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|By Marketwired .
Article Rating:
|January 7, 2013 11:29 AM EST
LAS VEGAS, NV -- (Marketwire) -- 01/07/13 -- OnLive, the leader of on-demand cloud gaming, will be demonstrating its OnLive® Game Service, now fully integrated with the LG Smart TV with Google TV (G3 Series), at this year's CES show in Las Vegas, Nevada.
Showing at the LVCC, Central Hall, LG booth (8204) from 8-11th January, OnLive combines the power of the Google TV platform with the speed of LG's dual-core chipset and a new user-friendly interface, making console-quality games instantly playable as part of the TV experience -- no discs, downloads or console required. The G3 Series is LG's first TV to make OnLive cloud gaming an integral part of an out-of-the-box offering and is currently available in the United States.
The growing OnLive catalog of over 300 titles includes games from more than 80 publishers, with everything from blockbuster new releases to classic franchises to family-friendly sports, racing and action-adventure games.
Unique to OnLive, an LG Google TV owner can play with (or against) friends on PCs, Macs and Android tablets. Users can even start a game on one device and continue playing on any other OnLive compatible device, with full saved game data intact in the cloud, whenever and wherever they want.
Games can be demoed free and purchased or rented a la carte. Players can also subscribe to the OnLive PlayPack for unlimited play of more than 200 games, with more titles added monthly. OnLive also offers free access to unique social features such as game spectating in the OnLive Arena, recording ten-second Brag Clip videos of players' best gaming moments, and sharing videos and Achievements with friends on OnLive and Facebook.
"LG's dual-core performance Google TVs, coupled with OnLive's innovative games-on-demand service, makes access to the latest videogame entertainment quicker and easier than ever before," said Georg Rasinski, Director of Home Entertainment Brand Management, LG Electronics USA. "By integrating cloud gaming into our powerful G3 series TVs, the entertainment possibilities for consumers are virtually limitless."
"Our partnership with LG demonstrates the exciting future for cloud gaming as an integrated offering for connected TVs," said Gary Lauder, OnLive Chairman. "OnLive's proven gaming on-demand technology enables consumers to enjoy a vast catalog of console-quality games, with no additional hardware necessary beyond an OnLive controller and LG's sophisticated and feature-rich Google TV."
To find out more about OnLive or to purchase a Universal OnLive Wireless Controller, visit www.onlive.com or www.onlive.co.uk.
About OnLive
OnLive is the pioneer of on-demand cloud gaming and computing services, delivering real-time interactive experiences and rich media over the Internet. With ground-breaking video compression technology, OnLive harnesses cloud computing to instantly deliver full-featured, media-rich applications and the latest, premium game titles to tablets, smartphones, PCs, Macs, HDTVs via the OnLive Game System, the VIZIO Co-Star, and the new G3 Series of LG Google TVs. The OnLive Game Service makes games playable instantly, with no need for lengthy downloads or waiting in line for a physical copy. OnLive is available in North America, the UK and Belgium. OnLive's technology is backed by hundreds of patents and patents pending worldwide. The company is headquartered in Mountain View, California. More information is available at www.onlive.com, www.onlive.co.uk and www.onlive.be.
OnLive and Brag Clip are trademarks or registered trademarks of OL2, Inc. in the United States and other countries. All other trademarks are the property of their respective owners.
Google and Android are trademarks of Google, Inc. in the United States and other countries.
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Media Contacts:
OnLive USA
Jane Anderson
[email protected]
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James Beaven
[email protected]
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The best-practices for building IoT applications with Go Code that attendees can use to build their own IoT applications. In his session at @ThingsExpo, Indraneel Mitra, Senior Solutions Architect & Technology Evangelist at Cognizant, provided valuable information and resources for both novice and experienced developers on how to get started with IoT and Golang in a day. He also provided information on how to use Intel Arduino Kit, Go Robotics API and AWS IoT stack to build an application tha...
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Whether your IoT service is connecting cars, homes, appliances, wearable, cameras or other devices, one question hangs in the balance – how do you actually make money from this service? The ability to turn your IoT service into profit requires the ability to create a monetization strategy that is flexible, scalable and working for you in real-time. It must be a transparent, smoothly implemented strategy that all stakeholders – from customers to the board – will be able to understand and comprehe...
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It's 2016: buildings are smart, connected and the IoT is fundamentally altering how control and operating systems work and speak to each other. Platforms across the enterprise are networked via inexpensive sensors to collect massive amounts of data for analytics, information management, and insights that can be used to continuously improve operations. In his session at @ThingsExpo, Brian Chemel, Co-Founder and CTO of Digital Lumens, will explore: The benefits sensor-networked systems bring to ...
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SYS-CON Events announced today that Venafi, the Immune System for the Internet™ and the leading provider of Next Generation Trust Protection, will exhibit at @DevOpsSummit at 19th International Cloud Expo, which will take place on November 1–3, 2016, at the Santa Clara Convention Center in Santa Clara, CA. Venafi is the Immune System for the Internet™ that protects the foundation of all cybersecurity – cryptographic keys and digital certificates – so they can't be misused by bad guys in attacks...
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Identity is in everything and customers are looking to their providers to ensure the security of their identities, transactions and data. With the increased reliance on cloud-based services, service providers must build security and trust into their offerings, adding value to customers and improving the user experience. Making identity, security and privacy easy for customers provides a unique advantage over the competition.
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SaaS companies can greatly expand revenue potential by pushing beyond their own borders. The challenge is how to do this without degrading service quality. In his session at 18th Cloud Expo, Adam Rogers, Managing Director at Anexia, discussed how IaaS providers with a global presence and both virtual and dedicated infrastructure can help companies expand their service footprint with low "go-to-market" costs.
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Security, data privacy, reliability and regulatory compliance are critical factors when evaluating whether to move business applications from in-house client hosted environments to a cloud platform. In her session at 18th Cloud Expo, Vandana Viswanathan, Associate Director at Cognizant, In this session, will provide an orientation to the five stages required to implement a cloud hosted solution validation strategy.
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Is your aging software platform suffering from technical debt while the market changes and demands new solutions at a faster clip? It's a bold move, but you might consider walking away from your core platform and starting fresh. ReadyTalk did exactly that. In his General Session at 19th Cloud Expo, Michael Chambliss, Head of Engineering at ReadyTalk, will discuss why and how ReadyTalk diverted from healthy revenue and over a decade of audio conferencing product development to start an innovati...
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The competitive landscape of the global cloud computing market in the healthcare industry is crowded due to the presence of a large number of players. The large number of participants has led to the fragmented nature of the market. Some of the major players operating in the global cloud computing market in the healthcare industry are Cisco Systems Inc., Carestream Health Inc., Carecloud Corp., AGFA Healthcare, IBM Corp., Cleardata Networks, Merge Healthcare Inc., Microsoft Corp., Intel Corp., an...
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Large scale deployments present unique planning challenges, system commissioning hurdles between IT and OT and demand careful system hand-off orchestration. In his session at @ThingsExpo, Jeff Smith, Senior Director and a founding member of Incenergy, will discuss some of the key tactics to ensure delivery success based on his experience of the last two years deploying Industrial IoT systems across four continents.
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Ovum, a leading technology analyst firm, has published an in-depth report, Ovum Decision Matrix: Selecting a DevOps Release Management Solution, 2016–17. The report focuses on the automation aspects of DevOps, Release Management and compares solutions from the leading vendors.
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When it comes to cloud computing, the ability to turn massive amounts of compute cores on and off on demand sounds attractive to IT staff, who need to manage peaks and valleys in user activity. With cloud bursting, the majority of the data can stay on premises while tapping into compute from public cloud providers, reducing risk and minimizing need to move large files. In his session at 18th Cloud Expo, Scott Jeschonek, Director of Product Management at Avere Systems, discussed the IT and busin...
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There will be new vendors providing applications, middleware, and connected devices to support the thriving IoT ecosystem. This essentially means that electronic device manufacturers will also be in the software business. Many will be new to building embedded software or robust software. This creates an increased importance on software quality, particularly within the Industrial Internet of Things where business-critical applications are becoming dependent on products controlled by software. Qua...
