, as well as for the possible development of relatively high-throughput drug-response assays for CSCs.
In the present work, using an approach to culture cells that display markers and characteristics of CSCs, we show that spheroid cultures (or colonospheres) generated from a limited number of human colon cancer-derived cell lines are enriched for cells that express colonic CSC markers. Widely accepted general characteristics of "stemness" include the expression of putative stem cell antigens, reduced cell cycle progression, and limited functional differentiation or dedifferentiation under the influence of the microenvironment [10, 44]. Currently, colon CSCs are defined by their expression of cell surface markers, such as Musashi, LGR-5, CD44, CD166, etc [11, 13–15]. Colonospheres generated under present conditions from the three different colon cancer lines show increased expression of colon CSCs markers along with pan-epithelial marker EpCAM, when compared with the corresponding parental cells. Additionally, the evidence for the presence of functional CSCs in colonospheres came from the ELDA that revealed a marked rise in frequency of sphere-forming ability of cells in colonospheres. Since under extreme limited dilution and in serum-free conditions (stem cell media), only stem-like cells are believed to divide to form spheroids, our observation of formation of colonospheres by a very small number of cells not only suggests the presence of functional CSCs in colon cancer derived cell line but also indicate the enrichment of CSCs in colonospheres [13, 20, 45].
CSCs have been shown to divide very slow and possess limited differentiation potential [10, 36]. The ratio between CSCs and differentiated cells has been suggested to depend on the rate of differentiation relative to the turnover rate of CSCs [46]. In view of this, it is reasonable to suggest that lower the propensity to differentiate greater the proportion of CSCs. Recent reports have also demonstrated low functional differentiation potential of colon cancer cell lines that contain a high proportion of CSCs [10]. Our current data demonstrate that colonospheres are poorly differentiated as evidenced by a marked reduction in alkaline phosphatase activity. Moreover, our observation that colonospheres also show increased H33342 efflux capabilities, accompanied by increased expression of ABC transporter protein ABCG2 suggests that they possess the ability to exclude drugs, a trait that has been demonstrated for CSCs [47, 48]. These observations and the fact that over 80% of colonospheres-derived cells are CD44 positive, a widely accepted colon CSC marker, and that the cells of colonospheres also show staining of EpCAM, which is believed to be a putative CSC marker in colon cancer cell lines, strongly indicate that colonospheres generated under present conditions are mainly composed of CSCs.
Increasing evidence suggests that many signaling pathways that are classically associated with cancer, including Wnt, Hedgehog, Notch and Bmi, may also regulate normal stem cell development. One particular interesting pathway that has also been shown to regulate both self-renewal of stem cell and oncogenesis in different organs is the Wnt signaling pathway [36]. Wnt proteins regulate development in several organisms and contribute to cancer when dysregulated through the activation of β-catenin (a downstream activator of the Wnt signaling pathway). The canonical Wnt/β-catenin pathway has previously been reported to play a role in hematopoietic, mammary and brain stem cell functions [28, 49, 50]. The Wnt pathway is also a major regulator of stem cells in gastrointestinal system as identification of many different Wnt/β-catenin target genes have been implicated in intestinal stem cell proliferation and carcinogenesis [37]. The best stemness-specific target of Wnt/β-catenin signaling known today is Lgr5 [51], while c-myc and cyclin-D1 are the other Wnt targets associated with proliferation of progenitor cells [29, 30]. Results derived from our current investigation suggest that Wnt/β-catenin pathway plays a critical role in regulating the growth and maintenance of colonospheres. Initial support for this inference comes from the observations that there is a marked loss of membrane bound β-catenin in colonosphere cells. Further, colonospheres formed by colon cancer HCT-116 (wt or p53-/-) and HT-29 show a marked reduction in the expression of phosphorylated form of β-catenin and axin-1 and increased levels of total β-catenin (non-phosphorylated form) and phosphorylated form of GSK-3β, compared to the corresponding controls. Moreover, to inactivate Wnt/β-catenin pathway by GSK-3β, axin is required for phosphorylation of β-catenin [30]. The observed decrease in expression of axin-1 and β-catenin phosphorylation, therefore, suggests that the increased level of total β-catenin is due to the inhibition of β-catenin degradation. Increased levels of β-catenin, following translocation into the nucleus, is likely to cause induction of the TCF family of transcription factors leading to increased synthesis of several proteins that are critically involved in cellular growth [52]. Indeed, our observation that colonospheres from HCT-116 wt and HT-29 cells shows higher levels of c-myc and cyclin-D1, compared to the corresponding parental cells, supports the contention that Wnt/β-catenin signaling plays a critical role in the formation of colonospheres. These findings were further corroborated by the observation that downregulation of β-catenin in the parent cell lines decreases the number of sphere-forming cells as demonstrated by ELDA. Furthermore, during serial passaging of colonospheres, downregulation of β-catenin causes reduction in CD44 positive cells and inhibition in transcriptional activation of TCF/LEF resulting in suppression of regeneration of secondary colonospheres formation. In contrast, overexpression of c-myc is associated with increased colonospheres formation. Taken together, the results strongly suggest that Wnt/β-catenin pathway has a pivotal role in the genesis, maintenance and expansion of CSCs enriched spheroid cultures.
