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70-338 - Lync 2013 Depth Support Engineer - Braindumps Information

Vendor : Microsoft
Exam Code : 70-338
Exam Name : Lync 2013 Depth Support Engineer
Questions and Answers : 114 Q & A
Updated On : July 17, 2018
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70-338 Questions and Answers

70-338 Lync 2013 Depth Support Engineer

Article by Killexams Microsoft Certification Experts


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Microsoft Lync 2013 Depth

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Nectar services Corp. Completes Qualification With Microsoft Lync and Chosen as Depth accomplice | killexams.com real questions with brain dumps

(Marketwire via acquire Media NewsEdge) FARMINGDALE, new york -- (Marketwired) -- 10/23/13 -- Nectar features Corp., a pacesetter within the development of network monitoring and management software for IP voice, video, facts, and superior purposes for the Unified Communications (UC) industry, today announced that the company's utility is qualified with Microsoft Lync and has also been chosen as a Depth partner within the Lync software defined Networking (SDN) API application.Nectar CMP and CEM (Cloud experience manager) convey the one/two punch to be sure the highest quality Microsoft Lync deployment and experience. building upon CEM's yr of success, Nectar brings new value to Microsoft Lync purchasers and furthers its capabilities with the Lync SDN API. Nectar can be concerned in the earliest prototypes of know-how concentrated on enhancing the Lync journey and intends to have its industrial-ready items and enhancements delivered simultaneous with Microsoft's authentic releases of the answer.

"we're very blissful Nectar has chosen to become a Depth accomplice within the Lync SDN software," commented Pascal Menezes, important software manager of Microsoft. "Nectar brings deep technical knowledge and experience in the box of UC service assurance monitoring. Nectar's Cloud adventure manager (CEM), a part of their Converged administration Platform (CMP) is a extremely powerful Lync monitoring solution; which has the means to take real-time performance monitoring of Lync deployments to a brand new level." Menezes further brought up, "Nectar's solution turned into qualified by way of Microsoft as being completely interoperable with Microsoft Lync Server 2010 and 2013, and is also the primary solution to be utterly appropriate with the Lync SDN API." "here is a really massive milestone and fulfillment for Nectar," said David Giangano, Nectar's CEO. "The Nectar CMP suite of solutions, along with Microsoft Lync Certification, grants interesting value and capabilities; vastly benefiting our channel companions and customers." Giangano added, "collaborating as a Depth partner within the Lync SDN program provides Nectar the capability to focus on an outstanding client adventure and align our Microsoft construction roadmap extra enhancing Nectar CMP and CEM." Nectar's Cloud journey supervisor (CEM), as a part of its Converged management Platform (CMP) answer, provides comprehensive perception into community concerns for quick problem resolution which consequences in a lower total charge of possession (TCO). Nectar is the first community monitoring and service assurance company to had been efficaciously proven, qualified and deployed with the Microsoft Lync SDN API. Nectar's tight integration, superior correlation and deep forensic monitoring the use of the true-time service assurance API, allows for root-trigger evaluation, identification and backbone in just a couple of clicks, with out the need for usual expensive probes. Nectar can cut back issue decision time with the aid of greater than 70% when in comparison to general community assurance equipment.

Nectar optimizes Lync UC service management and the user's experience with: automated correlation of all IP community session, content material and topology information -- taking pictures the whole "end-to-end" Lync event accurate root cause analysis identifies the "needle in the haystack" in precisely just a few clicks actual-time and ancient trending and reporting for enhanced predictability About Nectar functions Corp.Nectar gives you business price by using developing creative solutions that arm IT agencies with actionable counsel that helps them adapt to change, manipulate complexity, and provides quantifiable ROI and BI reporting.

Our flagship CMP providing improves carrier beginning across built-in voice, data, video and application options by means of proposing vital efficiency assistance to executives and technical components. Armed with this abilities, businesses now have the basis to align IT initiatives with business objectives, and to radically change their infrastructure and release vital elements. seek advice from www.nectarcorp.com for extra assistance.

Add to Digg Bookmark with del.icio.us Add to Newsvine Press Contact: Kelly Harman kharman@nectarcorp.com 646-355-0516 source: Nectar services Corp.

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Microsoft pulls September Lync safety replace | killexams.com real questions with brain dumps

Microsoft has withdrawn the 2982385 safety update that turned into launched as a part of  this month's protection updates . A revision to the protection bulletin states:

Microsoft revised this bulletin to handle a well-known subject that averted clients from efficaciously installing protection replace 2982385 for Microsoft Lync Server 2010. Microsoft is investigating habits linked to the setting up of this replace, and may replace this bulletin when greater information turns into attainable. As an delivered precaution, Microsoft has removed the download links to the 2982385 safety replace.

The 2982385 update is not a conventional security replace. The bulletin doesn't checklist any vulnerabilities addressed nor supply the update a severity rating. It says "...as a protection-in-depth measure, Microsoft recommends that shoppers of this software practice this security update to aid offer protection to towards any feasible new attack vectors recognized in the future." Microsoft has removed the down load links for the replace in addition to the KB article for it.

The other Lync Server 2010 replace (2982388) continues to be purchasable, as do several for Lync Server 2013. 

WindowsIT pro describes the manifestation of the difficulty as "... installers can be presented with a home windows security screen stating that the replace could not be put in as a result of windows could not investigate the 'writer of the motive force.'" This indicates a problem with code signing of the replace.

Microsoft has had to withdraw diverse updates in August and September because of problems. last week a September non-security replace for  OneDrive for enterprise was pulled . One security replace from August and a number of non-safety updates had been also pulled and  re-launched later on .


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70-338 Certification Brain Dumps Source : Lync 2013 Depth Support Engineer

Test Code : 70-338
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taking pictures the bull: a way to enrich your marksmanship | killexams.com real questions with brain dumps

in case you’re a dedicated marksman that fires heaps of rounds downrange each and every yr, then you definitely already possess the potential and potential that vicinity you well beyond the scope (pun meant) of today’s column. If, although, you accept as true with your self a typical hunter and shooter, then this column is for you.

We the great unwashed hordes of normal riflemen and ladies signify the large majority of nowadays’s looking neighborhood. “If that’s so,” you could be asking your self at the moment, “then what features will we have in general?” In regular, all of us possess a fundamental figuring out of the online game we pursue and have a good familiarity with the floor that we hunt. We even have a good familiarity with our firearms and might generally make correct photographs at pretty close levels under standard looking conditions.

What we lack, despite the fact, is the ability to judge distances beyond 2 hundred yards with any diploma of accuracy and the skill to make constant actual hits on game at those prolonged ranges. unfortunately, tons of of valuable video game animals undergo the consequences for these inabilities each year.

here’s the decent information: even though accidents and misjudgments do and may ensue within the container each season, there’s plenty that we will do to nudge our expertise from amateur to completed riflemen by studying and working towards basic principles of marksmanship. To begin, i would like to focus on a way to thoroughly sight-to your rifles and long-distance handguns to match your specific trend of hunting.

Let’s begin with the aid of dispelling a few commonplace misconceptions. We’ll beginning with a really customary myth: “The gun broking bore-sighted my ‘new’ rifle on the save, so it’s all set to take hunting appropriate out of the container.” How about this one, “If I hearth my rifle over the hood of my pickup and can nail lots of the beer cans that I set out at 25 yards, then my attractions may be ‘lifeless on’ at 200.” and even, “The ballistic chart on the lower back of my ammo carton says, that if I zero my gun at 100 yards, then the bullet could be drop precisely 48”at 500 yards.” Any of these tidbits of popular knowledge sound familiar?

in case you prepare your firearms for the genuine sighting-in procedure and choose the optimum ammo for the species and terrain you hunt, that you may in fact improve your marksmanship.

Tighten things down

begin by gathering all the correctly-sized screwdrivers, hex head or Torx wrenches that your gun and scope mounts require. Set every little thing out on a clean, neatly-lighted work floor. Many folks use shooting baggage or gun rests to at ease their firearms for this manner. once secured, verify the anxiety on all the gun’s action screws. If any are free, tighten them to manufacturing facility torque specs. next, seem cautiously on the barreled action to check whether it is competently seated within the stock. If the barrel is free-floated (as most factory rifles are at the moment), then run a thin sheet of paper down the barrel to make certain that it is not contacting the barrel channel any place alongside its length. if it is, both correct the issue yourself — in case you’re certified — or convey the firearm to your native gunsmith for adjustment.

verify the anxiety on the scope base and ring screws. Tighten if vital. loads of individuals want to region a drop of blue Loctite on the bottom mounts to aid insure that they don’t jar loose.

The ring screws may still be tightened firmly, however screwing too tight can pinch or bend the soft aluminum scope tube. Afterwards, i like to area a dot of fingernail polish at the junction of the scope and the edge of one of the most rings. Any inadvertent circulation of the scope will crack the dried nail polish and signal that your scope may be ‘out of zero.’

Bullet choices and selection

healthy the bullet to the game. select the bullet vogue, weight, and development premiere desirable to the species you pursue. as an example, when I lived In Wyoming, I hunted antelope, mule deer, and elk alongside the breaks of the Sweetwater River and canyons of the Wind River range. capturing distances the place always lengthy and obviously the sizes and relative durability of the three species varied significantly.

After relating to bullet manufacturers’ techniques, speakme with veteran hunters in the enviornment, gun shop clerks, and reasonably slightly of experimentation, I settled on the 225 grain Hornady Interlock bullet for my mannequin 70 .338 Win. Magnum. The sleek bullet carried its speed neatly at distance and its development gave it satisfactory expansion and weight retention to cleanly take a truck load of animals. For the chunky northern Idaho whitetail that I hunt now, I chamber all of a sudden expanding one hundred fifty grain bullets in my T/C undertaking.

when you’ve settled on the bullet weight and development gold standard suitable to your particular person needs, it’s time to head to the range with as a minimum three diverse manufacturing facility loadings to see which one your rifle prefers.


Three strikes: Reflections on a good LSU/South Carolina collection and a look at what may well be to come back | killexams.com real questions with brain dumps

1. WHAT A game; WHAT A series: depth usually raises throughout the stretch run of a baseball season. This weekend certainly didn't disappoint. Sunday’s mad conclude was a perfect conclusion to a brilliant series. South Carolina and LSU performed exciting baseball for most of their 28 innings at Alex box Stadium. Runners had been thrown out at the plate; a coach changed into ejected; pitchers yelled expletives after they escaped bases-loaded jams. It turned into an part-of-your-seat treat all weekend, and because LSU got here out of the knock-down-drag-out combat forward, it's going to go into the ultimate two weeks of the regular season just one game again in the Southeastern convention standings. There’s certain to be greater of this depth to come.