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text:
night critters
- Posted Dec. 23, 2011 by Mawey Viewed 560 times
lately, fatigue has become the lover i lost. the folly of sleep grants a kind of satisfaction that leaves me half numb, half energized for the day's duties. when i sink into the arms at last of the black beauty called night, i hear a high pitch tone, like singing of angels in the distance of dreams. that's where my grief is met by relief.
all darkness is warm. it swells, it allows me to breathe. i lay on my makeshift mattress of hay and moldening brushwood, the packbeasts aside me meditating on their ruminated foray through the moss; and i… and i - - - …there is body parts on me i feel pleasantly ruminating, too.
i awake in the middle of darkness, with the camp fires barely glowing. there is a buzz and stir and cricketing crackle, marking it's presence all over the place, as if my desires and wishes have grown tentacles out of my heart, filling the air with happy chit-chatter and flirring a dance of illusions in front of my eyes, that no blue salt could ever cause proper: the night critters are on, two by two, and "beautiful" is all i can think, is all that life, is all that is.
6 There are 6 comments, add yours!
Antonio Gil
Very good. Especially the way you combined photo and text. Examples: "if my desires and wishes have grown tentacles" and " night critters are on, two by two". Fantastic effort my friend.
Lennart Andersson
Very very fine.
Patricia Gonzales
Cool set!
nice shots
Loving the prose!!! You write really well! :)

subset: commoncrawl


text: How Rob Carrick's son plans to finish university debt-free Add to ... The Globe and Mail Published Tuesday, Sep. 10, 2013 3:29PM EDT Last updated Friday, Apr. 24, 2015 8:42PM EDT Personal finance columnist Rob Carrick and his 19-year-old son Will, a university student in Toronto, discuss their goal of graduating debt-free and how it is a team effort Watch More Video: How Rob Carrick's son plans to finish university debt-free Money Monitor: Choosing a commission-based or fee-based adviser Personal Investor: Earnings season myths I've been hiding money from CRA – how do I come clean? Personal Investor: The awesome power of the 'amateur' investor Comparing today's housing affordability to the high-rate days of the 80s Canadian consumer spending up 5% in seventh consecutive quarter of growth Turkey's coup attempt highlights emerging market risks Looking at changes to investment statements Millennials and ETFs: The perfect match Millennials, Gen Xers losing confidence in ability to meet financial goals: poll Money sense: How to make your savings last as long as you do MoneyTalk: Spending and saving tips for moms Ontario cafe finding success with a pay-what-you-can plan Why putting off tough financial choices usually make things worse Investors in the dark on fees: survey Housing affordability: Not just Toronto and Vancouver issues Why millennials should use a TFSA over an RRSP What's the smartest way to help my kids buy a house? Report: Big investment gap is stunting Canadian startups How Vancouver Island farmers have adapted to a longer growing season Comments AA More Related to this Story University costs to climb further after tripling over past 20 years, study says Let's Talk Investing Video: How society benefits from money-savvy kids Rob Carrick Video: Why schools need to teach financial literacy Follow us on Twitter: @GlobeMoney


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Watch: How One Cow Helped Make a Farmer's Life A Little More Fair
"...I have come from that poverty line, and I'll not go back again."
By joining our e-mail list, you'll learn about Heifer's work through inspiring stories & we'll send you info on how you can help move a family from poverty to self-reliance.
"...I have come from that poverty line, and I'll not go back again."
Watch a Heifer International project participant in Romania make cheese, then try making Heifer Farm's "Farmer's Cheese" Recipe!
Participants of the Rural Women on the Road to Prosperity project, formed six self-savings and loans committees to secure a sustainable source of financing for future undertakings and small business investments.
In a unique project with BlackRock, employees joined us in Cambodia to build chicken sheds and pig pens for participating families.
"I had an idea," she said. "I had seen a lot of coconut trees cut in my village and just left there." During the first break, Klang called her husband and asked him to gather all the discarded coconut trees. When he asked what they were for, Klang, in...
Davit Simonyan was born in a small, rural Armenian village and grew up in the country's capital city of Yerevan. As a young adult, he returned to his birthplace and has built a successful tourism business with a little help from Heifer's Advanced Rural Development Initiative.
The possibilities are endless, and we can't wait to see what's in store for 2016.
It's the last day of 2015, and everyone is taking stock of what happened this year. Check out Heifer's 2015 Annual Report for this year's financials, project data, a few of our best stories and a look towards what the future holds.
Make a year-end contribution to Heifer International and lessen your tax burden. At the same time, you will give a family the resources they need to escape hunger and poverty and live sustainable, self-sufficient lives.
Did you get or give Heifer this year? If so, what was your reaction or reason you chose to #Give Heifer? Let us know!
Need a last-minute gift? Look no further.


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It only takes a minute to get started. Fill out our uncomplicated sign up form and login to the website.
This step is noncompulsory but will drastically raise your chances of getting interest from other over 50s members on the site. Write a little about yourself, your interests and upload some good photographs of yourself.
Use our website's sophisticated search to locate over 50s members who meet your criteria. Or just look through listings and locate people that pique your interest.
Identified someone who you find interesting? Send them a few words or just a wink and find out if they're interested in you. With a little luck you'll be on your way to an amazing adventure.
It's quick and easy


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Getting Ready for NaBloPoMo
I found the blog roll here: Nablopomo and I'm not on it yet. I understand it takes a while to appear so I'm not too worried. I won't know if I registered my blog right unless it shows up on the list. If you see the name Redquill show up please let me know what number I am. It will help me to find it on the list which is almost to 300 already. I've never made a blog on Blogher before so I have 3 days to practice before the official start of this year's Noblopomo. I need to figure out how to add photos, because a blog without photos is like toast without bread.
I imagine I can use Photobucket so I'll go see. If a photo shows up here that means I've figured it out.
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Refreshing Drink Recipes Using Simply Beverages™
Simply Beverages™ use never-from-concentrate juices in all of their beverages. And they never add any preservatives, colors or artificial flavors. Read our bloggers' posts and find fun beverage recipes you can serve this summer! Plus get a chance to win $100. Read more
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Hong Kong holidays + Chinese food and drink
This neighbourhood may have cocktail bars and hotdog cafes but it hasn't forgotten its working class roots – for now
The foodie traveller The foodie traveller ... has congee rice porridge for breakfast in south-east AsiaThis rice-and-stock breakfast dish is eaten all over Asia, either in its plain form or with added tasty garnishes. And it's a great cure for hangovers, too
Hong Kong has some of the best restaurants in China – and they're not all Cantonese. From Sichuan to Manchuria, Susan Jung picks 10 great regional Chinese cuisines and the best places to try them in
There are plenty of fantastic restaurants in Hong Kong, says Susan Jung, food editor of the South China Morning Post. You can even get Michelin-starred dim sum for £5
From Michelin-starred restaurants to meatballs in a street market, OFM takes a whirlwind tour of true Cantonese cooking. Ox tendon optional
Territory enjoying its status as wine capital of Asia since cutting duty from 40% to zero two years ago


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Leon Isaac Moses
Mr. Leon Isaac Moses, 38, of 115 Belton Lane in Camden, died Thursday, July 21, 2016. Friends may visit at 2404 Salmond Street (Kirkwood) in ...
July 29, 2016 | | Obituaries
Outside the line?
SUMTER --- It may have well been the most dissected and discussed plume of white smoke this side of the Vatican.
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Hernandez tabbed as tourney's MVP
SUMTER --- It was seemingly appropriate that when the John Rateree Memorial Most Valuable Player award for the 2016 South Carolina American Legion State Tournament was ...
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Florence doubles up error-prone KC in winners' bracket battle, 12-6
SUMTER --- Call it the Monday blahs. Blame it on the heat or, chalk it up to the grind of a lengthy summer campaign.
July 29, 2016 | Tom Didato | Sports
Cobb's walk-off single sends KC into championip round
SUMTER --- Six days after having extended their season with a pair of play-in game wins, Kershaw County Post 17 used William Cobb's two-out RBI ...
July 29, 2016 | Tom Didato | Sports
Lee and Rutherford to Wed in October
Glenda and Darryl Lee of Cassatt, S.C. announce the engagement of their daughter, Ms. Celestine Denise Lee to Mr. Cedrick Tyrell Rutherford.
July 29, 2016 | | Weddings & Engagements
Robinson and Knight plan May wedding
Mr. and Mrs. William Thomas Robinson Jr. of Cassatt, S.C. announce the engagement of their daughter, Ms. Cassady Alexis Robinson to Mr. Joshua Thomas ...