In conclusion, our data demonstrate that colonospheres formed by colon cancer cells are predominantly composed of cells displaying the characteristics of CSCs. Wnt/β-catenin signaling plays a pivotal role in the growth and maintenance of colonospheres in that downregulation of β-catenin that decreases TCF/LEF transcriptional activity resulting in reduction of CD44 positive cells leading to a marked suppression of colonosphere formation and regeneration. In contrast, overexpression of c-myc, a downstream effector of Wnt/β-catenin signaling, reverses the situation. We suggest that the colonosphere formation technique is a useful in vitro method to enrich cultures for CSCs or as a surrogate for tumor formation. The data presented in this communication also provide a basis for identification of distinctive molecular pathway(s) for self-renewal of CSCs and for the growth and maintenance of colonospheres.
Cell lines and culture conditions
The human colon cancer cells HCT-116 (p53 wild type; K-ras mutant), HCT-116 (p53 null; K-ras mutant) and HT-29 (p53 mutant; K-ras wt) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cells were maintained in Dulbecco's modified Eagle medium (DMEM; 4.5 g/L d-glucose) supplemented with 10% FBS and 1% antibiotic/antimycotic in tissue culture flasks in a humidified incubator at 37°C in an atmosphere of 95% air and 5% carbon dioxide. The medium was changed twice a week, and the cells were passaged using 0.05% trypsin/EDTA.
Antibodies used for immunofluorescence, or Western blotting were as follows: rabbit anti-LGR5/GPR49 (Abgent, San Diego, CA), mouse anti-β-actin (Chemicon International, Billerica, MA), mouse anti-CD44 (Abcam Inc., Cambridge, MA), rabbit anti-Musashi-1 (Imgenex, San Diego, CA), mouse anti-cyclin-D1, rabbit anti-ABCG2, mouse anti-β-catenin (Santa Cruz Biotechnology Inc., Santa Cruz, CA); rabbit anti-c-myc, mouse anti-EpCAM, mouse anti-CD44, rabbit anti-axin1, rabbit anti-phospho-β-catenin, Cell Signaling, Danvers, MA) and FITC-conjugated mouse-IgG2b (Sigma-Aldrich Inc, St Louis, MO). Mouse anti-human PerCP-Cy5 IgG2b (isotype) or CD44 conjugated antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and mouse anti-human PE-Cy7 IgG2b (isotype) or CD44 conjugated antibodies (BD Pharmingen) were used for flow cytometry.
Western blotting
For all Western blot analyses, protein was harvested from adherent cells and colonospheres. Cell lysates were prepared by homogenizing the cells in lysis buffer as previously described [22]. Quantification of the proteins was carried out using a modified Bradford assay (Bio-Rad Laboratories). Protein samples for Western blotting were prepared by boiling after the addition of denaturing sample buffer. Proteins were separated using SDS-PAGE on an 8% or 15% gel and transferred to a polyvinylidene difluoride membrane (Millipore) by electroblotting. Antibodies were diluted in TBS and 0.1% (v/v) Tween with 5% nonfat dry milk after 1 h of protein blocking in the absence of antibodies. Membranes were incubated at 4°C overnight with the primary antibody, subsequently washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences) for 1 h at room temperature. Membranes were again washed, and protein bands were visualized using a commercially available enhanced chemiluminescence kit (Amersham Biosciences). When appropriate, membranes were incubated in stripping solution for 30 min at 65°C, washed, and re-probed with a second primary antibody for verification of loading control.