2. WHEN DUPLANTIS IS scorching … Duplantis has been, with the aid of his own admission, a streaky hitter this season. It has brought about some frustration to make sure. just remaining week, he changed into LSU’s No. 9 hitter towards Alabama, going through a bit of a stoop. but when he’s locked in, he’s been brilliant. Sunday’s 5-hit efficiency became his crew-leading seventh game with three or more hits. Sunday, his five hits went to each a part of the park, and none of them become cheap. When he’s in a groove, he’s in assault mode. That turned into the case on his online game-winner — he changed into looking slider, which he bought, however it was out of the strike zone. When he got forward 2-0 within the count number, he figured a fastball was on its approach. He turned into correct, and that became the ballgame.

three. CAN LSU maintain THIS GOING? It just looks that LSU has taken one step lower back for every step it's taken forward. notwithstanding LSU didn't play terrible baseball in any video game this weekend, a loss in Sunday’s finale would’ve felt like a step again. Wins and losses do not come with trend aspects. instead, LSU finds itself on the correct aspect of the razor-thin margin. The Tigers have received 5 of six, and they're appropriate where they are looking to be as they put together for a difficult closing stretch. here's removed from scientific, but the method this weekend’s series against South Carolina went down — huge crowds with full throats, cheering on dramatic wins in opposition t an excellent team — it simply seems like LSU is in for one other powerful conclude.

observe Luke Johnson on Twitter, @ByLukeJohnson. 


Why The Hague is an paintings lover’s dream | killexams.com real questions with brain dumps

Why go now?

most efficient standard because the domestic of the foreign criminal court, the highlight is on artwork this year in the Netherlands’ third biggest city. together with 17 other locations across the country, The Hague may be celebrating the one centesimal anniversary of De Stijl (‘The trend’), a Dutch inventive stream, all the way through 2017. Two predominant exhibitions that includes the vibrant work of Piet Mondrian, the art flow’s most noted exponent, are taking region at the Gemeentemuseum (1) between now and the conclusion of September: “Piet Mondrian and Bart van der Leck: Inventing a new artwork” (unless 21 may additionally) and “the invention of Mondrian” (three June to 24 September). however, this appealing metropolis, also the seat of the Dutch parliament, has a lot to present enthusiasts of ancient Dutch Masters and artwork Nouveau too. 

Get your bearings

The city centre is set 10 minutes’ stroll west of Den Haag (The Hague) Centraal educate station (2). En route you’ll circulate the white, high-rise metropolis hall, at Spui sixty eight, which is the place the vacationer office is discovered (three) (00 31 70 361 8860; denhaag.com). Opening hours are Monday 12-8pm, Tuesday to Friday 10am-8pm, weekends 10am-5pm. North of here is the Binnenhof (four), home to the Dutch parliament, and the Mauritshuis art gallery (5). simply west of the Mauritshuis you’ll locate the main looking area – on and round Hoogstraat (6) – and the hip Hofkwartier area. To the north of the browsing area you’ll find the Noordeinde Palace (7), the workplaces of the royal household. The Gemeentemuseum is about three kilometres north-west of the centre.

Mauritshuis residences Dutch Golden Age artwork (Getty/iStockphoto)

Day one

Take a hike

The Hague has a great deal to activity art fanatics as they stroll around the metropolis. one of the vital metropolis’s top-rated art Nouveau residences may also be discovered at Smidswater 26 (eight), simply north of the Binnenhof (four) – it’s private, and you may’t go in, however don’t pass over the cat-shaped letterbox. For greater of the equal, a self-guided going for walks tour, “artwork Nouveau within the Hague”, can also be picked up on the tourist office for €2.50. A seven-minute stroll south west of right here, there’s a cluster of tremendous galleries around the Hofvijver lake (9). For an offbeat experience, are trying Escher in Het Paleis at Lange Voorhout 74 (10) (00 31 70 427 7730; escherinhetpaleis.nl), a everlasting exhibition of intellect-increasing works through early 20th century graphic artist MC Escher, housed in a former royal palace dating from the 1760s. Entrance is €9.50. 

Binnenhof Palace is home to the Dutch parliament (Getty/iStockphoto)

The Museum Bredius at Lange Vijverberg 14 (11) (00 31 70 362 0729; museumbredius.nl), 5 minutes’ walk away, offers company the possibility to peer a one-time inner most assortment of nice Dutch paintings, in addition to silverwork and porcelain because it would have firstly seemed in a townhouse environment. Entry is €6. finish up at the Mauritshuis (5) at Plein 29 (00 31 70 302 3456; mauritshuis.nl), simply on the other aspect of the lake, which has a global-type assortment of Dutch Golden Age artwork, together with Vermeer’s girl with a Pearl Earring. Entry is €14. 

Lunch on the run

There’s been a fresh style in the Hague for Michelin-starred cooks opening chip stores, where the fries are made with good first-class potatoes. Friet District, a ten-minute stroll west at Prinsenstraat 73 (12) (00 31 70 889 1113; frietdistrict.nl), is owned by Niven Kunz of Restaurant Niven in Rijswijk. It presents trays from €three and you'll choose to devour in or remove – the garden of the Noordeinde Palace (7) is a brief walk north, and it’s free to move in.

Window searching

Friet District is appropriate in the core of the Hofkwartier, one of the crucial oldest materials of the city and now the hippest region for shopping (and ingesting). idea outlets with in-condo cafés are all of the rage within the Netherlands right now – one of the crucial most useful on the town is LISTed at Molenstraat 36 (13) (00 31 70 737 0577; listed-thestore.com), which sells little-general trend brands, artworks, books and accessories. On the contrary side of the street at Molenstraat 39 is Episode (14) (00 31 70 449 2882; episode.ecu), a old store with an ethical outlook stocking quirky objects and large manufacturers. 

Friet District serves up first-rate chips with additional-special toppings

An aperitif

A enjoyable place to delivery the evening is at Huppel the Pub at Oude Molstraat 21 (15) (00 31 70 360 9113; dehuppel.nl). It’s a “brown café” – a normal Dutch pub with smoke-stained, dark timber interiors – noted for stocking 150 forms of single malt whisky. There are additionally eight rotating taps, usually including the café’s personal brew, HuppALE, and 60 styles of bottled beer. The accompanying rock tune’s fairly decent, too.

Dinner with locals

youngsters The Hague has its personal Chinatown on and round Wagenstraat, the optimal Asian restaurant within the metropolis will also be discovered at Prinsenstraat 33, the place Michelin-starred HanTing cuisine (sixteen) (00 31 70 362 0828; hantingcuisine.nl) serves chinese language-French fusion dishes beginning at an astonishingly least expensive €37 for 3 courses (although that doesn’t include dessert). For anything completely distinct, Instock at Buitenhof 36 (17) (00 31 70 412 6192; instock.nl) offers a €27 three-course dinner made with leftover supermarket items.

Huppel the Pub is a standard Dutch brown residence (Huppel the Pub )

Day two

Take a ride

The Hague has an effective bus and tram system that takes you smartly past the city centre. One event fees €3.50 or a day ticket expenses €6.50. From the Centrum stop (18), Tram 1 whizzes passengers to the seaside lodge of Scheveningen, about 5 kilometres to the north, in below 20 minutes. right here which you could savour a bracing stroll alongside the promenade, stopping to check out the stores on the renovated pier (19) (00 31 6 10 38 sixty eight fifty nine; pier.nl) or take a 20-minute journey on the 40-metre Ferris wheel (20) (00 31 880 22 33 33; skyviewdepier.nl) for €9. 

Out to brunch

From the Scheveningen Slag cease with the aid of the pier (21), hop on Bus 22 (course Oude Waals or Duinzigt) and alight at Badhuisweg. It’s a two-minute walk south to Yon at Badhuisweg 191 (22) (00 31 70 352 3485; yonscheveningen.nl), a stylish modern café the place that you may savor a superb-cost buffet brunch, that includes the likes of bloodless meats, scrambled eggs and pastries, with espresso or tea and a glass of Prosecco for €9.95 (11am-2pm; reserving advised).

Take a stroll along the renovated pier at Scheveningen (Getty/iStockphoto)

A walk in the park

Head west from the café, go the canal and enter the Scheveningse Bosjes, or Scheveningen Woods, a park in a medieval woodland. The leading attraction here is Madurodam  at George Maduroplein 1 (23) (00 31 70 416 2400; madurodam.nl), a mannequin village that includes replicas of famous Dutch cities and points of interest. Entry is €14. The northern part of the park has a lake and a few struggle memorials, while the south is dense with bushes. It takes about 25 minutes to move from east to west on foot and also you’ll exit by way of the world discussion board convention centre on Johan de Wittlaan (24).

A cultural afternoon

This 12 months is all about the Gemeentemuseum (1) (00 31 70 338 1111; gemeentemuseum.nl), which has the area’s largest collection of works by Piet Mondrian. until 21 can also, friends can trap an exhibition on the origins of the De Stijl paintings movement entitled, “Piet Mondrian and Bart van der Leck – Inventing a new artwork”. It looks at how both artists developed the now instantly recognisable red, blue and yellow blocks with black strains. The famous person reveal is “the invention of Mondrian” from three June to 24 September, when all 300 of his works will go on reveal collectively for the primary time together with his closing, unfinished portray Victory Boogie-Woogie. operating alongside it from 10 June to 17 September, “structure and Interiors – The want for style” will explore the work of designers together with Gerrit Rietveld. Entry is €14.50. opt for up some Mondrian-inspired socks within the museum store on the manner out before catching Tram 16 (course Dorpskade) again to the centre.

The Gemeentemuseum is hosting a couple of dazzling exhibitions this yr (Getty)

commute essentials

Getting there

The simplest airways flying direct from the uk to Rotterdam The Hague Airport (25) (rotterdamthehagueairport.nl) are British Airways (britishairways.com) and Cityjet (cityjet.com), both from London metropolis Airport. however Amsterdam Schiphol is barely 28 minutes far from The Hague by using train, and has better hyperlinks with the united kingdom than anywhere else in the world, with connections from two dozen airports starting from Exeter to Inverness. The main carriers are KLM (klm.com), easyJet (easyJet.com) and Flybe (flybe.com).

alternatively, The Hague is effortlessly reached through educate from London. Take the Eurostar (eurostar.com) from St Pancras and change at either Lille or Brussels after which once again at Rotterdam – the journey takes around 4 and a half hours, and fares start from £122 from Voyages-SNCF (voyages-sncf.com). 

experience the 40m ferris wheel at Scheveningen (Getty photos/iStockphoto)

Staying there

Opened in 2010, the austere façade of the five-star Hilton The Hague (26) (00 31 70 710 7000; hilton.com) conceals 195 modern rooms, a Dutch-French restaurant and an upmarket cocktail bar. Doubles from €159, room handiest.

Housed in a former royal palace, De Salon (27) (00 31 70 221 0882; salondenhaag.nl), which opened ultimate year, is a boutique B&B with simply three spacious suites furnished with fashion designer add-ons. Breakfast is served in the subtle tearoom on the ground flooring. Doubles from €125, B&B.

a super-cool hostel-cum-resort for brief or lengthy stays, The pupil inn (28) (00 31 70 762 a thousand; thestudenthotel.com/the-hague) has minimalist rooms and a great latitude of facilities together with a video games area, bar-restaurant, fitness center and a co-working house. Doubles from €69, room best.