July 29, 2016 | | People & Places
Catoes celebrate 50th Anniversary
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July 29, 2016 | | People & Places
Julian R. Wilson
Julian Richard Wilson, 21, was born in Camden. He grew up in Lugoff where he graduated from Lugoff-Elgin High School in 2014. He had a ...
July 28, 2016 | | Obituaries
Margaret Coker Latimer
Margaret Coker Latimer of West Columbia entered into her heavenly home peacefully at Agape Hospice House on July 16, 2016 with her son and daughter ...
July 28, 2016 | | Obituaries
Ruth Stanley
Funeral service for Mrs. Ruth Clyburn Stanley, 91, of Camden, will be held Saturday, July 30, 2016, 2:00 P.M. at Saint John Baptist ...
July 28, 2016 | | Obituaries
Thomas Shannon, Sr.
Mr. Thomas Shannon,Sr., 68, of Fort Washington, MD, formerly of Camden, passed on Tuesday, July 19, 2016. Funeral services will be conducted today, July ...
July 28, 2016 | | Obituaries
Elizabeth Jackson
A funeral service to celebrate the life of Elizabeth Ann McCaskill Jackson, 77, of Elgin, will be held at 10:00 AM, today, July 29 ...
July 28, 2016 | | Obituaries
Charles Frost, Sr.
LUGOFF – Private services will be held for family and close friends of Charles J. Frost, Sr.
July 28, 2016 | | Obituaries
Ariail for July 26
July 26, 2016 | | Ariail
Archive site results
International winner
July 05, 2016 | | Education
Kershaw County to use SCAC program to collect debts
Kershaw County Council voted unanimously to adopt a resolution which would allow Kershaw County to collaborate with the South Carolina Association of Counties (SCAC) to collect debts from residents of Kershaw County. The vote came during council's June 28 meeting.
July 05, 2016 | Tenell Felder | Kershaw County
ATEC students sweep state computer service competition
Students from the Applied Technology Education Campus (ATEC) computer service technology program swept the state SkillsUSA competition and represented South Carolina in the national competition.
July 05, 2016 | | Education
JAMES HEAGLER - Bethune
Mr. James Heagler, 61, of 110 Pine Drive, Bethune, SC, passed on Thursday, June 30, 2016.
July 05, 2016 | | Obituaries
DORIS K. HUNTER - Lugoff
LUGOFF – Funeral services for Doris Kelly Hunter, 74, were held Monday, July 4, 2016. In lieu of flowers, memorials may be made to the Pine Grove Baptist Church Building Fund.
July 05, 2016 | | Obituaries
Msgt. ROBERT KELLY (ret.) - Lugoff
A funeral service to honor the life of MSgt. Robert Michael Kelly (Ret. US Army), 60, of Lugoff, SC, will be held at 11:00 AM, Friday, July 8, 2016 at Kornegay Funeral Home, Lugoff-Elgin Chapel. The family will speak to friends following the service at the funeral home. Burial will be held at 2:00 PM at Fort Jackson National Cemetery with full military rites.
July 05, 2016 | | Obituaries
STEPHEN TRUESDALE - Westville
A graveside service to celebrate the life of Stephen L. Truesdale, 57, of Westville, SC, was held at DeKalb Baptist Church Cemetery at 1:00 PM Sunday, July 3rd, 2016. Rev. Keith Coates officiated. Memorials may be made in Stevie's memory to The American Cancer Society, Relay for life of Kershaw County, c/o Gail Kirkland, 647 Lachicotte Road, Lugoff, SC 29078.
July 05, 2016 | | Obituaries
TAMMY WILSON - Cassatt
A memorial service to celebrate the life of Tammy Sue Dubbs Wilson, 55, of Cassatt, SC, will be held at 12:00 Noon, Saturday, July 9, 2016 at Providence Baptist Church. Pastor Richard Dubbs, Jr., will officiate. The family will receive friends at 11:00 AM, 1 hour prior to the service at the church.
July 05, 2016 | | Obituaries
Ariail for July 5, 2016
July 05, 2016 | | Ariail
Editorial: Green opportunities
The city of Camden has been doing quite a bit of work to boost tourism and generate more economic development. The Wayfinding Sign project is well underway and is really making the town look good. Thanks in large part to such incentives as the façade grant program, many buildings downtown are looking sharp. And thanks in large part to an economic development incentive ordinance passed in December 2015, a new 64 room hotel is coming to the I-20/Exit 98 area.
July 05, 2016 | | Editorials
Tatum: Nobody's right if everybody's right
As we hurtle toward intermission in this theater of the absurd we call the 2016 presidential election, I feel compelled once again to hearken back to the prescient words of the late, great George Carlin, who said the following nearly three decades ago:
July 05, 2016 | Jim Tatum | Columns
Parker: Bill-gate, again
WASHINGTON -- But of course Bill Clinton wants his wife to become president of the United States and make history as the nation's first female commander in chief.
July 05, 2016 | By Kathleen Parker Washington Post Writer's Group | Columns
Rich: If life were fair, pecan pie would have no calories
It happened, I suppose, when I was in the 4th Grade: my first strong recollection of the unfairness of life.
July 05, 2016 | | Columns
Teal: An untold Camden dueling story
Under the title of "The Iron Man", authors Kirkland and Kennedy in Chapter XIX of Historic Camden, Nineteenth Century chronicled the story of dueling in Kershaw District. They began with an 1804 grand jury presentment lamenting the prevalence of dueling at that time and concluded with the story of the last duel in S. C., that of Cash and Shannon in 1880.
July 05, 2016 | By Harvey S. Teal Provided by the Kershaw County Historical Society | Columns
Yesteryear - July 5, 2016
Helping to develop a breed of better informed, better qualified and more committed community leaders is a Kershaw County Chamber of Commerce goal, one it hopes to accomplish in the upcoming months.
July 05, 2016 | | Yesteryear
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West Hollywood Talent Agents
1. William Kerwin Agency
1605 N Cahuenga Blvd, Ste 202Los Angeles, CA 90028
From Business: By Appointment Only
2. L A Talent
7700 W Sunset BlvdLos Angeles, CA 90046
3. Go Casting
A real Talent agent will never ask for money. They will find a diamond in the rough. Talent is undiscovered everyday because we don't live in Califo…
4. Rival Agency
9157 W Sunset Blvd 205West Hollywood, CA 90069
5. Michael Mann Talent
121 N San Vicente BlvdBeverly Hills, CA 90211
6. Creative24
221 N Robertson Blvd Ste CBeverly Hills, CA 90211
7. Commercial Talent
9157 W Sunset BlvdWest Hollywood, CA 90069
8. Fusion Management
9200 W Sunset Blvd # 206West Hollywood, CA 90069
9. Gold Levin Talent
8424 Santa Monica Blvd Ste 706West Hollywood, CA 90069
10. Network Intl Models & Talent
215 S La Cienega Blvd # 205Beverly Hills, CA 90211
11. Abrams Artists Agency
9200 W Sunset Blvd # 1130West Hollywood, CA 90069
12. Rex Inc
6311 Romaine StLos Angeles, CA 90038
13. Cmt Talent Agency
8344 W 3rd StLos Angeles, CA 90048
14. Sid Levin Theatrical
8484 Wilshire Blvd Ste 745Beverly Hills, CA 90211
15. Alliance Talent
1626 Wilcox Ave Ste 726Los Angeles, CA 90028
16. Progressive Artists Agency
1041 N Formosa AveWest Hollywood, CA 90046
17. Click Models of Los Angeles Inc
9057 Nemo StWest Hollywood, CA 90069
18. The Magnet Agency
6363 Wilshire BlvdLos Angeles, CA 90048
19. R P M Talent Agency
I was represented by them for about a year. They did submit me. I went on auditions. I even booked something that I didn't audition for. However the…
20. Alvarado Rey Agency
Submitted headshots online and was immediately called the following day to stop by for an interview. The location is in the heart of West Hollywood…
21. Ford Models Inc
they never accept anyone! my friend is so beautiful but they denied her. she ended up going with elite models (much better and very efficient)
22. Sean Healy Presents Inc
7471 Melrose Ave # 17Los Angeles, CA 90046
From Business: By Appointment
23. Tilmar Talent
6404 Wilshire Blvd Ste 735Los Angeles, CA 90048
From Business: By Appointment
24. Association Of Talent Agents
9255 W Sunset BlvdWest Hollywood, CA 90069
From Business: ATA forged alliances ? this year?s passage of the American Jobs Creation Tax Bill Informative ATA Newsletters on WebsiteTHE ASSOCIATION OF TALENT AGENTS (ATA ) is a…
25. The Paradise Group Talent Agent
7211 Santa Monica BlvdWest Hollywood, CA 90046
26. Major Minors Management
8205 Santa Monica BlvdWest Hollywood, CA 90046
27. Huud Terry
853 N Spaulding AveLos Angeles, CA 90046
28. Adam Richmond Comedy Agency
7566 Romaine StWest Hollywood, CA 90046
29. Thomas Cole Events
7901 Melrose AvenueWest Hollywood, CA 90046
30. Factory Artist
8285 1/2 Santa Monica BlvdWest Hollywood, CA 90046


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He downplayed media emphasis on how this session might be different, given the historic Republican majority.