Formation of colonospheres
The ability of cell lines to form spheres in suspension was evaluated as described by Liu et al. [25], with slight modifications. Briefly, primary colonospheres were generated by incubating the limited number of parental HCT-116 (p53 wild-type; K-ras mutant), HCT-116 (p53 null; K-ras mutant) and HT-29 (p53 mutant; K-ras wild-type) cells at a concentration of 100 cells per 200 μL in serum-free stem cell medium (SCM) containing DMEM/F12 (1:1) supplemented with B27 (Life Technologies, Gaithersburg, MD), 20 ng/ml EGF (Sigma, St Louis, MO), 10 ng/ml fibroblast growth factor (Sigma), and antibiotic-anti-mycotic in 24-well ultra low-attachment plates (Corning Inc, Lowell, MA) for 10 days. The colonospheres formed at the end of the incubation period were centrifuged (1000 rpm), dissociated with 0.05% trypsin/EDTA using a 22 gauge needle and then passed through a 40 μM sieve to obtain single cell suspension, as described by Kakarala et al. [19]. The single-cell suspension derived from colonospheres that have undergone 15 or more serial passages were used for all experiments. For spheroids formation, an equal number of cells from adherent cell lines and colonospheres cells were plated at 200 cells/100 μL SCM in each 96 ultra low-attachment well (Corning Inc, Lowell, MA). The colonospheres formed after 5 days were evaluated for their number and size by light microscopy.
Extreme limiting dilution analysis
Extreme limiting dilution analysis (ELDA) was performed as described by Hu and Smyth [45]. Briefly, single cell suspension obtained from adherent or colonospheres-derived cells were plated at concentration of 1000, 100, 10 cells and 1 cell per 100 μl SCM (24 well for each dilution) in 96-well ultra-low attachment and incubated for 5 days. At the end of 5 days, the number of wells showing formation of colonospheres was counted. The frequency of sphere forming cells in a particular cell type was determined using ELDA webtool at http://bioinf.wehi.edu.au/software/elda.
Flow Cytometry Analysis
Single cell suspension from parental monolayer cell cultures and colonospheres were subjected to direct immunofluorescence staining followed by flow cytometry analysis according to our standard protocol [22]. Briefly, the cells were harvested and washed with PBS. Half a million cells were suspended in 90 μl of PBS containing 0.5% BSA. After 10 min at room temperature, 10 μl of fluorophore conjugated anti-human CD44 antibody was added and incubated for 30 min in dark at room temperature. The samples were then washed and analyzed using a FACS DiVa (BD, San Jose, CA). The cells stained with mouse IgG2b (isotype-negative control) served as gating control. The proportion of CD44 positive cells was determined on the basis of fluorescence intensity-spectra of CD44-conjugated PE-Cy7 or PerCP-Cy5.
Immunofluorescence cytochemistry
Single cell suspension, obtained from parental and colonosphere, was washed in PBS and fixed in 2-4% formaldehyde for 10 min at 37 °C. They were washed and re-suspended in 0.5% BSA-PBS (blocking buffer) for 10 min, subsequently incubated in primary antibodies at appropriate dilution for 1 h at room temperature. After rinsing with incubation buffer, the cells were resuspended in fluorophore-conjugated secondary antibodies diluted in incubation buffer according to the manufacturer's recommendations and incubated for 30 minutes at room temperature. The cells were then resuspended in PBS after washing with the incubation buffer. Appropriate aliquot of cells were mounted on glass slides immediately before examining under fluorescence microscope.
Hoechst 33342 dye exclusion assay
Single cell suspension obtained from parental cell lines and colonospheres were washed with PBS (3 times) and stained with Hoechst 33342 or H342 (5 μg/ml, Sigma-Aldrich Inc, St Louis, MO) for 45 minutes at 37°C in HBSS buffer, vortexing gently every 15 min. As a control, a sample was treated with verapamil (Sigma, 50 μM) for ten minutes at room temperature prior to the addition of H342. The stained cells were collected, washed with PBS and resuspended in 3 ml of PBS containing 2 μg/ml of propidium iodide, and subsequently analyzed by flow cytometer-FACS Vantage SE/DiVa SORP (BD Biosciences, San Jose, CA) with all-digital electronics and octagon- and trigon-shaped detector arrays. Excitation of 100 mW at 488 nm was provided by a 177-G argon ion laser (Spectra-Physics, Mountain View, CA) and 200 mW of all-lines UV (351-365 nm) was provided by an Innova 90-5 argon ion laser (Coherent, Palo Alto, CA). Forward and side laser scatter was detected from 488 nm excitation. H342 and propidium iodide fluorescence from UV excitation was split into "blue" and "red" wavelengths by a 505 nm long pass dichroic with a 450/50 bandpass (425-475 nm) filter in front of the "blue" detector and a 630 nm long pass filter in front of the "red" detector. Cell population showing H342 Bright (H342Br) and H342 Low (H342Lo) was determined and the ratio of H342Lo/H3342Br was calculated to evaluate the dye-efflux capacities of the cells. The gating of H342Lo and H342Br cells was based on a verapamil control. Dead cells were gated out based on positive staining with propidium iodide.