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SDI [3 Certification Exam(s) ]
See-Beyond [1 Certification Exam(s) ]
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References :


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Dropmark : http://killexams.dropmark.com/367904/10847498
Issu : https://issuu.com/trutrainers/docs/70-338_2
Scribd : https://www.scribd.com/document/352134160/pass4sure-70-338-lync-2013-depth-support-engineer-exam-braindumps-with-real-questions-and-practice-software
Dropmark-Text : http://killexams.dropmark.com/367904/12567375






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Microsoft 70-338 Exam (Lync 2013 Depth Support Engineer) Detailed Information

Microsoft Certification Program benefits
Learn about the benefits of the Microsoft Certification Program. Find answers to frequently asked questions regarding program benefits and Microsoft accounts.
Program benefits
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Q. What are the benefits of achieving a Microsoft Certification?
Microsoft Certification is an industry standard that is recognised worldwide. After you earn your Microsoft Certification, you have access to a number of benefits, which can be found on your benefits and exams dashboard.
Q. What will I find on the benefits and exams dashboard and who has access?
Individuals who have passed a Microsoft exam have access to the benefits and exams dashboard.
On the site, you will find:
A downloadable version of your certificate (you can order a printed copy); MOS members will need their Certiport login information
Your official Microsoft Certification downloadable transcript and access to the transcript-sharing tool
A tool to create and download certification logos
Your contact preferences and profile
Your Certification Planner
A sign up for the MCP Flash newsletters
Promotional offers, discounts and additional services
Q. What is a Charter certificate?
Charter Members are the pioneering group of individuals who achieve a certification within six months following the retail release date of the certification. (People who pass the beta exams will receive the Charter certificate after the certification is commercially released.) Charter Members are recognised by being given the Charter version of the certificate acknowledging their early adoption of the technology solution. The Charter version of the certificate includes the word "Charter".
Q. Which certifications are eligible for Charter status?
If you have any of the following certifications, you are eligible for the Charter certificate: Microsoft Certified Solutions Associate (MCSA), Microsoft Certified Solutions Expert (MCSE), Microsoft Certified Solutions Developer (MCSD), Microsoft Certified Technology Specialist (MCTS), Microsoft Certified IT Professional (MCITP), Microsoft Certified Solutions Master (MCSM), or Microsoft Certified Professional Developer (MCPD). Exceptions include those certifications that are released when the technology covered is no longer the latest version of the technology. These include the MCSA: Windows 7, the MCSA: SQL Server 2008 and the MCSA: Windows Server 2008.
In addition, Microsoft Specialist (Specialist) certifications released in and after September 2015 are also eligible for Charter status, including all Windows 10, Big Data Analytics and Cloud Data Platform Specialist certifications.
Q. How much time do I have to order my Charter certificate?
You can digitally download or order your Charter certificate at any point after it is earned.
Q. When does the Charter certificate period start for localised exams?
The six-month "clock" start time is based on the earliest time the certification can be earned, which is typically based on English-language exam availability. For example, if an exam is available in German two months after the English version is available, the German candidate has only four more months to earn a Charter certificate.
Q. I have heard that Microsoft sponsors an Elevate America veterans initiative to help our country's veterans and their spouses acquire the skills and resources that they need to be successful in today's workplace. What is this initiative?
Through this initiative, Microsoft convenes a coalition of public, private and non-profit organisations that are interested in contributing expertise, cash and in-kind resources to help U.S. veterans and their spouses build the skills and access the resources that they need to be successful in today's workforce.
Accessing the benefits and exams dashboard
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Q. How do I qualify for access to the benefits and exams dashboard?
The benefits and exams dashboard is available if you have passed a Microsoft qualified exam. It provides customised resources specific to your achievements and certifications.
Sign in to the benefits and exams dashboard
Q. How do I access the Microsoft Certification benefits and exams dashboard for the first time?
After you pass your first Microsoft Certification exam, you will receive a welcome email message outlining the steps needed to gain access to the benefits and exams dashboard. Please check your junk folder to ensure that the automatic email message is not blocked by your spam filter. Here are the instructions you will receive in the email message.
Create a Microsoft account if you don't already have one.
On your first visit, click on the link provided in the email and use your Microsoft account to log in to the benefits and exams dashboard.
Note This must be done within 90 days of receiving the email to meet security requirements.
In some cases, you may be required to enter your Microsoft Certification ID (MC ID) and temporary access code, which will be supplied in the email message.
On future visits, log in to the benefits and exams dashboard using the same Microsoft account.
For assistance, go to Microsoft training and certification help.
Q. I do not know my Microsoft Certification ID (formerly MCP ID). What should I do?
Your MC ID is shown in your welcome email as well as in the profile information on the benefits and exams dashboard. If you are unable to access the benefits and exams dashboard, contact your Microsoft Regional Service Centre for assistance.
Q. What is the access code required for the benefits and exams dashboard?
An access code is a unique code that allows first-time access to the benefits and exams dashboard and, if required, is provided in your welcome email. You can only use the access code to sign in to the benefits and exams dashboard for the first time. If you misplace your code or it has retired, contact your Microsoft Regional Service Centre for assistance.
Q. How long does it take for my data to update after accessing the benefits and exams dashboard for the first time?
After you register, you must wait 24 hours before accessing your program benefits.
Q. I am a returning user and cannot access the benefits and exams dashboard. How do I gain access?
If you cannot remember the Microsoft account information associated with your account, or you have other difficulty accessing the benefits and exams dashboard, contact the Microsoft Regional Service Centre in your area.
Note If you have a Hotmail account, MSN email account or Microsoft Passport, it is your Microsoft account.
Q. I saved the URL to my Microsoft Certification Program benefits in my Favourites, but when I navigate there I am redirected to a sign-in page, not the page I am trying to access.
Because online benefits are available only to eligible Microsoft Certification Program members, you must enter your Microsoft account credentials to access your online benefits. After your account is authenticated, you will be redirected to the page you are trying to access.
Q. Why is my name on the benefits and exams dashboard different from what it is in my Microsoft Personal Profile?
You are required to use your legal name for the benefits and exams dashboard. The Microsoft Personal Profile is associated with your Microsoft account.
Q. How do I update my legal name?
To update your legal name, contact your Microsoft Regional Service Centre.
Q. How do I update the information in my Microsoft Personal Profile?
You can update your Microsoft Personal Profile in the Profile Centre.
To change the name that is associated with your Microsoft Certification ID (formerly, MCP ID), contact your Microsoft Regional Service Centre.
Q. I updated my email address at the Profile Centre, but it was not changed on my Microsoft Certification transcript. What is wrong?
It can take up to 48 hours for changes in your profile to appear on your Microsoft Certification transcript. Check your transcript later to verify that your email address has been updated.
Q. I entered an email address in my Microsoft Personal Profile, but the email field is still blank. What happened?
After you update your Personal Profile, a message is sent to the email address noted in your profile. For the changes to be saved, you must follow the instructions in the email message to confirm the update. Otherwise, the email address in your profile will be removed because it has not been confirmed.
Q. How do I use the Microsoft Certification logos, and what are my rights and responsibilities when I use them?
When you access the logo tool, you are required to read and accept the MCP logo guidelines to correctly promote your relationship with Microsoft and to protect the integrity of the logos. You must sign in to the benefits and exams dashboard in order to access these guidelines.
Q. How can I download electronic files of Microsoft Certification logos?
Logos are available for download from the benefits and exams dashboard.
Q. I passed a test a week ago and I want to print my logo, but I cannot select it for printing.
The test that you passed might not qualify you to print that specific logo. If you believe this is an error, contact the Microsoft Regional Service Centre.
Q. How do I report misuse of a Microsoft Certification logo?
Contact the Microsoft Regional Service Centre.
Microsoft account and Profile Centre
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Q. What is Microsoft account?
Microsoft account (formerly Windows Live ID) enables you to simply use your email address and password to sign in. After you create your Microsoft account, you can access all Microsoft account sites and services. If you already have an MSN Hotmail, MSN Messenger or Passport account, it is your Microsoft account.
Learn more about Microsoft account.
Q. If I do not have a Microsoft account, do I need to set one up to access the benefits and exams dashboard?
Yes. You can sign up for a Microsoft account now. You will not be able to access the benefits and exams dashboard without one.
Q. If I already have a Microsoft account, can I use it to access the benefits and exams dashboard?
Yes.
Q. I do not remember the Microsoft account I used to access the benefits and exams dashboard. What should I do?
If you cannot remember the Microsoft account that you used to access the benefits and exams dashboard, contact your Microsoft Regional Service Centre for assistance.
Q. Will my Microsoft account expire?
If your Microsoft account is unused for one year, your Microsoft account may expire, and you may need to re-establish a Microsoft account.
Q. How do I update my profile?
Log in to the benefits and exams dashboard and, under the Account menu in the top right corner of the page, click Profile settings.
Q. What should I regularly update in my profile?
Update the following information to ensure that you receive communications from Microsoft:
Your contact preferences: Specify whether you want to receive promotional information about Microsoft products, services and events.
Your personal information: View, add or edit your personal contact information, such as your email address, business or personal address and phone numbers.
Your business information: View, add or edit information about your job and your organisation so that we can personalise content and recommend materials that match your job, organisation or industry.
Your technology preferences: View, add or edit information about your technology interests so that we can personalise content and recommend materials that match your interests.
Q. What if I do not want to receive email messages from Microsoft?
You are under no obligation to receive email messages from Microsoft. However, if you indicate that you do not want to receive communications from Microsoft through email, you cannot receive e-newsletters, such as the MCP Flash and MCT Flash. These communications provide up-to-date information on changes in training and certification resources, such as discontinued exams, Microsoft Certified Professional (MCP) benefits and special offers.
Q. How do I ensure that I receive communications by email?
Visit the Microsoft Profile Centre to indicate that you want to receive email messages from Microsoft.
Sign in to the Microsoft Profile Center to view and update your profile
Profile Center screenshot
Click My Contact Preferences, located on either the Profile Centre home page (shown above) or on the left-hand side of the page (shown below). Select E-Mail Address, and then click Save.
Note You can always unsubscribe from selected e-newsletters. However, if you do not select the E-Mail Address option, Microsoft cannot send you any information by email, including product notifications and news.
My Contact Preferences screenshot
Sign-up for e-newsletters: Select Manage Subscriptions on the left-hand side of the page. Tick all of the subscriptions that you want to receive, and then click Subscribe. You can also unsubscribe from publications.
Accessing your transcript
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Q. Where can I find my transcript or see which exams I have passed?
Access your transcript from the benefits and exams dashboard.
Under the section called Transcript, click on the View link.
Q. How do I give my employer or others access to my transcript via a secure connection that they can trust?
Microsoft offers a tool called Transcript Sharing, which can be accessed from the benefits and exams dashboard. Under the section called Transcript, click the Share link.
You will be asked to create an Access Code (which can be changed at any time) and given a Transcript ID. You will then provide these two codes to your selected audience, along with a URL to view your transcript.
Q. I have certifications from multiple providers; can I combine all my credentials into one transcript?
Microsoft has worked with the IT Certification Council (ITCC), along with other industry certification providers, to provide MCPs with the opportunity to create one transcript across providers. Under the Transcript section of the benefits and exams dashboard, click Create a multi-vendor transcript. You will then be asked to provide your transcript sharing codes. The transcript sharing code can be found by clicking Share your transcript.
Q. What if I cannot print or access my transcript?
If you cannot print or access your transcript, contact your Microsoft Regional Service Centre.
To ensure a prompt response:
Use the email address associated with your Microsoft Certification ID (MC ID), if possible.
Have your Microsoft Certification ID number available.
If you do not know your Microsoft Certification ID, have other information available, such as your address, telephone number, exam numbers and completion dates.
Q. I viewed my transcript but it is missing data. Why can't I see all my data?
It can take up to two weeks for Microsoft to receive and process your exam records. If it has been more than two weeks since you took your exam, and the results still do not appear on your transcript, or if you notice any other problems, contact your Microsoft Regional Service Centre.
Training and certification from Microsoft
Fulfill your potential and make the most of your investment in Microsoft technology with certification, classroom training, e-learning, and books from Microsoft and Microsoft Learning Partners.
Certifications
A Microsoft Certification validates your expertise in a Microsoft technology. As a Microsoft Certified Professional, you’ll have access to community resources and tools that allow you to exchange ideas with peers, increase your knowledge and skills, and broaden your career opportunities.
Microsoft Certified Trainers
Microsoft Certified Trainers (MCTs) are classroom and e-learning instructors, training and certification consultants, authors, conference presenters, and user group leaders who combine their expertise, experience, and passion for training and leadership to help Microsoft customers and partners realize their full learning potential.
Microsoft Learning Partners
Microsoft Partners with the Learning competency (Learning Partners) are training companies that meet stringent Microsoft qualifications to train IT professionals and developers on Microsoft technologies.
Microsoft Imagine Academy
The Microsoft Imagine Academy program connects the world of education to the world of work by enabling faculty and students to acquire new technology skills in an academic setting.
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Quick News

NCPL offers computer classes

Natrona County Public Library offers basic computer classes to help participants navigate the computer, the Internet and basic programs like Microsoft Word.