"This session will not be that different. Fellow Arkansans selected us and gave us the task of acting in the best interest of our fellow men and women," Beebe said.
UPDATE: Full speech transcript here.
* JOBS: Beebe said he'd be asking the legislature for help landing "one of the biggest projects the state has ever seen." No details yet.
(I asked Matt DeCample for more details. His response:
"Believe me, if we had more details to release, they would have been in the speech.")
UPDATE: Arkansas Business speculates and maybe arches an eyebrow a tiny bit at the billion-dollar value being tossed around. That's more than a Toyota assembly plant in Texas. State is indicating state-backed bonds would be involved. Hope it's not for a steel mill like the big one that flopped in Alabama.
* SALES TAX CUT: Beebe said he couldn't propose elimination of the small sales tax remaining on food "without endangering needed services." But he said he'd propose legislation to dedicate any future savings from what he said was the inevitable end of state payments in the Pulaski County desegregation case to removing the sales tax on food. He said that might be a year, two years or more away.
* MEDICAID AND NURSING HOME CARE: He said numbers were still in flux on what's necessary to fully fund the existing Medicaid program, though it should be smaller than what has been expected and level 3 nursing home care now seems sure to be protected. Folks will not be thrown out of nursing homes, he said. He touted the smallest growth in Medicaid funding in years.
But he said cuts will still be necessary to balance the books on the program and they will affect "real people." He then turned to the working poor who are without health care insurance. He said federal law provides an expansion of Medicaid to cover them, but it will be up to each state to decide whether to do it. He recited the familiar facts — coverage for 250,000 people at no cost to the state for three years and then a rising cost that tops at 10 percent in 2020. It would provide "immediate savings" in Medicaid that would cover shortfalls in the current program. It would benefit 40,000 hospital workers and "tens of thousands" more workers in related health businesses. A failure to expand the program will continue the "hidden tax" of unpaid services that drive up the cost of health care and insurance for everyone — an average $1,500 a year in premiums to pay for uncompensated care.
Beebe said a conservative estimate of Medicaid expansion's cost in 2020, should the state have to pay it, after factoring in the economic benefit of increased federal dollars both in direct state aid and circulating through the taxed economy, would be tiny, $5 million a year.
He said he, too, was worried about the national debt. But he said that should be done in Washington, as a president from Arkansas once did. Arkansas shouldn't sacrifice its share of the money to other states, Beebe said.
* SCHOOLS: Beebe said the legislature might have to revisit school funding on account of a recent state Supreme Court ruling that Beebe has criticized for ending an equal tax millage contribution to the state aid fund by all school districts. He'll propose a "modest" contribution to reducing inequities in higher education spending.
* STATE EMPLOYEES: He'll propose a 2 percent raise for state employees.
* BIPARTISANSHIP: "We must resolve not to let Washington's animosity seep in and poison our well of civil discourse." Lots of applause. Subsequent action will be a better barometer of receptiveness to that viewpoint that today's clapping.
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Capitals 5 Flyers 4 (SO)
WASHINGTON, D.C. Alex Ovechkin scored with 58 seconds left and goalie Phillip Grubauer off for an extra attacker to cap a late three-goal rally and Washington beat Philadelphia in a shootout.
Mike Green cut Washington's deficit to 4-2 with 8:40 left, and Dmitry Orlov pulled the Capitals to one with 3:31 to go. Ovechkin tied it with his 28th goal.
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Lindholm cleared to return for AHL Norfolk
Defenseman Hampus Lindholm, the Ducks' first-round pick in this year's draft, is cleared to return to action for the American Hockey League's Norfolk Admirals after missing three games due to an upper-body injury.
Norfolk coach Trent Yawney confirmed, through a team spokesman, that Lindholm should be able to play on Thursday against the Hershey Bears at Verizon Center in Washington.
Lindholm was injury on Nov. 24 against Connecticut when the Whale's Blake Parlett drilled him in the latter stages of a 5-2 home victory over the Admirals. Parlett received a charging major but was not suspended.
As the sixth overall pick in the 2012 draft, the 18-year-old Lindholm has a goal, four assists and a plus-2 rating. The native of Helsingborg, Sweden is playing his first season in North America.
Meanwhile, Yawney also said that both Frederik Andersen and Igor Bobkov will continue to share the workload in goal for the Admirals and that "there is no clear cut No. 1 guy." Andersen has started four of the past five games and won consecutive starts over the weekend, allowing only one goal in each.
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Wilfried Zaha and Alex Buttner posed in a Ryan Giggs mask to wish Man United teammate Happy Birthday [Pictures]
- By Dov Rawson
- November 29, 2013
- Comments0
Legendary Manchester United winger Ryan Giggs today turns 40.
The landmark reached by few footballers is something youngsters should be in awe of as they embark on their Premier League careers.
On Friday morning, new Manchester United youngster Wilfried Zaha and Alexander Buttner wished Ryan Giggs happy birthday on Instagram.
They donned a Giggs mask for the picture.
Despite a rough start to his United career, Zaha will no doubt he hoping to follow in the footsteps of the Welsh wizard.
Grab more information on: Manchester UnitedRyan GiggsWilfried Zaha
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If you are interested in following my blog, I would be more than happy to help you.
I haven't been great about tweeting, but I'll try and get better: My twitter feed is at …Joana_Smith
Also, you can friend me on facebook!
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July's Full Moon in Capricorn: The Heart wants what it Wants. The 4 Stages of a Good Divorce. How to Love a Woman who Scares You. Our Soulmates are Rarely Who We Expect. Men, Let's Stop Fooling Ourselves: Size Matters. To the One Who Tried to Break Me. A Letter to my Children: You do not come from a Broken Home. An Open Letter to the Fixers. How your Stored Memories in the Amygdala can lead to PTSD. How My Sister's Death Transformed my Self-Perception.


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Nintendo of America has released a new trailer for Pokemon Black and White 2, showcasing the World Tournament and the gym leaders that are involved in it. The game will be releasing October 7.
New Pokemon Black and White 2 Trailer
August 1, 2012 / 8:14 PM
By: Zack O'Neill
Tagged with: DS, Game Freak, Nintendo, Pokemon Black and White 2
About the author /
Zack O'Neill
Zack started working at VGTribune at 15 years old and has been gaming since the the age of 2. He is currently in college studying film and technology with plans to continue in the game industry. He's also really good at Counter Strike.
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Hi job seekers! Click on this link to find some current hospitality jobs available in Brisbane this week. From seek.com.au. Remember that we have a weekly Job Workshop every Tuesday to help you with your resume, cover letters, interview skills, etc. Good luck!
Welcome Tokyo High School!
This week we have a great group of students from Tokyo. They will be at Lexis Brisbane for 2 weeks improving their English skills, as well as having fun sightseeing in and around this beautiful city of Brisbane. Welcome guys, enjoy!!
This Week's Fantastic Four
Today we welcomed 4 new students. Please say hello to (from left to right) Lucas from Brazil, Melody from Spain, Jihee from South Korea and Samantha from Colombia.
We hope you have a great first week at Lexis Brisbane and make the most of this experience in Australia.
Welcome New Students!
This week, we welcomed six new students from five different countries! Let's give a warm welcome to Marta and Hubert from Poland, Maria from Colombia, Asumi from Japan, Rebecca from Italy and Lucas from Brazil. We are so happy to have you all her at Lexis Brisbane, and we wish you all the best for this fantastic English language study adventure.