Determination of cellular growth
Changes in cellular growth, an assessment of proliferation, of parental and colonosphere-derived cells were assessed by 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay as described previously [22]. Briefly, 4000 cells were plated per well in 96-well plates in 200 μl medium. At each time point (0, 24, 48, 72, 96, 120 h), 20 μl MTT solution was added to each well and the plate was incubated for 1 h at 37°C. Medium was then aspirated from each well, and 100 μl DMSO was added. The intensity of the color developed, which is the reflection of number of live cells, was measured at a wavelength of 570 nm. All values were compared to the corresponding controls. All assays were performed with 6 replicates.
Measurement of Alkaline phosphatase activity
Status of differentiation was determined by measuring the alkaline phosphatase activity using SensoLyte, pNPP alkaline phosphatase assay kit (Anaspec, San Jose, CA) according to the manufacturer's instructions and measuring the absorbance at 405 nm.
β-catenin siRNA transfection
For the transfection of siRNA into the parental and colonosphere derived cells, Oligofectamine reagent (Invitrogen Corp., Carlsbad, CA) and serum-free Opti-MEM (Invitrogen Corp., Carlsbad, CA) medium was used to prepare transfection complexes according to the manufacturer's instructions. Briefly, single cell suspension of colonospheres was plated in 10 cm tissue culture plates with normal growth medium overnight to achieve 25-30% confluence. Next day the medium was removed, washed twice with serum-free Opti-MEM (Invitrogen Corp., Carlsbad, CA) medium prior to adding the complexes containing non-targeted or β-catenin siRNA (Integrated DNA Technologies Inc., Coralville, IA). After 3 days of transfection, the cells were collected and analyzed for protein expression of β-catenin using Western blot and for colonosphere formation assay using SCM.
Activation of relative TCF/LEF-Dual Luciferase assay
The activation of transcription factor TCF/LEF was evaluated by using Cignal TCF/LEF reporter assay kit (SA Biosciences, Frederick, MD). The cells were grown to 25-30% confluence as described above and co-transfected with TCF/LEF reporter constructs and either non-targeted or β-catenin siRNAs (Integrated DNA Technologies Inc., Coralville, IA) using SureFECT transfection reagent (SA Biosciences, Frederick, MD) according to manufacturer's instructions. The TCF/LEF reporter used a mixture of an inducible β-catenin-responsive luciferase construct and a constitutively expressing Renilla element (40:1). At the end of 16-24 h incubation period Opti-MEM medium was changed to DMEM 10% FBS for parental cell lines or SCM for colonosphere derived cells. The cells were allowed to grow for another 3 days, collected and analyzed for TCF/LEF activity using a dual-luciferase assay kit (Promega-Biosciences, San Luis Obispo, CA) following the instructions outlined by the manufacturer.
Overexpression of c-myc gene
Single cell suspension of HCT-116 parental cells was plated in the tissue culture plates to achieve a 90% confluence. Once the 90% confluence is achieved, the adherent cells were transfected using Lipofectamine 2000 and PLUS reagent (Invitrogen Corp., Carlsbad, CA) with plasmid vector-pCMV6-Neo (Origene, Rockville, MD) containing human cDNA clone for c-myc gene or empty plasmid PCMV6-vector in OPTI-MEM medium according to the manufacturer's instruction. After 3 days of transfection, the cells were analyzed for c-myc protein expression by Western blot and re-plated for colonosphere formation assay.
Statistical analysis
Unless otherwise stated, data are expressed as mean ± SD of six observations. Where applicable, the results were analyzed using analysis of variance followed by Fisher protected least significant differences or Scheffé test. p < 0.01 was designated as the level of significance.