Upcoming classes are: “Intro to Computers” at 5 p.m. Wednesday, Sept. 3; “Intro to the Internet” at 10 a.m. Thursday, Sept. 11; “Intro to PowerPoint” at 1 p.m. Monday, Sept. 15; “Intro to Genealogy” at 10 a.m. Tuesday, Sept. 16; and “Intro to Microsoft Word” at 1 p.m. Monday, Sept. 17.

Stop by the library’s Reference Desk to sign up for classes, or call 577-READ, ext. 2.

Citizenship class in need of immigrants

A free class for individuals who are within one year of being eligible to apply for citizenship will begin on Monday, Sept. 8, at Casper College.

“Anyone who knows how to speak, read, and write some English and is interested in becoming a United States citizen is urged to sign up for this free citizenship class,” said Lisa Mixer, Casper College tutor coordinator and ABEGED co-director.

The class is scheduled to run for 14 weeks, each Monday from 6:30-7:30 p.m. It is sponsored by the Casper College Adult Basic EducationGED Center.

Pre-registration is required and can be done through Sept. 12 by calling Mixer at Casper College at 268-2453.

Defensive driving courses offered

Defensive driving courses for people 55 years and older are conducted on the second Tuesday and Wednesday of each month, sponsored by the Central Wyoming Senior Citizens Center.

A course is scheduled from 2-6 p.m. on Sept. 9 and 10 at the Natrona County Senior Center, 1831 E. Fourth St. Each session is taught by AARP-trained instructors.

After successful completion, participants will receive a certificate that can qualify them for a 10 percent reduction in car insurance (liability andor collision) over two years.

The cost is $10. For reservations or more information, call Georgia at 265-4678, ext. 12.

CC Greenhouse hours announced

New fall hours for the Casper College Greenhouse, featuring a variety of plants, birds and reptiles, have been announced.

According to Evert Brown, greenhouse director and Casper College biology instructor, the facility will be open to the public from noon to 3 p.m. Monday through Friday.

Many types of plants are grown in the greenhouse, representing several different climates from desert to tropical.

ors also are likely to see several varieties of birds that live and fly within the confines of the greenhouse, and also some turtles.

The greenhouse, which is located on the west side of the Loftin Life Science Center, is free and open to the public.

Bookmobile schedule

For more information, call 577-READ, or log on to

Thursday, Sept. 4: 9:30-10 a.m., Garden Square (1950 S. Beverly); 10:15-10:45 a.m., Office Max . (441 Landmark Drive); 11-11:30 a.m., Pineview School Area II (1190 S. Forest Drive); 11:45 a.m. to 12:45 p.m., Cottonwood School (2300 Bellaire Drive); 3-3:30 p.m., Park Place (1930 E. 12th); 3:40-4:10 p.m., New Directions (LifeSteps Campus); 4:40-5:15 p.m., Evansville School.

Monday, Sept. 8: 9:30-10:10 a.m., Giggles & Wiggles (1720 S. Poplar); 10:25-10:55 a.m., Sunshine Corner (2303 E. 15th); 11:05-11:45 a.m., Sagewood School Area (1230 E. 22nd); 3:20-4 p.m., Apple Tree Learning Center (60 Magnolia); 4:15-5 p.m., Robertson Road Area (Whispering Springs & King Salmon).

Tuesday, Sept. 9: 11-11:30 a.m., Powder River School; 11:35 a.m. to noon, Powder River Post Office; 3:45-4:20 p.m., Paradise Valley I (Daffodil Street); 4:30-5 p.m., Paradise Valley II (Glendo Street).

Wednesday, Sept. 10: 10-10:30 a.m., Big Tree Area (1712 S. Oak); 10:40-11:20 a.m., Mtn. Road Christian Academy (2657 Casper Mtn. Road); 11:30 a.m. to 12:30 p.m., Willard School (129 N. Elk); 2-2:30 p.m., Prince of Peace School area (South Beverly & Eighth streets); 2:45-3:20 p.m., Sagewood School Area II (1901 E. 24th); 3:30-4:10 p.m., Leaps and Bounds Preschool (615 S. David); 4:25-5:15 p.m., Boys & Girls Club (1701 E. “K” St.).

Buffalo hunt raffle benefits CWRM

An all-inclusive guided buffalo hunt to benefit the Central Wyoming Rescue Mission has been donated by various sponsors.

The winner also will receive a Pre-64 Winchester Model 70 338 Win. Mag. rifle, 3-9 Zeiss scope, gun case, sling and ammo, as well as meat processing.

Only 250 tickets will be sold at $100 each. Tickets are available at the rescue mission at 230 N. Park St., City Service Electric, Rescued Treasures and Bar-D Signs.

Checks, written to CWRM, can be sent to PO Box 2030, Casper 82602. Be sure to write “buffalo hunt” in the memo. All proceeds will go to the mission.

For more information, call Risa or Deb at 268-4474.

Counseling association sets fall conference

The Wyoming Counseling Association has scheduled its annual fall conference for Oct. 9-11 at the Parkway Plaza in Casper.

The theme is “Wyoming Winds of Change,” and Dr. Susan McCabe will be the distinguished keynote speaker.

McCabe, a nationally known expert in psychiatric mental health nursing, will be speaking at the pre-conference on “Mind and Brain: Understanding Mood States Across the Lifespan.”

She serves as an associate professor at the Fay W. Whitney School of Nursing at the University of Wyoming and is the recipient of numerous awards, including the 2007 University of Wyoming Faculty Senate Lectureship Series Award, the 2004 Basham Faculty Fellowship Award and many more.

Mental health professionals may earn up to 15 hours of continuing education at the conference. The early-bird registration deadline is Sept. 14, and payment is by check or agency voucher.

For more information about the conference, call Becky Gurtler at 265-7545, or go to

Kids’ Grief Support Group returns

After a break for the summer, the monthly meetings of the Kids’ Grief Support Group will begin again Saturday, Sept. 13.

The group will meet from 11 a.m. to 1 p.m. at Central Wyoming Hospice, 319 S. Wilson, for water balloon volleyball and pizza. They also will make plans for upcoming meetings.

The Kids’ Grief Support Group meets the second Saturday of the month throughout the school year. Kids age 6-16 who have experienced the death of someone important in their lives are encouraged to attend. Parents also are welcome.

For more information or to RSVP, call Dama at 577-4832.

Family Fun Fest raffle tickets available

Central Wyoming Hospice & Transitions will raffle numerous quilts and gift baskets at its Fourth Annual Family Fun Fest, Saturday, Sept. 20, from 11 a.m. to 3 p.m. at the Central Wyoming Fairgrounds Industrial Building.

Each year, area quilters donate their handiwork for this event. Also, area businesses pitch in by filling gift baskets with family-friendly gifts.

Tickets for quilts and for baskets are sold separately. The cost is $3 each or two for $5. The tickets are available at CWHTP, 319 S. Wilson, and at Kalico Kat Quilt Shop, 350 W. Collins, also will be available at the event.

The Family Fun Fest is Casper’s largest indoor picnic and will feature games for all ages and prizes.

For more information, call Denise at 577-4832.

Book drive under way

Joan Anderson of Casper is conducting a book drive for the Natrona County Detention Center.

Anderson is asking for donations of paperbacks, in decent condition, of all genres: classics, action, Bibles, mysteries, romance, everything.

She noted that books in Spanish especially are needed.

Anderson plans to donate books to the detention center in September. Anyone with donations is asked to call Anderson at 472-3720.

Platte River Revival announced

The Second Annual Platte River Revival will be held on Saturday, Sept. 20, from 9 a.m. to noon, with check-in at Mike Lansing Field.

Residents are encouraged to start forming a team, perhaps with colleagues or neighbors.

“Volunteers will help remove debris and Russian olive branches, and plant trees in designated locations along the North Platte River,” said Jolene Martinez, Keep Casper Beautiful director.

In preparation for volunteers, Bureau of Land Management fire crews began cutting Russian olives, a non-native, invasive species, recently in what is expected to take a few weeks to complete.

Last year, volunteers removed 381,380 pounds of debris from the river and its banks, including appliances, tires and vehicles in the area just east of Bryan Stock Trail to the White Water Park.

Eleven native species trees were planted in Riverview Park.

“This year, the Revival’s operations committee is looking at areas along the river from Casper’s east city limit to just past Morad Park,” Martinez said.

A barbecue will follow, and there will be a group picture of all volunteers.

For more information or to sign up to volunteer, visit the Web site link at

The Platte River Revival is a Keep Casper Beautiful event in conjunction with National Public Lands Day and is organized by a partnership of public and private organizations.

Bereavement support groups in Glenrock

North Platte Home Health and Hospice are conducting Bereavement Support Group meetings in Glenrock to provide support, discussion and conversation for those who have lost a loved one.

Meetings are held the third Friday of the month at the Glenrock Senior Center, 615 W. Deer. Led by Chaplain Gayle Unruh, the meetings are held from noon to 1:30 p.m.

Lunch is provided, and the public is invited to attend these free meetings.

Hospice is a health care program that provides physical, emotional and spiritual support with life-limiting illnesses to enable the best quality of life for the patient.

It provides educational and emotional support to the patient's family to make their remaining time with the patient as meaningful as possible. Additionally, hospice provides grief support to family members following the death of the patient.

North Platte Home Health and Hospice is owned by Amedisys ., a provider of home health and hospice services at more than 480 sites across the United States and Puerto Rico.