What's on in Brisbane this weekend! :D
July is packed full of fun and exciting activities, and there is plenty more to see and do this weekend. There's even a Medieval Festival coming up that I'm super excited about!! So if you want to know more, then keep on reading!
We just LOVE our food here in Brisbane, and the entire month of July is all about food with the return of the Good Food Month event. For the next 3 weeks, restaurants, cafes and bars around the city will be showcasing the very best they have to offer to you guys – the public! There's something for everyone; breakfast lunch and dinner. Take a look at the official website for more information and timetables.
If you haven't heard about the Brisbane Winter Festival by now, where have you been?! The giant snow toboggan in King George Square is a big give away! This weekend is your final chance to enjoy this beautiful Brisbane Winter event before it's gone forever!! (I just wish we had REAL snow here…) :(So be sure to at least try ice-skating over in front of the clock tower this weekend! There will be plenty of other icy events on as well.
This week also marks the coming of NAIDOC Week, which celebrates our native Aboriginal and Torres Strait Islander people. Head on over to Musgrave Park in South Brisbane this Friday for a fun-filled day with rides, stalls, traditional foods, story telling and much more! Let's celebrate our beautiful Indigenous Australian history and people together.❤ Entry is only a gold coin donation. If you want to learn more about NAIDOC Week, click here.
Keeping in the theme of celebrating culture, this Friday the Brisbane French Festival (or Le Festival) is back and bigger than before! Running all weekend, South Bank will be painted red, white and blue with all things French! Come on down to celebrate this colourful, arty culture, enjoy a croissant or two and sip on fine French wine! Major landmarks around town like the Treasury Casino will also be coloured in the traditional French colours to show its support! Take a look at the website here for more info!
Sharpen your swords, and saddle your horses – the Abbey Tournament is back!! The gates to Abbeystowe will be opened at 8:45am this Saturday, soon followed by the official Grand Parade Opening Ceremony. Get in on all the fun by dressing up as a knight, a gypsy or even a dragon, as cosplaying is highly encouraged! There will be plenty of Medieval themed stalls, games, and food available on both days, so be sure to bring plenty of gold coins! Tickets to the annual Jousting competition will also be on sale both Saturday and Sunday, so don't miss out! For more information about this event, and how to get there, visit the official website. To save time at the front gate, purchase your tickets online. See you there!!
That's all for now, folks! As always, have a fun yet safe weekend! Remember to keep warm, and plan your journey ahead of time to avoid any unnecessary problems! See you next week!!😀
Job Search Tuesday!! :)
Freshly picked from Seek are this week's job listings that are perfect for students looking to make some extra moolah. $$ Look no further, Job Search Tuesday is here!!😀
The Emporium Hotel in Fortitude Valley are looking for their next all-rounder, to cover a range of shifts within the bar/dining and coffee making. 20+ hours on offer for this part-time role. Must have at least 1 year experience in similar roles, and hold a current RSA*. Click the Apply Now button here on the job ad.
*RSA (Responsible Service of Alcohol) is required by law to work with and around alcohol throughout Australia. You can get yours today by completing a short online course. Bundle and save, with courses starting from just $18. For more information, visit the Hospitality Institute of Australasia's website here.
The Brisbane International Hotel in Windsor are currently searching for experienced Food & Beverage attendants to join their busy Italian style restaurant. 15-20 hours on offer per week, but only those with at least 4 years experience in a professional hospitality environment will be considered for the job. Must have your own transport. Send your resumes to Bec on: firstname.lastname@example.com. Click here to read more.
The Kenmore are hiring new Bar, Bistro and Gaming Attendants to join their newly renovated venue. Duties include (but not limited to) serving customers, following RSA and RSG* laws thoroughly and precisely and knowledge on food and drink products is a must. 20-30 hours a week are on offer to the right people! To apply, click here.
*RSG (Responsible Service of Gaming) is similar to the RSA, but is required to work with gaming products within licensed venues. Visit the above Hospitality Institute's website for course details. Bundle with an RSA course and SAVE!
Bar Pacino over on Eagle Street here in the city are looking for professional bar tenders who have a passion for making cocktails! The right person for the job should be able to work independently, offer outstanding customer service, and of course be a cocktail mastermind! If this sounds like you, and you have your RSA ready to go, then why not apply?
Experienced bar tenders also needed at a busy venue in Brisbane's West End. Variety of shifts available. All training will be provided, with knowledge on cocktails a bonus, but not necessary for applying. For more info, click here.
The Tinderbox Kitchen in Fortitude Valley have an opportunity available to bartenders with at least 2 years experience in hospitality. This casual position offers 30-35 hours per week to those who are willing. For more info and to apply, click here.
Just over in Ann Street next to Lexis is The Cutting Board, and they're looking for a new barista to join their team. Must have at least 2 years experience brewing the perfect coffee! If this sounds like you, click here to apply.
Another busy cafe here in Brisbane CBD are looking for an experienced barista to start immediately. To be considered for the job, you should be able to work independently, have cash handling experience, and have an outgoing and friendly personality. This casual position offers 20-25 hours a week, Monday to Friday only, (no weekends – yey)!! If this sounds like the job for you, click here.
There is a retail position available at the University of Queensland – St Lucia Campus at their famous Lolly Shop! As a former UQ student, I highly recommend this shop, so how cool would it be to work there?!!😀 This is the perfect opportunity for you to get a taste of what University life is like here in Australia while getting working experience, and you'll also have the chance to meet hard working students like yourselves! So if you've got the retail experience to back up your application, apply today!!
Those are just a few jobs that are on offer this week. You can always do a more defined search using the Seek website. Another good website to check out is Gumtree, especially if you're looking for cleaning jobs, (just click on the "Jobs" tab, and choose your profession).
Don't forget that today is also our Jobs Workshop day! It has moved back to Room 3 again this week, so be sure to check it out if you need that little extra help. Starts at 2pm (after Option Class). See you there!
Good luck with the job hunt, everyone!
New Students – 4/7/16
Yesterday we welcomed 6 lovely new students to the Lexis Family!❤ Despite being a little sleepy, everyone was very excited to begin their new adventure! Please say a big hello to…:
Issui from Japan, Vlastimil from the Czech Republic, Miu from Japan, Fabio from Italy, and Rikako also from Japan. Also joining us this week was Koji, who transferred to us after spending some time at Lexis Perth!
Welcome, everyone! We hope you have an enjoyable first week. As always, if you have any questions, come and see one of the friendly staff!


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The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: MAC JMB. Performed the experiments: MAC AAS. Analyzed the data: MAC AAS MSHK JMB. Contributed reagents/materials/analysis tools: MAC MSHK. Wrote the paper: MAC JMB.
Current address: HPV Research Group, Division of Pathology, School of Molecular and Clinical Medicine, University of Edinburgh, Edinburgh, United Kingdom
The authors have declared that no competing interests exist.
Detection of low-level, lineage-specific transcription aids in the identification of lineage-primed populations of ES cells provides a new framework for pluripotency.
ES cells are defined as self-renewing, pluripotent cell lines derived from early embryos. Cultures of ES cells are also characterized by the expression of certain markers thought to represent the pluripotent state. However, despite the widespread expression of key markers such as Oct4 and the appearance of a characteristic undifferentiated morphology, functional ES cells may represent only a small fraction of the cultures grown under self-renewing conditions. Thus phenotypically "undifferentiated" cells may consist of a heterogeneous population of functionally distinct cell types. Here we use a transgenic allele designed to detect low level transcription in the primitive endoderm lineage as a tool to identify an immediate early endoderm-like ES cell state. This reporter employs a tandem array of internal ribosomal entry sites to drive translation of an enhanced Yellow Fluorescent Protein (Venus) from the transcript that normally encodes for the early endodermal marker Hex. Expression of this Venus transgene reports on single cells with low Hex transcript levels and reveals the existence of distinct populations of Oct4 positive undifferentiated ES cells. One of these cells types, characterized by both the expression of the Venus transgene and the ES cells marker SSEA-1 (V+S+), appears to represent an early step in primitive endoderm specification. We show that the fraction of cells present within this state is influenced by factors that both promote and suppress primitive endoderm differentiation, but conditions that support ES cell self-renewal prevent their progression into differentiation and support an equilibrium between this state and at least one other that resembles the Nanog positive inner cell mass of the mammalian blastocysts. Interestingly, while these subpopulations are equivalently and clonally interconvertible under self-renewing conditions, when induced to differentiate both in vivo and in vitro they exhibit different behaviours. Most strikingly when introduced back into morulae or blastocysts, the V+S+ population is not effective at contributing to the epiblast and can contribute to the extra-embryonic visceral and parietal endoderm, while the V−S+ population generates high contribution chimeras. Taken together our data support a model in which ES cell culture has trapped a set of interconvertible cell states reminiscent of the early stages in blastocyst differentiation that may exist only transiently in the early embryo.