Authors' information
Shailender S. Kanwar, Ph.D.: Postdoctoral Research Fellow, Department of Internal Medicine and Veterans Affairs Medical Center, Wayne State University, Detroit, MI 48201, USA. E-mail: email@example.com
Yingjie Yu, M.D., Research Assistant Professor, Department of Internal Medicine and Veterans Affairs Medical Center, Wayne State University, Detroit, MI 48201, USA. E-mail: email@example.com
Jyoti Nautiyal, Ph.D.: Postdoctoral Research Fellow, Department of Internal Medicine and Veterans Affairs Medical Center, Wayne State University, Detroit, MI 48201, USA. E-mail: email@example.com
Bhaumik B. Patel, M.D.: Staff Oncologist and Assistant Professor, Department of Internal Medicine, Veterans Affairs Medical Center and Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA. E-mail: firstname.lastname@example.com
Adhip P.N. Majumdar, Ph.D., D.Sc.: Professor and Senior Research Career Scientist, Department of Internal Medicine, Veterans Affairs Medical Center and Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA. E-mail: email@example.com
This work was supported by grants (APNM) from the National Institutes of Health/National Institute on Aging (AG014343) and the Department of Veterans Affairs.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


text:
If you're looking for nothing but fine artisanal cuisine in Iloilo City, then this might be your next stop.
From among the many restaurants in the city, Deo. Optimo. Maximo. attracted me most since when talking about 'Change', they embrace it. What I mean by that 'change' is the way they organize every meal into a gastronomical gourmet. They don't particularly stick to the basics and one certain menu--they change the whole game. Aside from being seasonal, which means their menu varies highly from the abundant harvest of certain crops and seasonal produce, they are valuing the concept of putting art in every dish. The only constant in their menu, to be honest, is change. --And that's why it was love-at-first-visit.
Come to think of it, most restaurants just offer you the same menu for years. Yes, you may have your go-to meal and all, but sometimes, you tend to get bored, looking for what's new in their menu (sometimes, looking forward to what's new) but end up with the same dish, on a different day.
Of course, there's no problem with that, some people can even eat the same dish every day in a year. But what I'm pointing out here is that, if you want to turn your every meal into an experience, then you might want to try this restau out.
Respect for food- that's what they live for. And that's exactly what matters. You're not only paying for the food, the service, the place, you're also paying respect to the bounty of nature. Sure, that's not monetary, but that's priceless even.
|Complementary bread with Eggplant dip
Talking about the highlight of the restau-food. We had our complementary bread with eggplant dip. It was good. At first, it'll taste like a little exotic (probably just my tongue not being used to this dip), but after a few more bites, they'll just go well together. And you won't even taste the eggplant overpowering the whole thing.
|Sisig Balls:
(Pork Cheek, Market Greens, Sriracha, Cocoa Aoli, Nori, Sesame, Katsuobushi)
And for a fresher take, their sisig specialty was not served in a usual sizzling plate, instead it was on just a different plate, rolled into balls and served with market vegetables. It was topped with Katsuobushi, a (Japanese) dried skipjack tuna. It was a refreshing look for the usual sisig plate and it tastes fine too. It was too big for two people though. :)
|Holiday Chicken: Roast Chicken, Bacon & Sourdough Stuffing, Mangosteen Jelly and Gravy
Visiting this place, especially this month, you shouldn't miss out on ordering this dish because it's one of their specialties for the holidays. And with that point aside, it tastes really good you won't mind you're just devouring chicken all along. As they'd say, it melts in your mouth making this one my favorite from among the dishes I tried.
|Flat Iron Skewer
Since we can't have enough of their dishes, we ordered extra skewers off the grill. We tried out the Flat Iron Skewer. We were then asked to choose from various sauces which were also their specialty blend.
|That's our table.
Had thoughts of having too many wonderful dishes to try; yet so little time and tummies. (lol!)
We also tried their Dalandan Tsokolate juice, the Cucumber-ade juice, and a scoop of their homemade frozen yogurt for dessert. Their juices didn't disappoint. While it tasted like chocolate energy drink with a citrus aftertaste, I was amazed how they came up with the Dalandan Tsokolate idea after all. The cucumber drink was really refreshing. And for their fro-yo, it was a little bit powdery, I don't know how it became such with its texture but the taste was also winning.
|Homemade Frozen Yogurt scoop with caramel syrup
Below are the snaps that I managed to save from my snapchat. :)
True to their concept, I can attest to their idea of thinking globally about their food but staying rooted in the local. I am 100% sure that I'm visiting this place again. In fact, I'm already looking forward to the next set of dishes. (though I'm also hoping they'd retain the Holiday chicken or have banh mi's again). ;)
For the love of food, I highly suggest you visit them out. Find helpful deets below and feel free to leave a comment if you have anything you want to share.
- Deo.Optimo.Maximo., also known as "D.O.M." is located at Westland Gateway Complex, Donato Pison Ave., Brgy San Rafael, Mandurriao, Iloilo City.