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Discovery of immunodominant T-cell epitopes reveals penton protein as a second immunodominant target in human adenovirus infection

Human adenovirus (HAdV) infection constitutes a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). The incidence of HAdV infection ranges from 5 to 30 %, with pediatric recipients showing the highest rates of infection with up to 83 % lethality [1–6]. Monitoring for HAdV infection and therapeutic intervention (reduction of immunosuppression, antiviral treatment) may reduce mortality due to HAdV in pediatric HSCT recipients [7]. However, antiviral treatments for HAdV infection with agents like cidofovir and ribavirin are associated with toxicity and may result in delayed immune reconstitution. Previous studies clearly indicate that T cells, the most potent effectors of the human immune system, are crucial for HAdV clearance [2]. It was demonstrated that children with HAdV-associated mortality had no HAdV-specific T cells, whereas patients who cleared HAdV infection showed HAdV-specific T-cell responses [2, 8]. Adoptive transfer of HAdV-specific T cells offers an effective and non-toxic immunotherapeutic strategy to reduce or prevent the clinical manifestation of HAdV in HSCT recipients with no or low numbers of HAdV-specific T cells [2, 8–12]. Monitoring HAdV-specific T-cell immunity may improve risk assessment in HSCT recipients and enhance treatment efficacy by determining the optimal time point for adoptive T-cell transfer. The median time between the first detection of HAdV DNA in the blood and the onset of symptoms is 3 weeks, which therefore seems to be the optimal time point for adoptive T-cell transfer [2, 13, 14]. Since the generation of short-term in vitro generated virus-specific T-cell lines takes about 3 weeks including quality controls, the production should start even earlier at the time of high viral load in stool (>106 copies) [12, 15].

The 70 different human HAdV types identified to date are divided into seven species (A to G) [16, 17]. Type 31 (of species HAdV-A) and HAdV 1, 2, and 5 (of species HAdV-C) are the most prevalent types in HSCT recipients [4–7]. Occasionally, types of species HAdV-B can be observed in adult HSCT recipients [18]. The major capsid protein hexon serves as an immunodominant target antigen across the different HAdV types, but few hexon-derived epitopes have been identified as immunodominant so far [13, 19–23]. Most of these epitopes are highly conserved, demonstrating that HAdV-specific T cells can cross-react across HAdV species and may therefore provide protection against a wide range of HAdV types [20]. HAdV-specific T-cell responses to the recombinant hexon protein, the overlapping peptide pool covering the complete hexon sequence, HLA-restricted peptides, and whole viral lysates have been investigated. A study by Feuchtinger et al. revealed that 10.5 % of donors had a specific T-cell response to the whole adenovirus but no response to the hexon protein, while 17 % of donors had no detectable T-cell response to HAdV [11]. Moreover, Zandvliet et al. detected specific CD8+ T cells in 616 healthy donors (37.5 %) after stimulation with the 15-mer hexon peptide pool, but only 316 donors (18.8 %) had specific T cells for known CD8+ hexon epitopes [24]. Sukdolak et al. observed a specific T-cell response to the 15-mer hexon peptide pool in 73 % of HAdV seropositive healthy donors, while 30 % were classified as high responders and 43 % as low responders [25]. Interestingly, 27 % of all HAdV seropositive healthy donors tested showed no response to the hexon peptide pool. These results underline the need to identify more immunogenic T-cell epitopes to improve the selection of HAdV-specific T cells for adoptive transfer and the immunomonitoring of high-risk patients.

T-cell epitopes can be identified by direct or reverse immunology. Various computer algorithms have been developed over the past years that allow for the prediction of peptide binding to MHC class I and II molecules, proteasome cleavage patterns and transporter associated with antigen processing translocation [26]. Naturally presented CD8+ T-cell epitopes are usually among the top-scoring 2 in 80 % of all predictions, whereas the reliability of CD4+ T-cell epitope prediction is much lower due to the more variable pocket binding behavior of MHC class II molecules [27]. SYFPEITHI [26, 28, 29], BIMAS [26, 30] and NetChop [31] are the most widely used algorithms to identify cytotoxic T lymphocyte (CTL) epitopes in viral, microbial, and tumor antigens. These well-established algorithms, which have been validated and compared [26], were employed in this study to predict new HAdV epitopes.

The major focus of this study was to identify and evaluate novel immunodominant HAdV-specific T-cell epitopes by analyzing the main structural proteins, hexon and penton. HLA-A*01-, A*02-, A*03- and B*08-restricted peptide epitopes within conserved protein regions (Table 1) were pre-selected based on the predictions of several established computer algorithms. Immunogenicity of the top-ranked epitopes was investigated by established methods: IFN-γ-based EliSpot, cytokine secretion assay (CSA), peptide MHC (pMHC) multimer staining and multicolor flow cytometry. Four of the selected peptide candidates were classified as low immunodominant and two as high immunodominant according to the number of responders in the healthy donors and HAdV-infected HSCT recipients. This paper describes for the first time the immunogenic potential of penton-derived epitopes and demonstrates that the penton, as an immunological target, it is not secondary to the hexon. Expanding the repertoire of immunodominant HAdV-specific T-cell epitopes will enable more precise immunomonitoring and more effective multi-epitope-based T-cell therapy by targeting epitopes presented in a broader array of HLA molecules. Table 1

Predicted peptide candidates used for HAdV-specific T-cell screening in healthy donors

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Award-Winning Chef Elizabeth Falkner Reveals Her Struggle with Atopic Dermatitis to Highlight the Physical and Psychological Impact of the Disease

CAMBRIDGE, Mass. and TARRYTOWN, N.Y., July 12, 2016 PRNewswire -- Celebrity chef, restaurateur, and media personality Elizabeth Falkner has teamed up with Sanofi Genzyme, Regeneron Pharmaceuticals, ., the National Eczema Association, and the Dermatology Nurses Association to launch Understand AD, a national awareness campaign focused on educating people about moderate-to-severe atopic dermatitis (AD), a potentially serious, chronic inflammatory skin disease.1 Falkner is speaking out for the first time about her own struggle with the disease to drive awareness about the physical impact and effects on quality of life for people living with atopic dermatitis, and to encourage others to speak up about their experience.

"I have been living with the challenges of atopic dermatitis for more than 20 years. At its worst, my atopic dermatitis causes constant, unbearable itching, scabbing, visible rashes on my body and even bleeding, and that's only the physical part," says Elizabeth Falkner. "Having atopic dermatitis can affect many aspects of a person's life – physically and emotionally – and yet many people don't understand the severity and impact. I joined Understand AD to empower people to have more open conversations with their doctors and loved ones about the impact this disease has on their lives."

Atopic dermatitis is a chronic, systemic inflammatory disease characterized by rashes and can include intense itching, skin dryness, cracking, redness, crusting and oozing.1,2,3 Though symptoms can appear on the surface of the skin all over the body,4 advances in research have provided new insights on the cause of atopic dermatitis.5 Scientists now believe AD is caused in part by systemic allergic inflammation that results from a malfunctioning immune system.4,6 The physical symptoms are challenging and impact people's sleep and daily lives and the disease can also make people feel self-conscious and embarrassed about their appearance.7,8,9

"Understand AD aligns with our mission to educate the public and support patients impacted by atopic dermatitis," says Julie Block, President and CEO, National Eczema Association. "Unfortunately, there's a misperception that atopic dermatitis is just a 'skin condition' that people can deal with on their own, but in reality, it's an immunological disease that has a huge impact on patients' lives. We want people living with this disease to know that they're not alone and that we're committed to advocating for better care and treatments, providing support and raising the level of awareness about this serious, and often overlooked, disease."

An estimated 1.6 million adults in the United States live with uncontrolled moderate-to-severe atopic dermatitis.10 Researchers continue to discover more about atopic dermatitis and there is still a need for additional treatment options for atopic dermatitis.

"Our community of nurses on the front lines see people every day who are suffering with atopic dermatitis," says Donna Beyer, MSN, RN, DNC, President of the Dermatology Nurses Association. "But there is still a gap in public awareness about this disease and a clear need for continued education and supportive resources for patients. We're excited to join Understand AD to help educate about the disease and to drive the dialogue that atopic dermatitis is more than skin deep."

.UnderstandADm to learn more about moderate-to-severe atopic dermatitis, get connected with advocates such as the National Eczema Association and Dermatology Nurses Association, and hear from award-winning chef, media personality and restaurateur Elizabeth Falkner who has lived with atopic dermatitis for the past 20 years.

About SanofiSanofi, a global healthcare leader, discovers, develops and distributes therapeutic solutions focused on patients' needs. Sanofi is organized into five global business units: Diabetes and Cardiovascular, General Medicines and Emerging Markets, Sanofi Genzyme, Sanofi Pasteur and Merial.

Sanofi Genzyme focuses on developing specialty treatments for debilitating diseases that are often difficult to diagnose and treat, providing hope to patients and their families.

Genzyme® is a registered trademark of Genzyme Corporation. Sanofi® is a registered trademark of Sanofi. .

About Regeneron Pharmaceuticals, .Regeneron is a leading science-based biopharmaceutical company based in Tarrytown, New York that discovers, invents, develops, manufactures, and commercializes medicines for the treatment of serious medical conditions. Regeneron commercializes medicines for eye diseases, high LDL cholesterol and a rare inflammatory condition and has product candidates in development in other areas of high unmet medical need, including rheumatoid arthritis, asthma, atopic dermatitis, pain, cancer, and infectious diseases. For additional information about the company, please visit .regeneronm  or follow Regeneron on Twitter.

About the National Eczema AssociationThe National Eczema Association (NEA) is a non-profit 501 (3) patient advocacy organization whose mission is to improve the health and quality of life for individuals with eczema through research, support, and education. In the United States alone, over 10% of the population has some form of atopic dermatitiseczema. NEA was founded in 1988 by a group of patients, medical professionals, and parents to help individuals and families living with this skin disease live healthier lives. Through a variety of educational materials, including a quarterly patient-oriented magazine, a monthly electronic newsletter, and trustworthy website, the NEA reaches out to a diverse audience that includes eczema patients, caregivers, medical professionals, and other stakeholders. NEA also conducts patient conferences and participates in a wide-variety of medical symposiums. NEA is active year round to promote eczema awareness, break through stereotypes and address issues critical to patient care. Advocacy efforts include advancing increases in skin disease research funding through the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) of the National Institutes of Health, as well as increasing public understanding regarding the burden of eczema. NEA provides a network of support groups, an up-to-date website with the latest research and treatment information, a Seal of Acceptance program for over-the-counter products to help eczema patients navigate the myriad of products necessary for their daily skin care regimen, and a research program to advance scientific knowledge and care.  All NEA programs and services result in benefits for eczema patients and their families. NEA does not endorse specific products. For more information about the National Eczema Association, visit .nationaleczema, contact at infonationaleczema, or call 1-800-818-7546.