Embryonic stem (ES) cells are karyotypically normal, embryo-derived cell lines that are pluripotent, i.e. capable of generating all the cell types of the future organism, but not the extra-embryonic lineages. What gives ES cells this unique capacity? Here, we use a fluorescent reporter cell line that employs translational amplification to visualize single ES cells expressing low levels of lineage-specific genes. With this reporter we split ES cell cultures into two fractions that both express certain stem cell markers but only one of which expresses low levels of an endodermal marker gene. Following purification, single cells from either fraction are equally competent to re-establish a heterogeneous culture. However, when challenged to differentiate immediately after purification, each exhibits strong lineage bias, with the endoderm marker-expressing fraction unexpectedly able to contribute to the extra-embryonic endoderm in chimeric embryos. These data suggest that ES cells expand under steady-state conditions as a heterogeneous mix of lineage-biased—but not lineage-committed—cell types. We propose that these observed uncommitted substates exist temporarily in vivo, but are perpetuated in vitro under the selectively self-renewing conditions of ES cell culture. Our findings suggest that pluripotency is determined by the capacity of a mixed population of lineage-biased intermediates to commit to different cell fates in specific contexts.
ES cells are an in vitro cell line derived from the inner cell mass (ICM) of the early mammalian blastocyst
ES cells can be described based on a characteristic morphology, the presence of cell surface markers such as SSEA-1 and Pecam1, or the expression of the key transcription factors such as Oct4, Sox2, Nanog, and a number of ES cell-specific transcripts (ECATs)
Recent observations suggest that there may be lineage-specific markers expressed in sub-populations of ES cell cultures. In particular, the expression of the ICM markers Nanog, Rex1, and Stella has been shown to be heterogeneous
ES cells are derived from a stage of development in which key early lineage specification events are occurring. ICM cells are formed from the inner cells of the morula as the outer cells form the first extra-embryonic or trophoblast lineage. A day later, at implantation (4.5
When injected into host blastocysts, cells derived directly from the ICM of an expanded blastocyst stage can contribute to the PrEn as well as the fetus
One of the earliest markers of anterior asymmetry in the AVE is the homeobox transcription factor Hex. While Hex is discretely expressed in the VE on the anterior side of the embryo, it is initially expressed throughout the early PrEn
To generate a reporter cell line that gives real time read outs of low-level early endodermal gene expression, we introduced a synthetic internal ribosomal entry site (IRES) designed to amplify translation upstream of a fluorescent reporter
(A) Schematic representation of the gene targeting strategy. Hex cDNA tagged with a recognition site for the bacterial BirA ligase (B), followed by an artificial IRES sequence composed of a tandem array of reiterated 9 bp elements from the Gtx promoter and DNA encoding the fluorescent reporter, Venus, was inserted into the first exon of the
To confirm that the expression of the Venus allele reflects endogenous Hex expression
The low levels of Hex transcript observed in undifferentiated ES cells (
(A) Flow cytometry of two independent HV clones (HV 5.1 and HV 16.1) cultured either under self-renewing conditions or in the absence of LIF show the presence of a subpopulation of cells positive for Venus and/or the ES cell surface marker SSEA-1. Gates for expression of Venus and the presence of SSEA 1 were based on unstained E14 ES cells. Upon the removal of LIF for 3 d, the percentage of cells negative for SSEA 1 increased in both HV clones and the E14 cell line. (B) Fluorescence microscopy of the HV cell line in the presence or absence of LIF. Cultures were differentiated as (A). Note the brighter intensity of Venus in the tightly apposed pavement-like cells in the LIF negative culture (white arrows). Venus expression is absent from giant flat cells (white arrowheads). (C) Expression of the Venus transgene is similar to the low-level expression of the Hex cDNA. RNA was prepared from self-renewing cultures of three HV clones, parental R26BirA cells, and Cgr8 cells. Quantitative PCR analysis was carried out to monitor levels of mRNA derived from both targeted and untargeted alleles of Hex (1f, 2r) or targeted allele only (Bf, 1r). The schematic diagram depicts the different primers used. Values for each primer set used were normalised to the levels of Actin value obtained for each sample.
As we were initially surprised by these observations, we asked whether the expression level of Venus RNA was equivalent to that generated by endogenous Hex. Using quantitative PCR, we compared the levels of Hex transcript from the wild-type and transgenic alleles to those from the transgenic allele only. We found that the endogenous Hex was expressed at extremely low levels with the transgene representing between 50% and 75% of this value (
Since V+ cells were found abundantly in the SSEA-1 positive population, we asked whether this population expressed other markers of the undifferentiated state. Antibody staining for Nanog and Oct4, imaged alongside YFP/Venus fluorescence, indicated that while the Venus positive cells were also Oct4 positive, they expressed low levels of Nanog (
(A) Venus positive cells express Oct4, but not Nanog. Colonies of HV cells were fixed and immunostained for both Oct4 and Nanog. Primary antibodies specific to Oct4 and Nanog were detected using Alexa 568 conjugated secondary antibodies (red). Images include Venus fluorescence, antibody staining, overlay of Venus and antibody, and bright field for each cell line and the indicated antibodies. (B) Quantitative RT-PCR showing the relative expression of endodermal and pluripotency genes between Venus positive and negative cells obtained from the SSEA-1 positive fractions of two HV clones following flow cytometry. Quantitative PCR analysis was performed to compare transcript levels of Venus with PrEn (
To further address what the co-expression of these markers represented, we purified populations of cells from ES cell culture based on the expression of the Venus transgene and SSEA-1 by flow cytometry. Quantitative real time PCR based on RNA extracted from both SSEA-1 positive fractions revealed that while Oct4 levels remained constant, the Venus positive fractions from two different clones expressed higher levels of the PrEn markers Gata4, 6, Dab2, Sox7, and Hnf4α and lower levels of ICM markers such as Nanog, Klf4, Stella, and Rex1 (
To test the notion that this low level of transcription at the Hex locus producing the V+S+ fraction in ES cell culture represented an immediate early state in PrEn differentiation, we examined global differences in gene expression. RNA was isolated from all four fractions (V−S+; V+S+; V−S−; V+S−) in two independent clones of HV ES cells and hybridised to NIA Mouse 44K Microarray chips v2.3 (GEO Accession GSE13472)
Analyses of global gene expression in fractions defined by expression of the Venus transgene and SSEA-1. HV ES cells grown under self-renewing conditions were fractionated by flow cytometry into four fractions based on Venus (V) and SSEA-1 (S) expression. RNA was isolated from the following fractions: V−,S+; V+,S+; V−,S−; V+,S− and hybridised to a NIA Mouse 44K Microarray v2.1. (A) Heat map illustrating hierarchical clustering of differentially expressed genes identified in a pair-wise analysis of all four fractions. Significant changes in the expression of 2,169 genes (FDR <0.05) resulted in the identification of three to four expression groups, depending on whether clonal variation is taken into account. (B) Pair-wise comparisons (FDR <0.05, >1.5-fold expression levels) of the two ES cell populations, V+S+ and V−S+ depicted alongside the comparison between differentiated PrEn V+S− fraction and the Venus negative ES cell fraction (V−S+). (C) Gene expression changes characteristic of PrEn, ICM/pluripotency, neurectoderm, and mesoderm genes (expression of individual markers are included as supplementary,
We analyzed overrepresentation of Gene Ontology (GO) terms in the non-redundant genes that were overexpressed in the V+S− and V+S+ fractions based on 1.5-fold change with a 0.05 FDR (
An equally consistent pattern of gene expression is observed in the set of ICM markers (contained within Group 2 in
To confirm that early differentiation pattern exhibited in the V+S+ fraction was indeed an early state in PrEn differentiation, rather than a metastable pro-differentiation state similar to that described for the Oct4 positive populations that do not express Nanog, Rex1, or Stella
As Nanog is rarely expressed in the Venus positive cells, we asked whether enforced Nanog expression would suppress baseline transcription at the Hex locus and thereby reduce expression of the Venus reporter. Nanog was misexpressed in HV ES cells under control of the CAG promoter driving an IRES puro cassette
(A) Western blot demonstrating Nanog overexpression from the CAG promoter in two clones of HV cells. Control clones were derived in parallel with an empty vector. (B) Nanog overexpression makes HV ES cells resistant to LIF withdrawal. Nanog overexpressing and control cell lines were cultured in the absence of LIF for 10 d and assessed for ES cell-like morphology. (C) Nanog overexpression suppresses the V+S+ population. Expression of Venus and SSEA-1 were quantitated by flow cytometry in two independent clonal lines and compared to both control and parental cells.