- Contact Number: 09173196699
- Opening Hours: Monday 12NN-3PM //5:30PM-10PM
- Wednesday-Sunday 12NN-3PM //5:30 PM-10PM
- Cash Only (Payment Method)
- Accepts Walk-Ins, Reservations, Take out, Catering and Waiter Service


text:
Day: July 2, 2015
The Consent Of Our Conscience
It will always be okay to sin as long as we can obtain the consent of our conscience. If our conscience agree with our actions, then it becomes easier to engage in activities which we know are contrary to God's Word, and thereby broadening the breach between us and God. Paul says in his Epistle to the Romans,
"But we are sure that the judgment of God is according to truth against them which commit such things (ref. 1:18-32)…Which show the work of the law written in their hearts, their conscience also bearing witness, and their thoughts the mean while accusing or excusing one another…" (2:2,15).
Sin, in any way, shape, form or fashion, disrupts our relationship or our potential relationship with God. God is holy and He requires us to live lives of holiness in order to be in relationship with Him. In order to be in a relationship with someone, we must be able to "relate" to the other person. God, in His holiness, cannot relate to sin. In our sinful state, we cannot relate to God. In order for a relationship to be established, either we must begin to lean toward living lives of holiness or God must lean toward becoming mired with sin. The latter, we know, will never occur as it would mean that God would cease to be God and there would no longer be a standard to which He would be able to hold us.
We cannot continue to manufacture excuses to continue in sin, especially when God has provided a way out of a life of sin through faith in the atoning work of His Son Jesus Christ. We cannot allow our conscience "…to be seared with a hot iron…," but we must
"…purge your (our) conscience from dead works to serve the living God…(and) draw near with a true heart in full assurance of faith, having our hearts sprinkled from an evil conscience, and our bodies washed with pure water…For if we sin willfully after that we have received the knowledge of the truth, there remaineth no more sacrifice for sins, But a certain fearful looking for of judgment and fiery indignation…" (1 Tim. 4:2; Heb. 9:14; 10:22,26,27)…#thinkonthesethings
Any redistribution or reproduction of part or all of the contents in any form is prohibited, except with the written consent of The Joshua Generation Ministries.


text: Jean offers a national perspective on healthcare. She is recognized for bringing innovation and strategic vision to a wide variety of healthcare planning and design projects. Jean specializes in leading project teams for health science centers, teaching hospitals and academic medical centers worldwide. Throughout her 25-year career, clients have benefited from her consistent involvement in the total design process, a hands-on approach to programming and planning, and unmatched energy and enthusiasm. Jean also serves on the firm's Board of Directors. Recent project successes include the implementation of the New Clinical Building at The Johns Hopkins Hospital, planning and design for Swedish Medical Center in Seattle, master planning for the University of California Davis Hospital and Cedars-Sinai Medical Center in Los Angeles.


text: A simple twist gives it one surface, flowing from inside to outside and back again. Inscribed all around with the prayer Jesus taught his disciples ("Our Father who art in Heaven…"), it's a statement of faith and a symbol of infinity. Bangle is ¼" at widest point, 83/8" around. Story card included. Made in USA. Allow 3-4 weeks for Gold. No rush delivery or gift box. Specify King James Version, New International Version, Gaelic, Spanish, or Latin. Item can only be shipped within the Continental US.


text:
By Elsa Maxey
With the General Election this year now a part of our history, in a nutshell here's what the early voting results showed in Fort Bend County. According to John Oldham, the county's elections administrator, the total voter turnout before Tuesday's votes were cast was 153,090. Not quite the reported three quarters of the votes before Election Day this go-round, which Oldham said had been the case in 2008. Of the total number of registered voters, almost half of them or about 45 percent voted early.
This was predicted to be one of the highest early voter and General Election turnouts in Fort Bend County with far more registered voters. "We had 41,000 more registered voters than in 2008, so the percentage may not be the highest," said Oldham about this year's outcome. That means that although the voter registrations were up from that of the past 4 years, the smaller percentage turnout can mean more voters casting ballots. Most of the early votes, he said, came from Cinco Ranch during the scheduled period from Oct. 22 ending in Nov. 2, which included Sunday voting in some locations.
About any challenges or confusion on Election Day, "so far none related to locations that I am aware of," said Oldham on Tuesday morning several hours after voting had begun. But, "voters call constantly asking where they vote."
Prior to the close of Election Day this Tuesday, Oldham said he expected the voter turnout to be probably 58,000 to 60,000." After the official count, that could mean that for the 2012 election, Fort Bend County could have seen a 62 percent voter turnout.