About the Dermatology Nurses AssociationThe Dermatology Nurses Association (DNA) is a professional nursing organization comprised of a diverse group of individuals committed to quality care through sharing knowledge and expertise. The core purpose of the DNA is to promote excellence in dermatologic care. Members include nurse practitioners, registered nurses, licensed practical and vocational nurses, medical assistants and others associated with dermatology nursing, who work in a variety of settings including clinics, academic institutions, private practice, public health centers, and government facilities. DNA offers education and training in fundamental and cutting-edge dermatology care and treatment through its annual convention, local chapter meetings, dermatology nurse and nurse practitioner certification review courses and expert workshops. Members of the DNA's Nurse Practitioner Society are afforded tools, resources and education focused on the needs of the advanced nurse practitioner. The DNA Focus Newsletter and official journal, the Journal of Dermatology Nurses Association, extend the DNA's informational and education presence with association and practice news, learner-paced continuing education and timely resources.

s Sanofi:

Media Relations                                                     Carrie Brown                                                           Tel: (908) 981-6486                                           carrie.brownsanofim                                           

s Regeneron:

Media Relations                                                     Ilana Tabak                                                              Tel: (914) 847-3836Mobile: (914) 450-6677                                                ilana.tabakregeneronm

1 World Allergy Association 2004:

 

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NF-κB Regulates Mesenchymal Transition for the Induction of Non-Small Cell Lung Cancer Initiating Cells

Abstract

The epithelial-to-mesenchymal transition (EMT) is a de-differentiation process that has been implicated in metastasis and the generation of cancer initiating cells (CICs) in solid tumors. To examine EMT in non-small cell lung cancer (NSCLC), we utilized a three dimensional (3D) cell culture system in which cells were co-stimulated with tumor necrosis factor alpha (TNF) and transforming growth factor beta (TGFβ). NSCLC spheroid cultures display elevated expression of EMT master-switch transcription factors, TWIST1, SNAI1Snail1, SNAI2Slug and ZEB2Sip1, and are highly invasive. Mesenchymal NSCLC cultures show CIC characteristics, displaying elevated expression of transcription factors KLF4, SOX2, POU5F1Oct4, MYCN, and KIT. As a result, these putative CIC display a cancer “stem-like” phenotype by forming lung metastases under limiting cell dilution. The pleiotropic transcription factor, NF-κB, has been implicated in EMT and metastasis. Thus, we set out to develop a NSCLC model to further characterize the role of NF-κB activation in the development of CICs. Here, we demonstrate that induction of EMT in 3D cultures results in constitutive NF-κB activity. Furthermore, inhibition of NF-κB resulted in the loss of TWIST1, SNAI2, and ZEB2 induction, and a failure of cells to invade and metastasize. Our work indicates that NF-κB is required for NSCLC metastasis, in part, by transcriptionally upregulating master-switch transcription factors required for EMT.

Citation: Kumar M, Allison DF, Baranova NN, Wamsley JJ, Katz AJ, Bekiranov S, et al. (2013) NF-κB Regulates Mesenchymal Transition for the Induction of Non-Small Cell Lung Cancer Initiating Cells. PLoS ONE 8(7): e68597. doi:10.1371journal.pone.0068597

Editor: Srikumar P. Chellappan, H. Lee Moffitt Cancer Center & Research Institute, United States of America

Received: January 14, 2013; Accepted: May 30, 2013; July 30, 2013

: 2013 Kumar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Work was supported by National Institutes of Health grants R01CA132580, R01CA104397 (to M.W.M.), and R01CA136705 (to D.R.J.). All funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Cancer development from early pre-malignant neoplasm to full metastatic disease is a multistep process that involves tumor epithelial-stromal interactions, angiogenesis, and infiltration of tumor-associated pro-inflammatory cells [1], [2]. An emerging hypothesis proposes that this milieu of cell-cell interactions, growth factors, and cytokines known as the tumor microenvironment, stimulates morphogenesis within tumor cells referred to as the epithelial-to-mesenchymal transition (EMT) [3]–[5]. EMT induces a redistribution of intracellular architecture, decreased cell-cell adhesion, and loss of cellular polarization. Carcinoma cells that have undergone EMT are characteristically motile, invasive and highly metastatic. Over the past several years, EMT has also been recognized as a de-differentiation program attributed to generation of tumor-initiating or cancer-initiating cells (CICs) that are important in the maintenance of cancer “stemness” [6]–[9].

Although multiple cytokines and growth factors induce EMT, one of the best studied factors is transforming growth factor beta (TGFβ) [2], [3], [10]–[13]. Stimulation of cells with TGFβ results in expression of the EMT master-switch transcription factors, TWIST1, SNAI1Snail, SNAI2Slug, and ZEB2Sip1 that together differentially regulate genes to promote the mesenchymal phenotype [10], [12]. While extensive research details the ability for TGFβ to induce EMT, evidence indicates that tumor necrosis factor (TNF) further potentiates the transition [14], [15]. During cancer progression, secretion of TGFβ within the tumor microenvironment occurs through many different cell types, including tumor-associated fibroblasts, while secretion of TNF originates from tumor-associated M2 macrophages [3], [16], [17]. A prevailing hypothesis in the field is that exposure of cancer cells to these cytokines within the tumor microenvironment promotes EMT, de-differentiation, and the formation of CICs [2], [5], [17].

TNF is a powerful pro-inflammatory cytokine that stimulates signaling cascades to activate nuclear factor kappa B (NF-κB). As a transcription factor, NF-κB plays a key role in the expression of genes involved in cancer initiation and progression. Upregulation of NF-κB activity often occurs in primary solid and hematological tumors, directly correlating with de-differentiated morphology, advanced tumor stage, and poor clinical prognosis [18]. Importantly, NF-κB has been linked to mammary CICs [19], [20]. NF-κB induces and maintains EMT in model systems through two mechanisms, upregulation of EMT master-switch transcription factors [21]–[24] and stabilization of Snail [25]. NF-κB is composed of five Rel family members: RelAp65, RelB, cRel, p50 and p52. In unstimulated cells, inhibitory IκB subunits associate with NF-κB dimers and sequester them in the cytoplasm. Upon cellular stimulation by pro-inflammatory cytokines, IκBα is phosphorylated by the IκB kinase (IKK) complex, ubiquitinated by the SCF-type E3 ligase, E3RSIκBβ-TrCP and degraded by the 26S proteasome [26]. Liberated NF-κB then translocates to the nucleus to activate gene expression by recruiting transcriptional coactivators [27]. Our laboratory has shown that posttranslational modifications on RelA are required for full NF-κB transcriptional activity [27]–[30].

Although EMT in breast cancer models requires NF-κB activity [31], the role of this transcription factor in stimulating EMT and developing CICs in NCSLC has not been thoroughly examined. However, strong evidence exists for the presence of NSCLC stemprogenitor cells in primary adenocarcinomas and established cell lines [32]–[35]. Here, we demonstrate that coordinated activation of TNF and TGFβ signaling cascades effectively induces EMT and the expression of genes related to de-differentiation and stemness. Further, we show that mesenchymal NSCLC cells possess constitutively active NF-κB, and that inhibition of NF-κB decreases EMT, CIC formation, and metastatic potential.

Materials and Methods Cell culture and reagents

NSCLC lines A549, H359, H1299, and H157 were obtained from ATCC and maintained as 2D cultures in DMEM (CellGro), 10% FBS (Invitrogen) and penicillinstreptomycin (Invitrogen). The antibodies used include: E-cadherin (BD Pharmingen- 610404), N-cadherin (BD Pharmingen- 610920), Vimentin (V6630), Fibronectin (BD Pharmingen- 610078), α-Tubulin (Sigma T6793), HMGA2 (Biocheck 59170AP), Twist1 (Cell Signaling 4119), Snail1 (Cell Signaling 4719), Sip1 (SCBT sc-48789), Slug (Abcam ab27568), IκBα (pS32, Cell Signaling 2859), IκBα (SCBT sc-371), RelA (pS536, Cell Signaling 3031), RelA (SCBT sc-372), and M2-Flag (Sigma F1804). Baculogold protease inhibitors were obtained from BD Biosciences. TGFβ (PHG 9204) and TNF (PHG 3015) were purchased from InvitrogenLife technologies. All other chemicals were from Sigma.

Three-dimensional multicellular spheroid cultures

Three-dimensional multicellular spheroid cultures were created using a modified hanging droplet method [36]. Cells were grown to approximately 80% confluence on standard tissue-culture plates. The cells were subsequently trypsinized, resuspended in DMEM10% FBS, and counted. To create 25,000 cell spheroids, the cell suspension was diluted to a concentration of 1,000,000 cellsml, and 25 µl of the cell suspension were pipetted onto the underside of a sterile 10 cm tissue-culture plate lid. Each lid holds approximately fifty droplets. After loading the droplets, the lid was placed onto a tissue culture plate containing 6 mL of sterile PBS and incubated for 48 hours to facilitate cellular aggregation and spheroid formation. The freshly formed spheroids were then transferred into 10 cm suspension plates containing DMEM and 2% FBS to prevent cell attachment to the dish. Suspension plates were made by adding 8 ml of poly-HEMA solution (Sigma-Aldrich P3932, 10 mgml) in 95% ethanol to sterile polystyrene petri dish plates (Fisher Scientific). The plates were then incubated for 24 hours in a sterile environment to allow the ethanol to evaporate. Prior to use, plates were washed with sterile PBS to remove any residual ethanol or other contaminants. Each suspension plate holds up to 100 spheroids. After transfer, the spheroids were treated with vehicle or with 10 ngml TNF and 2 ngml TGFβ, and incubated for 48 hours. After incubation, cells were subjected to a second treatment of vehicle or TNF and TGFβ, and incubated an additional 48 hours. The spheroids were then collected and analyzed by various assays.

Immunofluorescence Microscopy

A549 cells were seeded on glass coverslips and subjected to EMT induction or left untreated. After induction, the cells were fixed in 100% methanol and subsequently incubated with primary antibodies to the extracellular domain of E-Cadherin (SCBT, sc-7870). An AlexaFlour-conjugated, goat anti-rabbit antibody (Invitrogen) was used as a secondary antibody, and indirect immunofluorescence of E-Cadherin was imaged using a Nikon E3800 fluorescence microscope.

Migration and Invasion

In vitro migration and invasion assays were carried out according to the manufacturer's protocol (BD Biosciences). 2D and 3D cultures were disaggregated by trypsin and subsequently counted. 1×105 cells (migration) or 1×104 cells (invasion) were seeded in plain DMEM in the top well of a transwell control plate (BD 354578) or Matrigel invasion plate (BD 354480). The bottom well was loaded with DMEM containing 10% FBS as a chemoattractant, and the plates were incubated for eight hours (migration) or twenty-four hours (invasion) at 37°C and 5% CO2. Afterwards, cells on the upper side of the membrane were removed, and the remaining cells were fixed in 100% methanol and stained with 0.1% crystal violet. The stained cells were imaged and quantified using Adobe® Photoshop.

Tumor model

Monolayer (2D) and 3D A549 cultures that had been left untreated or treated with TNF and TGFβ were trypsinized, resuspended in DMEM0.5% FBS, and carefully counted and diluted in the appropriate volume for injection. Cells were subcutaneously (SC) injected into female outbred Crl:NUNU nude mice (Charles River). Five mice were injected per experimental condition. All animal studies were performed as three independent experiments. Mice were sacrificed forty days post-injection. The primary SC tumors were removed and weighed. Additionally, the lungs were removed, fixed in formalin, and surface lung metastases were counted. To quantify the amount of total tumor burden in the formalin fixed lung tissue, genomic DNA was extracted [37] and assayed for the presence of human genomic material as described using quantitative real time-polymerase chain reaction (QRT-PCR) primers specific to human endogenous retrovirus-3 (ERV3, Table S1) [38], [39].