Nanog overexpressing HV cells were grown in the presence of LIF and the fraction of these cultures that expressed the amplified Venus transgene quantitated by flow cytometry. In two independent clones we observed a dramatic reduction in V+S+ population (3–6-fold,
The ability of Nanog to suppress early Hex positive endoderm states is consistent with both the mutually exclusive nature of Nanog and Gata6 expression in vivo
(A) FGF signalling modulates Nanog and Gata6 expression in HV cells. Inhibition of FGF signalling with PD173074 (10 nM) increases the levels of Nanog gene expression in two HV clones while slightly reducing low-level Gata6 expression. Conversely, potentiation of this pathway with the phosphatase inhibitor Sodium Vanadate (50 µM) in aggregate cultures (EB + Na3VO4) reduces the levels of Nanog while increasing those of Gata6. Transcript levels were assessed by qPCR and normalised to the TBP value obtained for each sample. Normalised values are related to the untreated sample for each clone. (B) The V+S+ fraction responds to FGF signalling. Cells grown as in (A) were subject to flow cytometry. Inhibition of the FGF pathway by PD173074 or culture in 2i reduces the extent of Venus expression, while Sodium Vanadate stimulates it. (C) Measurement of Phospho-Erk levels in V+S+ and V−S+ fractions shows an enrichment of activated Erk with Venus positive ES cells. (D) Venus cells persist in 2i culture. Immunocytochemistry of HV cells in 2i culture show the persistence of some Venus cells that have lower levels of Nanog expression, whereas Venus and Oct4 co-express.
We confirmed the ability of Fgf signalling to regulate the V+S+ population by treating suspension cultures with the phosphatase inhibitor sodium vanadate to stimulate the FGF/Grb2/Mek pathway. Treatment of cell aggregates with sodium vanadate in the presence of LIF has been shown to repress Nanog and stimulate PrEn differentiation
Heterogeneous ES cell states have been observed with respect to Nanog, and while the Nanog expression state appears reversible, there are significant differences in the ability of Nanog positive and negative cells to clonally reconstitute each other in vitro
(A) Reconstitution of Venus distribution from single V+S+ or V+S− cells. HV cells cultured under self-renewing conditions were subjected to flow cytometry to separate Venus positive and negative subpopulations within the SSEA-1 positive fraction. A sample purity check is shown in the top panel. Representative clones produced from each fraction plated at clonal density and imaged by fluorescence microscopy are shown. (B) Flow cytometry on cells from each fraction 24 h after plating. (C) Flow cytometry on cells plated at single cell density in 96 well plates from each sorted fraction. Cells were cultured for 10 d following plating and 12 wells derived from each fraction were subjected to flow cytometry. All appeared identical and a representative image of each is shown in the figure.
|Number of Clones Obtained
|% of Clones Fluorescent by Microscopy
|Venus−, SSEA1+
|Venus+, SSEA+
To determine the length of time required for the two states to interconvert we purified populations V+S+ and V−S+ cells and examined the extent to which the original distribution was re-established and observed significant changes in both populations within 24 h of plating (
The ability of these populations to interconvert in vitro combined with their subtle differences in gene expression lead us to ask if there was any functional significance to this low level of PrEn gene expression. As ES cells are defined based on their ability to contribute to all tissues of the future conceptus in chimeras, we asked whether the embryo contribution activity of ES cells was contained in either V+S+ or V−S+ fraction or both. Initially we injected purified fractions of HV ES cells into Rosa26 blastocysts that constitutively express β-galactosidase (β-gal) and examined embryos at 9.5
The loss in ability to contribute to blastocysts generated in this transient PrEn-like state was interesting, but we wanted to establish if these cells had gained new properties. To ascertain this we generated cell lines that both contained the HV cassette and constitutively expressed β-gal as a lineage label. We used this cell line for morula aggregation and obtained the chimeric embryos shown in
(A) V+S+ and V−S+ ES cells contribute differently to embryos in morula aggregation. HV cell lines constitutively expressing β-Geo from the CAG promoter (HV lacZ) were fractionated into V+S+ and V−S+ and their ability to contribute to chimeric embryos assayed by morula aggregation. Within an hour of separation by flow cytometry, cells from each fraction were aggregated with wild-type F1 morulae. Following transfer into pseudo-pregnant mice, resultant embryos were harvested at E6.5 and subjected to X-gal staining. Representative embryos derived from each population are shown. White bars indicate the plane of section shown in the panel beneath specific embryos. Black arrows show the presence of LacZ positive cells in the visceral endoderm. Black arrowheads show the presence of LacZ positive cells in the parietal endoderm. (B) Only V−S+ fraction forms normal spherical EBs. Fractionated HIV ES cells were cultured for 4 d as aggregates in the absence of LIF. (C) V+S+ cells contribute to the presumed visceral endoderm in chimeric EBs. When V+S+ cells were recombined with V−S+ cells immediately following sorting, they formed normal EBs and the V+S+ cells move preferentially to the outside to form the presumptive visceral endoderm. The V−S+ fraction of HVlacZ cells was combined with an equivalent number of V+S+ HV cells (top) or V+S+ HVlacZ cells recombined with V−S+ HV (bottom). EBs were stained with X-gal and representative sets shown. The bottom panel shows sections through representative chimeric EBs. (D) Sections of EBs grown under the same conditions as in part (C), showing that the outer layer consists of visceral endoderm as marked by
|Venus+, SSEA+
|Venus-, SSEA1+
|No contribution
|VE/PE contribution
Table shows the numbers of resultant embryos from aggregation with wild-type morulae following fractionation based on Venus and SSEA1 expression by flow cytometry. Percentages of embryos with LacZ positive cells detected in the Visceral or Parietal (VE/PE) endoderm are shown.
Interestingly, while the V+S+ cells did not effectively contribute to the epiblast, V+S+ ES cells were found in both the visceral and parietal endoderm (
In this paper we have used translational amplification to detect an immediate early and reversible state in PrEn differentiation that appears an inherent component of standard ES cell culture. The existence of ES cell precursors to this lineage is supported by the observed heterogeneous expression of other PrEn genes, Lefty1, Cerl, and Gata6 in the ICM of blastocyst stage embryos, the stage from which ES cells are derived
A number of recent studies have suggested that ES cell cultures are heterogeneous and can be split into two developmental states, one that resembles the ICM and the other early epiblast or PrEc. Thus it has been suggested that ES cell cultures can be split based on Rex1 and Oct4
The schematic diagram depicts the potential cell subtypes that make up ES cell culture. The red line represents the boundary established by the culture conditions. We depict an early PrEn precursor cell defined by the V+S+ phenotype in light yellow, expressing low levels of PrEn determinants such as Hex and Gata6. This cell type is shown in equilibrium with an ICM-like cell. A hypothetical PrEc cell implied by the findings of others is indicated in blue.