On Election Day, a total of 341,544 persons were registered and eligible to vote.
Were there any international monitor sightings in Fort Bend County? No reports and Oldham said that to his knowledge he did not know of any to be sent here.
There were 18 counties in Texas to have international observers monitor the election polls. Texas was among 10 states in the U.S. including Alaska, Arizona, Georgia, Louisiana, Mississippi, New York, North Carolina, South Carolina, and South Dakota to be have observers at polls from the Organization for Security and Co-operation in Europe (OSCE).
But even before early voting started, the Texas Attorney General sent a letter warning the organization's representatives not to enter a voting site and to keep their distance from the polling place within a 100 feet in accordance with state law.
Reportedly, representatives of this group of the European and Central Asian nations have been observing U.S. elections since 2002. According to information about the Department of Justice, the Voting Rights Act provides for the appointment of federal observers by order of a federal court. Also reports indicate that members of the NAACP, the League of Women Voters, the ACLU and other organizations requested the measure which involved the deployment of 44 observers from countries such as Albania, Estonia, Russia, Slovenia and Turkey throughout the U.S.
For detailed election results from Fort Bend County, check the county's website. For findings from the OSCE, a preliminary report is scheduled for Nov. 7.


text:
Lilly Drumeva reports from Bulgaria:
Dear musician friends,
Just wanted to inform you that Lilly of the West is currently recording a new album. It will be ready in March 2011. The repertoire is country & swing from the 1930s-40s. That's why we did a 'retro photoshoot'. You may see some funny photos here.
Lilly of the West has had a constant lineup for the past two years, featuring Lilly Drumeva (vocal, guitar, fiddle! - I'm getting better and play on many songs), Yasen Vasilev (vocal, guitar), Misho Shishkov (Dobro, harmonica, piano, vocals), Body Bozduganova (upright bass, vocals).
Otherwise, we'll be on tour end of March (Czech Republic) and end of May/first two weeks of June (Germany and Holland). More info on our MySpace. Hope to see you around,
All the best,
Lilly Drumeva
Lilly of the West
2019 Bluegrass Conference
22 hours ago


text:
Arches National Park
Map and Climate
Overview | Description | Things To Do | Lodging/Camping | Nearby | Video
Map In a PDF format Arches National Park - PDF
Arches National Park is located on the Colorado Plateau of eastern Utah in the northeast corner of what is called Canyon Country. It runs about 15 miles north-to-south and about 10 miles east-to-west, encompassing 114 square miles ( 73,379 acres).
The Colorado River creates the southern boundary of Arches National Park. On the opposite bank is the Moab Valley and the town of Moab. To the east, across the Colorado River, the La Sal Mountains rise to over 12,000 feet. The park is adjoined on the west by the Moab Canyon. Salt Valley runs through the park from northwest to southeast.
In summer, June through September, temperatures may exceed 100 degrees F; in winter, December through February, temperatures often drop below 32 degrees F. Temperatures may range 50 degrees in a 24-hour period. Dress accordingly. Very dry! Carry drinking water at all times.
Getting There
Major airlines fly into Salt Lake City, Utah (5 hours by car) or Grand Junction, Colorado (2 hours by car). A commuter airline, Alpine Air, flies into the Canyonlands Airport, 15 miles from the park, and into Salt Lake City.
Greyhound Bus stops 50 miles northwest of the park in Green River, Utah.
Amtrak stops 40 miles northeast of the park in Thompson Springs, Utah. Rental vehicles and taxis can be arranged.
By Auto
Arches National Park entrance is 5 miles north of Moab, Utah on U.S. Route 191, 3 miles north of its junction with Utah Highway 128. From Interstate 70 traveling east, take U.S. Route 191 south from Crescent Junction, 30 miles south to the park. Traveling west on Interstate 70, take Utah 128 southwest from the Cisco exit, 48 miles to the park entrance.
Overview | Description | Things
To Do | Lodging/Camping | Nearby | Video
Arches National Park
P.O. Box 907
Moab, UT 84532
Phone: (435) 719-2299
Related DesertUSA Pages
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- Desert Survival Skills
- How to Keep Ice Cold in the Desert
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text: So, I am thinking of starting a blog called Hallam Heim. Yes, I know it's a play on words, but it is totally appropriate. I want to put genealogy stories for both families on that site. For those of you who don't know, my maiden name is Heimberger, and the word "Heim" means home. So, what do you think? There is so much information out there and I have so much I can share, like pictures and stuff. Also, if you are or should be on this family tree, you could definitely add to it's growing branches. So, here is a sampler of what I am working on. The first person in this scenario is my father. Of course this is just a snapshot and only his mother's side of the family, but I think it gives you a good idea of what we can work with. I also have some pictures. Can a gedcom be embedded here?