This study was carried out in strict accordance with recommendation from the Animal Care and Use Committee (ACUC) of the University of Virginia. The protocol was approved by ACUC Number 3914. All experiments were terminated after 40 days at which time SC tumors were less than 1.0 cm3 in size; thus, restricting tumor burden. All efforts were made to minimize pain and suffering.

QRT-PCR, Immunoblots, and Electrophoretic mobility shift assays (EMSAs)

QRT-PCR and immunoblot experiments were carried out as previously described [28]. PCR primers are shown in Table S1. Nuclear extracts were prepared using spheroids from A549.V and A549.I cell lines treated with or without TNF and TGFβ. EMSAs and supershift assays were performed as described previously [40].

Statistics

Where appropriate, comparisons between experimental groups were carried out by performing a one-tailed Student's t test in Microsoft excel. Data for all experiments was considered statistically significant when p<0.05.

Results A model to study EMT in NSCLC

TNF has been shown to potentiate TGFβ-mediated EMT through the activation of co-stimulatory pathways [15]. To confirm this observation in our three-dimensional (3D) model, a timecourse was performed using both cytokines in tandem and alone. Multicellular spheroid cultures were created using a modified hanging droplet method [36]. After two days, spheroids were suspended in poly-HEMA coated plates and treated every two days with the indicated cytokines to induce EMT (Figure 1A). Samples were collected from untreated (0 days) and cytokine-treated cultures (1–8 days). Epithelial (E-cadherin) and mesenchymal (N-cadherin, Vimentin, and Fibronectin) markers were measured by immunoblot. Treatment with TNF resulted in a modest increase in N-cadherin and Fibronectin, but failed to show differences in other markers (Figure 1B). Consistent with the induction of EMT, TGFβ treatment resulted in a loss of E-cadherin expression and an increase in N-cadherin, Vimentin, and Fibronectin. Moreover, co-stimulation with TNF and TGFβ yielded a more mesenchymal phenotype and persisted throughout the eight day time course (Figure 1B). Importantly, stimulation with TNF and TGFβ effectively induced EMT in both A549 and H358 cell lines within four days of treatment, compared to H1299, which already shows changes in E-cadherin and vimentin (Figure 1C). Based on results in Figure 1, we used the four day timeframe throughout our remaining experiments.

Figure 1. Establishment of three-dimensional multicellular culture model for EMT studies.

(A) A timeline illustrates the procedure used to create a three-dimensional mesenchymal cell population from confluent monolayers. (B) Spheroid cultures of A549 cells were treated, with TNF, TGFβ, or both cytokines every forty-eight hours for the indicated times. Immunoblot analysis measured changes in epithelial (E-cadherin) and mesenchymal (N-cadherin, Vimentin, and fibronectin) markers over an eight day timecourse. (C) 3D cultures of multiple NSCLC cell lines (A549, H358, H1299) were incubated for ninety-six hours in the absence or presence of TNF and TGFβ. Epithelial and mesenchymal markers were subsequently measured by immunoblot. Results from Figure 1B and 1C are representative examples from at least three independent experiments; α-tubulin acts as a protein loading control.

3D cultures undergo EMT more efficiently than 2D cultures

To determine whether 3D A549 cultures undergo EMT more efficiently than two-dimensional (2D) monolayer cultures, we measured expression of epithelial and mesenchymal markers in response to stimulation with TNF and TGFβ as described in Figure 1. Following cytokine treatment, 3D cultures show significant loss of CDH1E-cadherin expression when compared to 2D cultures (Figure 2A). Moreover, the spheroids also possess increased expression of mesenchymal markers VIM, HMGA2, and the EMT master-switch transcription factors, TWIST1, SNAI1Snail1, SNAI2Slug and ZEB2Sip1 (Figures 2A and 2B). Immunoblot analysis of spheroid cultures confirm that the differential mRNA expression resulted in a corresponding change in protein levels (Figure 2C). Additionally, we examined changes in cellular morphology and E-cadherin localization by microscopy. Both 2D and 3D cultures were treated with cytokines as described, trypsinized, re-plated on glass coverslips, and indirect immunofluorescent staining was carried out eighteen hours later. As expected, untreated monolayer and spheroid A549 samples showed robust E-cadherin expression, though the junctional localization appeared diminished in cells from the 3D cultures (Figure S1). Furthermore, cells derived from cytokine-treated spheroids displayed enhanced loss of E-cadherin when compared to 2D treated samples, suggesting that 3D cultures underwent more efficient EMT. Results shown in Figure 2 and Figure S1 illustrate significant EMT induction in 3D cultures as measured by changes in mesenchymal markers, EMT master-switch transcription factor expression, and cellular morphology.

Figure 2. Three-dimensional cultures show enhanced sensitivity to cytokine treatment.

(A and B) Monolayer (2D) and 3D cultures of A549 cells were left alone (No Add) or treated with TNF and TGFβ (TNFTGF) for ninety-six hours. Expression of epithelial markers (CDH1), mesenchymal markers (VIM, HMGA2), and EMT master-switch transcription factors (TWIST1, SNAI1, ZEB2, SNAI2) were measured by QRT-PCR. (C) Immunoblot analysis of 3D A549 cultures, left alone (No Add) or treated with TNF and TGFβ (TNFTGF), was performed on E-cadherin, Vimentin, HMGA2, Twist1, Snail1, Sip1, Slug, and α-tubulin. Results in Figure 2A and 2B were normalized to GAPDH, and are calculated mean ± S.D, *p<0.05, N = 3. Immunoblots in Figure 2C are representative example from at least three independent experiments.

Mesenchymal NSCLC cells are invasive and endogenously express genes known to promote stem-like properties

Phenotypically, mesenchymal cells have high migration rates and secrete enzymes that degrade extracellular matrix to facilitate cellular invasion. Using in vitro transwell assays, we measured the migration and invasion characteristics of A549 cells grown as either 2D or 3D cultures. Interestingly, untreated 3D spheroid cultures showed higher migration rates than 2D monolayer cultures (Figure 3A, left). However, treatment of 3D cultures with TNF and TGFβ further potentiated migration when compared to untreated 3D cultures. Spheroids treated with cytokines invaded through Matrigel more effectively than any other condition (Figure 3A, right). Additionally, cytokine treated A549 spheroids displayed upregulated expression of MMP9, LOX, and COL22A1 (Figure 3B), genes known to potentiate invasion [41], [42]. These results demonstrate that culturing 3D spheroids in the presence of TNF and TGFβ establishes a highly invasive mesenchymal population. Finally, cytokine-treated spheroids showed endogenous upregulation of markers associated with de-differentiation and maintenance of CICs [43]–[48], including KLF4, SOX2, POU5F1Oct4, MYCN, and KIT (Figure 3C). Data shown in Figure 3 indicate that co-stimulation of spheroids with TNF and TGFβ promotes phenotypic changes in A549 cells that result in increased invasion and expression of gene products associated with stem-like properties.

Figure 3. Efficient induction of EMT promotes invasion and the expression of genes required to maintain CICs.

Monolayer and 3D A549 cultures were left alone (No Add) or treated with TNF and TGFβ (TNFTGF) for ninety-six hours. (A) Cells were disaggregated and subsequently subjected to migration and invasion assays. (B and C) Expression of invasion (MMP-9, LOX, COL22A1) and stem cell markers (KLF4, Sox2, POU5F1, MYCN, and KIT) was measured by QRT-PCR. Results in Figure 3 are calculated mean ± S.D, *p<0.05, N = 3. Results from 3B and 3C were normalized to GAPDH.

Mesenchymal cells are highly metastatic and display cancer initiating phenotypes

To examine whether induction of EMT promotes the development of CICs in vivo, we utilized a xenograft tumor model in nude mice. TNF and TGFβ treated 2D and 3D cultures were disaggregated and cell suspensions were SC injected into the right flank of nude mice. Forty days later, animals were sacrificed and SC tumors were resected and weighed while the lungs were excised and scored for surface metastases. To our surprise, TNF and TGFβ-treated cells did not form SC tumors to the same extent as cytokine-treated 2D cultures (Figure 4A, left). However, examination of the lung surface in these mice revealed extensive metastasis (Figure 4A, right). The only plausible explanation for these results is that mesenchymal cells from 3D cultures invaded and metastasized to the lung without developing SC tumors.

Figure 4. Cytokine-treated 3D cultures contain CICs with increased metastatic potential.

(A) Monolayer and 3D A549 cultures were treated with TNF and TGFβ for ninety-six hours. Cells were disaggregated and SC injected into nude mice (1×106 cellsanimal). Forty days later, the primary SC tumors were resected and weighed. Additionally, the lungs were excised and the number of surface metastases were determine. (B) Monolayer and 3D A549 cultures were either left untreated or treated with TNF and TGFβ and limiting cell numbers (1×103animal) were SC injected into nude mice to evaluate the presence of CICs. Metastasis was evaluated by surface lung tumor count and lung tumor burden was evaluated using genomic QRT-PCR to detect human DNA in total lung tissue. Weight and lung metastases data from Figure 4 are mean ± S.D. of five mice per condition, *p<0.05, N = 3 independent experiments. Genomic QRT-PCR data from Figure 4B are normalized to total lung tissue (mg).

Measuring the extent of metastasis under limiting cell dilution proves a reliable test for the presence of enriched CICs in epithelial-derived tumors [49]. Therefore, experiments were repeated using one-thousand cells per SC injection. Cell suspensions, derived from TNF and TGFβ treated spheroids, produced more surface lung metastases under limiting cell dilution than cytokine-treated monolayers or untreated 3D cultures (Figure 4B left). Limiting cell dilution assays indicate that induction of EMT in 3D cultures produces a CIC population that effectively metastasizes to lung. As expected, analysis of DNA isolated from mouse lungs confirmed the presence of metastatic burden and verified that the lesions were of human origin (Figure 4B right). We conclude from the experiments in Figure 4 that de-differentiation, CIC formation, and metastatic potential are all significantly enhanced in EMT-induced spheroid cultures.

NF-κB is constitutively active in 3D cultures and is required for induction of EMT

TNF, a potent NF-κB activator, enhances induction of EMT in NSCLC cell lines. Therefore, we assessed whether EMT induction results in activation of NF-κB signaling by immunoblot. Interestingly, mesenchymal A549 spheroids displayed constitutive IKK activity as measured by phospho-specific antibodies that detect IκBα pS32 and RelA pS536 (Figure 5A and Figure S2A). Change in E-cadherin and Vimentin levels confirmed efficient EMT in the cytokine-treated spheroids. Moreover, QRT-PCR experiments demonstrated increased expression of NF-κB-regulated genes IL8 and BIRC3cIAP2 in mesenchymal 3D cultures (Figure 5B). Collectively, these data indicate that cytokine-treatment of 3D A549 cultures results in the increased phosphorylation of IKK-regulated substrates and constitutive NF-κB transcriptional activation.

Figure 5. Mesenchymal cells display constitutive NF-κB activity.