In addition to expressing slightly increased PrEn gene expression, V+S+ cells also contain almost all the phospho-ERK activity in our ES cell cultures (
The notion that a Nanog positive, ICM-like population of high probability self-renewing cells is a developmental ground state is supported by the expansion of this state in the presence of a blockade on the major signalling pathways known to promote ES cell differentiation, the MAP kinase/ERK cascade and GSK3β
Based on the equivalent regenerative capacity of V+S+ and V−S+ cells, the small number of significant gene expression changes, and the identical morphology, we assume that these two cell states have not drifted significantly apart. Rather these states may represent distinct reversible transcriptional signatures affecting key lineage regulators. Comparison of the differences in gene expression between the V+S+ and V−S+ fractions supports this idea. Every PrEn marker present in our data set increased in the differentiated V+S− cells and importantly showed small but consistent increases when the V+S+ fraction was compared to V−S+ cells. As a result we believe that ES cells in culture consist of a mixture of early self-renewing precursors that can alternatively express low-level transcription of different lineage-specific promoters related to the states surrounding the early blastocyst (
The model in
Our observation that the V+S+ fraction preferentially contributes to the VE when mixed with more ICM-like cells indicates that low-level lineage-specific changes in gene expression have functional consequences. That we have observed a direct contribution of ES cells to both visceral and parietal endoderm also has implications for canonical definitions of pluripotency. Pluripotency is defined based on the ability of ES cells to contribute to the embryonic but not extra embryonic lineages and our observations suggest this definition may need to be somewhat modified. Alternatively it might be more appropriate to consider ES cells as closer to totipotent, but that the pluripotent ICM fraction of ES cell cultures has a competitive advantage when tested in chimera generation. In support of this idea, Beddington and Robertson originally observed ES cell contribution to all the extra-embryonic lineages, but in particular to parietal endoderm
The observation that some ES cells retain the capacity to contribute to the extra-embryonic lineages begins to resolve a number of conflicting observations. Why should ES cells be able to generate PrEn in vitro but not in vivo? Moreover, as it has recently been shown that VE can contribute to the embryonic gut
The capacity of V+S+ cells to colonize the exterior of EBs and extra-embryonic endoderm in chimeras is similar to the properties of extra-embryonic endoderm (Xen) cells derived from the mammalian blastocyst
We recently performed a genome wide screen looking for Hex targets in ES cells and found a number of genes with ICM expression patterns
ES cells were cultured on 0.1% gelatin-coated flasks or plates (IWAKI) in Glasgow modified Eagle's medium (Gibco) containing non-essential amino-acids, glutamine and sodium pyruvate, 0.1 mM mercaptoethanol, and 10% Fetal Calf Serum (FCS) together with LIF
ES cells were differentiated toward ADE in aggregation culture according to
The 5′ and 3′ arms used for homologous recombination were described by Martinez Barbera et al.
Cells grown in 12 well plates were washed 2× in PBS before fixation in 4% paraformaldehyde. Cells were then permeabilised in PBST (1× PBS, 0.1% Triton X (Sigma)). Blocking was performed by adding 1% Bovine serum albumin (Sigma) in PBST solution to the fixed cells for 30 min at room temperature (rt). Primary antibodies were added at a dilution of 1∶1000, and incubation continued overnight (o/n) at 4°C. Following 3×10 min washes in PBST, Alexa568 conjugated secondary antibodies diluted (1∶1000) in blocking solution were added to the cells and incubation took place at rt for 1 h. Also included at this step was DAPI solution (1∶1000). Finally, cells were washed 3 times, then stored in PBS. Primary antibodies used were mouse anti-Oct3/4 (Santa Cruz) and rabbit anti-Nanog peptide specific antibodies (a gift from Ian Chambers)
ES cells or EBs were collected into Cell Dissociation Buffer (Gibco) and incubated at 37°C for 10 min. Single cells suspension was achieved by gentle repeated pipetting. Following washes in PBS, cells were resuspended in 500 µl FACs buffer (1× PBS, 10% FCS) and 7AAD solution (BD Pharmingen, 5 µl/1×106 cells) to exclude dead cells. Analysis of fluorescence took place in a FACSCalibur flow cytometer (BD Biosciences). Dotplots were generated using CellQuest software (BD Biosciences).
In the case of additional labelling of specific cell surface proteins, primary antibodies were added at a dilution of 1∶1000 to cells resuspended in FACs buffer. Incubation took place for 10 min on ice. Following three washes in FACs buffer, cells were resuspended in fresh FACs buffer containing appropriate conjugated antibody at a dilution of 1∶1000 and incubated as before. After three washes in FACs buffer, cells were finally resuspended in 500 µl FACs buffer and analysed as above.
For collection of populations, cells were prepared as above and subjected to flow cytometry using the MoFlo MLS high speed sorting apparatus (DakoCytomation). Cells were collected in FACs buffer and stored on ice for further analysis.
Chimera mouse generation was performed by morula aggregation with or injection of ES cells into host blastocysts. Injected or aggregated blastocysts were then transferred into pseudopregnant recipient mothers. Embryos were dissected at the stages indicated in the figures and imaged by fluorescent and conventional microscopy.
X-gal staining of embryos and EBs was performed as follows. Embryos and EBs were washed in PBS solution (80 mM sodium phosphate, 15 mM potassium phosphate, 27 mM KCl, and 1.37 M NaCl), then fixed with X-gal fix solution (1× PBS, 2 mM MgCl2, 5 mM EGTA, 1% paraformaldehyde, 0.2% Glutaradehyde, 0.02% NP-40) at 4°C for 20 min. Following 3×20 min washes in PBS they were then stained with X-gal staining solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCI, 0.01% sodium deoxycholate, 0.02% Nonidet P-40 (NP-40) in PBS) o/n in the dark at rt. Following 3×5 min washes in PBS, stained embryos or EBs were then fixed in 4% paraformaldehyde.
X-gal stained, paraformaldehyde fixed embryos were embedded in paraffin wax and sectioned transversely in a microtome at 7 micron intervals. X-gal stained or unstained EB or embryos were also cryosectioned. Samples were sunk in 30% sucrose in PBS, frozen in Tissue Teck, and sections were cut on a Cryostat (Leica). Sections were collected on poly lysine microscope slides (VWR International), air-dried for 30 min to 1 h, and stored at −20°C until used. Immunocytochemistry was performed essentially as described above for cells.
RNA was extracted from different cell populations using Trizol™ (Invitrogen) and precipitated with isopropanol. Biological and technical replicates for each population were hybridised to NIA Mouse 44K Microarray v2.3 (whole genome 60 mer oligonucleotide probe; manufactured by Agilent Technologies, #014951)
Pairwise comparisons were performed using standard statistical conditions (FDR <0.05, >1.5-fold expression levels) to unveil genes up-regulated or down-regulated between the populations. Log intensity plots for each gene were created to find pattern matches between those of similar tissue origin.
We thank Ian Chambers, Tariq Enver, Tilo Kunath, Alfonso Martinez Arias, Jenny Nichols, Sally Lowell, Austin Smith, and Val Wilson for discussions and critical comments on this manuscript; Bill Stanford, Shankar Shrinivas, Tetsuya Tanaka, and Ian Chambers for reagents and advice; and Jeanette Astorga, Yulan Piao, Dawood Dudekula, Simon Monard, Rosa Portero, Janice Ure, and Yong Qian for technical assistance.
anterior visceral endoderm
embryoid bodies
ES cell specific transcripts
embryonic stem cell
false discovery rate
gene ontology
Hex RedStar
hex venus
inner cell mass
internal ribosomal entry site
primitive ectoderm
primitive endoderm
visceral endoderm
extra-embryonic endoderm cells


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War vet/amnesiac/accused murderer Adrien Brody (pictured) is committed to an asylum with a questionable level of care — it involves trippy drugs, a straitjacket, and a body drawer in the morgue. Keira Knightley and Kris Kristofferson costar.
Posted February 10 2005 — 12:00 AM EST
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Blogs HNN "ISRAAID IN AMERICA"Sep 27, 2005 3:31 pm
To be honest, I have just discovered the Israaid website and it could not make me happier. It is not easy to hold on to the old time humanitarian traditions when one serves as a Scapegoat to the World and you need to fight the same battles over and over again. But it is never as important as during those times.
I have recently attended a private briefing with a senior Israeli diplomat with a extensive intelligence background. He was grilled about Israel's repeated hapless efforts to reach an accomodation with the Palestinians. His strategic answers left much to be desired and he knew it.
Finally he lost his temper and shouted:"It says 'Peace, Peace you shall chase!' Who are we if we don't do that?"
All I could think is, these are my people, these remain my people and wipe a secret tear.
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Sitka assembly votes to continue litigation against land sale ballot sponsors. Pros and cons of Coastal Zone ballot initiative discussed at Sitka Chamber of Commerce. Crews fight invasive weeds on Admiralty Is.
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Crouching Yeti APT Actor Still Targeting Industrial Sectors: Kaspersky
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