text:
This is a sponsored article on behalf of Squarespace.
We like a challenge here at Londonist.
So when the guys from Squarespace suggested we could build a beautiful London blog in under an hour, we wanted to try.
Taking inspiration from the 'other side' of Peckham, we decided to build a showcase for the greener bits of our beloved part of town.
Setting up a Squarespace site was hugely satisfying and surprisingly easy.
Their beautifully designed interface actually inspired us to create a similarly gorgeous site: with great templates to choose from, building a high-quality, well-designed website felt simple.
After registering for a free trial and choosing the template design, we selected the site goals.
In this case, as we only had an hour, we plumped for memories and ideas, but with more time, we can imagine getting really excited about using a Squarespace site to promote a business or sell products.
Throughout the process, we felt well supported by the Squarespace team: emails like the one below gave us the confidence to get it right.
Within the hour, we were able to upload four separate blog posts about our beloved Peckham.
Adding images through the intuitive interface was straightforward, knowing Squarespace would take care of resizing and displaying our pictures to make them look, frankly, better than our original snaps.
Connecting our fledgling site to our existing social media channels was also easy to do: within a few clicks, our Instagram and Flickr accounts were linked up.
And here's the final product: within an hour, we'd built a (surprisingly) beautiful homage to Peckham.
We can only imagine what someone with more time could achieve: we'd love to see what you guys are able to come up with.
Start creating your website with Squarespace now. Get 10% off using the code LONDONIST.


text:
British Cycling West Midlands Go-Ride coach Jess Potts delivered a three hour women-only coaching session on Saturday 23rd February at Stourport Cycle Track.
Coaches Craig Ansell, Jess Potts and Regional Manager Nick Yarworth were there to lead the session, attended by 19 women keen to improve their racecraft. Jessie Ansell, a local young volunteer was also on hand to give support and advice to the women.
The majority of the women were novice riders with little or no racing experience. They all expressed an interest in competing over the upcoming season, so the session was based around racing skills and techniques.
The day was dry but bitterly cold, so the session quickly got started to keep the riders warm. "Having only competed in a couple of cross races so far, the exercises to promote cycling close to other riders has boosted my confidence in cycling in a group."
Jo Barker, Halesowen A&CC, who attended the session
This was the first time many of the riders had ridden in a large bunch and they took to it with ease. Coaches demonstrated techniques such as close group riding, cornering and correct gear selection around the circuit.
Skills such as passing a water bottle through the group and riding with your hand on a partners shoulder were all factors in increasing the confidence of the women, whilst riding closely side by side.
A quick tea break and then it was back outside to progress their skills even further. As the pace increased, coaches emphasised the importance of riding as a group, to ensure no riders got left behind.
The day finished with a chaingang style activity to give them a final blast around the closed road circuit.
Jo Barker from Halesowen A&CC said the following of the session:
"The women's only training session was a great introduction to cycle racing for me. Having only competed in a couple of cross races so far, the exercises to promote cycling close to other riders has boosted my confidence in cycling in a group. The cornering training was of particular benefit, and my handling of the bike improved over the session. I was motivated to set myself targets for the season, and I have already entered 2 races for this coming weekend!"
Future women's sessions are being planned, so please keep an eye out for dates and venues right here on the British Cycling website.
Related: British Cycling Coaching Homepage


text:
are many small producers of real cider and perry
scattered around the UK. The area deservedly most
famous for scrumpy is the West Country - notably
in Somerset, Devon, Gloucestershire and Herefordshire
- but many counties have cider farms. The number
of smallscale cider producers has been declining
over the years for a variety of reasons, although
it is encouraging that recently new producers have
been appearing. This page attempts to list all active
cider producers across the country.
Scrump's Cider Map containing all active cider producers
listed on this website. Please note that not all
are open to the public, so please check with the
listing, and if neccessary call the farm before
list British cider farms arranged by name,
by county
- and now by
reputation (as voted by the readers
of Old Scrump's Cider House).
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Fatal error: require_once(): Failed opening required '../mysql_connect.php' (include_path='.:/usr/lib/php:/usr/local/lib/php') in /home/ziderco/public_html/ciderfarm-byreputation.php on line 81