Monolayer and 3D cultures of A549 cells were incubated with cytokines for ninety-six hours. (A) Mesenchymal A549 cells display constitutive NF-κB activated pathways, as determined using phospho-specific antibodies to IκBα and RelA. (B) Untreated and TNF and TGFβ stimulated 2D and 3D cultures of A549 cells were harvested and analyzed for expression of NF-κB regulated genes by QRT-PCR. (C and D) Three dimensional cultures of A549.V (vector control) and A549.I (SR-IκB) were incubated for ninety-six hours in the absence or presence of TNF and TGFβ. (C) Immunoblots confirm the expression of the Flag-tagged SR-IκBα in the A549.I line, which successfully blocked nuclear translocation and DNA binding, as measured by EMSA. (D) QRT-PCR confirmed the inability of A549.I cell to upregulate NF-κB-regulated genes following TNF and TGFβ treatment. Immunoblots in Figure 5A are a representative example from three independent experiments. Results in Figure 5B and 5D are calculated mean ± S.D, *p<0.05, N = 3. RNA values were normalized to GAPDH.

To determine the importance of NF-κB activity during induction of EMT in NSCLC cell lines, stable clonal pools expressing the super-repressor IκBα (SR-IκBα) were generated. The SR-IκBα is resistant to proteasomal degradation, and consequently sequesters NF-κB in the cytosol. Cells expressing the SR-IκBα protein therefore display an inhibition of NF-κB-mediated transcription [50]. Figure 5C (top) confirms expression of Flag-tagged SR-IκBα in A549 stable cells (A549.I) compared to empty vector control cells (A549.V). Furthermore, nuclear protein extracts from A549.I spheroid cultures, treated with TNF and TGFβ, lacked NF-κB DNA binding activity as compared to A549.V extracts (Figure 5C, bottom). Supershift experiments confirm that the NF-κB activity is composed predominantly of a RelA-p50 heterodimer complex (Figure S2B). QRT-PCR assays show repressed cytokine-mediated induction of IL8 and BIRC3cIAP2 in A549.I cells when compared to control cells A549.V (Figure 5D). In contrast to high doses of TNF (100 ngml), low doses (10 ngml) did not result in a loss of cell viability in A549.I lines, since expression of the house keeping gene, HPRT, did not change and was used for normalization in Figure 5D. These data verify that SR-IκBα expression in the A549.I cell line effectively blocks NF-κB transcriptional activity.

Characterization of NF-κB in potentiating the mesenchymal phenotype

NF-κB has been shown to regulate the expression of EMT master-switch transcription factors in multiple model systems [21]–[24]. Therefore, we hypothesized that inhibiting NF-κB activity in the A549.I cell line would dampen EMT induction. Immunoblot analysis confirmed that A549.I cells fail to down regulate E-cadherin expression or upregulate mesenchymal markers (Vimentin, N-cadherin and Fibronectin) compared to control cells (Figure 6A). Moreover, cytokine-treated A549.I cells showed only minimal upregulation of TWIST1, ZEB2 and SNAI2 gene expression following TNF and TGFβ treatment (Figure 6B). These results indicate that NF-κB is required to upregulate TWIST1, ZEB2 and SNAI2, while expression of SNAI1 appears independent of NF-κB-dependent transcription in the A549.I cell line. These results suggest that the expression of critical EMT master-switch transcription factors requires NF-κB activity.

Figure 6. NF-κB is required for the maintenance of CICs and lung metastasis.

(A) A549.I cells fail to show changes in mesenchymal markers, as determined by immunoblot analysis. (B) NF-κB is required to upregulate mRNA expression of master-switch transcription factors. (C) Spheroid cultures of A549 and H157 cell lines, expressing empty vector or the Flag-IκB super-repressor, were left alone (No Add) or treated with TNF and TGFβ (TNFTGF) for ninety-six hours. The cells were disaggregated and subjected to invasion assays. (D) A549.V and A549.I 3D cultures were left alone (No Add) or treated with TNF and TGFβ (TNFTGF) for ninety-six hours. The cells were disaggregated and SC injected into nude mice (1×106animal). Forty days later, animals were sacrificed and the number of surface lung metastasis were determined. In addition, SC tumors were excised and wet tumor weight determined. Weight and lung metastases data from Figure 6 are mean ± S.D. of five mice per condition, *p<0.05, N = 3 independent experiments. The graphs in Figure 6 are mean ± S.D., *p<0.05, of three independent experiments. Data with P values greater than 0.05 were considered not significant (ns). QRT-PCR experiments are normalized to GAPDH expression.

Next, we assessed whether NSCLC required NF-κB for invasion using transwell assays. Inhibited NF-κB activity in A549.I cells abolished invasion through Matrigel when compared to the control lines (Figure 6C). This effect was not cell-line specific since another NSCLC line expressing the SR-IκB (H157.I) showed similar results as A549.I cells. Because data shown in Figure 6 indicate that NF-κB is required for NSCLC to undergo EMT, we tested the A549.V and A549.I cell for their ability to metastasize to lung using a nude mouse model. As expected, cytokine-treated A549.I cells failed to form lung metastases (Figure 6D, left). The inability of these cells to metastasize to lung was not due to a loss of cell viability or an inability to form primary tumors, since untreated A549.I formed SC tumors with similar growth rates as A549.V cells (Figure 6D, right). Thus, data shown in Figure 6 indicates that TNF and TGFβ treated 3D NSCLC cultures require NF-κB to upregulate master-switch transcription factors, induce EMT, and promote invasive properties. Moreover, without NF-κB transcriptional activity A549 cells lose their ability to metastasize to lung without impacting primary tumor growth.

Discusson NF-κB regulates EMT to potentiate metastatic progression of NSCLC

We implemented a simple and relatively quick 3D culture system to examine the importance of NF-κB signaling during EMT induction and CIC propagation within NSCLC cell lines. In response to TNF and TGFβ exposure, A549 spheroid cultures displayed a loss of E-Cadherin and elevated expression of mesenchymal markers, N-Cadherin, Vimentin, and Fibronectin. The increased expression of mesenchymal protein markers likely occurs due to induction of the EMT master-switch transcription factors, TWIST1, SNAI1, SNAI2 and ZEB2. Furthermore, spheroid populations of mesenchymal A549 cells show elevated expression of endogenous transcription factors known to potentiate dedifferentiation, including KLF4, SOX2, POU5F1, MYCN and KIT. Interestingly, mesenchymal A549 cells from spheroid cultures failed to generate large SC tumors, compared to 2D cultures. Despite this effect, cytokine-treated 3D A549 cells displayed elevated lung surface metastatic lesions. These results support the hypothesis that CICs extravasated into the circulatory system and metastasize to the lung without forming SC tumors. We further demonstrated that EMT-induced A549 3D cultures effectively metastasize to lung under limiting cell dilutions, confirming the presence of an enriched “stem-like” CIC population. Thus, our results suggest that EMT induction effectively selects for self-renewing CIC with metastatic potential, a phenotype described by Dieter and colleagues as self-renewing long-term tumor initiating cells responsible for color cancer metastasis [51]. Since IKK and NF-κB pathways have been linked to EMT and development of CICs [21]–[25], [31], we examined whether mesenchymal A549 cells upregulate NF-κB transcriptional activity. Surprisingly, EMT-induced spheroid A549 cultures displayed chronic IKK activity as measured by phosphorylation of IκBα(pS32) and RelA(pS536), and by constitutive expression of IL8 and BIRC3 transcripts. Moreover, cytokine-treated spheroid A549 cultures maintained the activation of IKK signaling pathways well beyond the half-life of the TNF and TGFβ cytokines added to the culture media. These results suggest that mesenchymal A549 spheroid cultures must produce autocrine factors capable of maintaining the EMT phenotype. Importantly, constitutive NF-κB activity proves essential for effective EMT partially through its ability to upregulate the master-switch transcription factors TWIST1, ZEB2 and SNAI2. As a result, the loss of NF-κB activity prohibited cytokine-treated spheroid A549 cells from becoming invasive and also abolished lung metastasis in the mouse xenograft model. This work firmly establishes a role for NF-κB in the induction of EMT and for the development of NSCLC CICs that promote metastasis.

Spheroid models and the propagation of CICs

Various 3D culture models have been developed that more accurately mimic tumor biology, such as cell-cell contacts, extracellular matrix composition, and nutrient accessgradients [52]–[54]. The advantage to using the hanging drop technique, over other techniques, is that 2D cultures can be quickly expanded to form multicellular aggregates that share similar size and shape, and mesenchymal populations are generated within six days. Data provided in Figure 1C demonstrate that multiple NSCLC cell lines form compact spheroids that undergo highly reproducible EMT when exposed to TNF and TGFβ. Moreover, these spheroids possess increased sensitivity to TNF and TGFβ compared to monolayer cultures (Figures 2, 3, and 4). Therefore, we utilized this 3D system to examine EMT and CIC formation in NSCLC cell lines. Surprisingly, A549 spheroids show increased migration without requiring exposure to TNF and TGFβ and despite expressing epithelial markers (Figure 1B, 2A, and 3A). This indicates that phenotypic changes occur in 3D cultures prior to exposure to EMT-inducing cytokines. However, increased invasion is restricted to cytokine-induced A549 spheroid cultures and corresponds with the upregulation of matrix and extracellular remodeling enzymes known to induce invasive properties [41], [42]. Therefore, spheroid cultures are poised to respond to TNF and TGFβ cytokines and are able to sustain EMT reprogramming. Together, we establish that CIC populations formed from EMT induction of 3D NSCLC cell lines provide a useful tool for further characterization of cancer progression in the lung.

Mesenchymal A549 cells show constitutively active NF-κB signaling pathways

Constitutive NF-κB activation occurs in many different types of hematopoietic and epithelial-derived carcinomas. However, mutations that result in chronic activation of NF-κB signaling are extremely rare in epithelial cancers [19]. Thus, activation of NF-κB most likely results from autocrine and paracrine signaling within the tumor microenvironment rather than genetic alterations [2], [19]. Our data support this hypothesis by showing that TNF- and TGFβ-treated A549 spheroid populations both undergo EMT and maintain constitutive NF-κB signaling (Figure 5A and 5B). Rather than using co-culture systems, which introduce contaminating cell types other than NSCLC cells, we chose to treat A549 spheroids with EMT-inducing cytokines. TNF and TGFβ were selected because within the tumor microenvironment, TNF is believed to be produced predominantly by tumor-associated macrophages, while TGFβ is secreted by fibroblast and endothelial cells. This combination of cytokines not only effectively and reproducibly induces EMT, but also facilitates a reprogramming event that results in chronic NF-κB signaling. The molecular mechanism by which this occurs is currently unknown, but most likely is due to an increase in NF-κB-regulated gene products that function in an autocrine-dependent manner to maintain active NF-κB.

Inflammatory regulatory circuits that drive constitutive NF-κB activation

In the past two years, evidence has emerged that an epigenetic switch occurs during breast cancer transformation in which inflammatory circuits involving IL6 and IL8 mediate self-renewal of CICs [55]–[57]. Ginesteir and colleagues showed that breast CICs upregulate the IL8 receptor CXCR1 to potentiate self-renewal, tumorigenicity and metastasis [57]. Additional studies indicate that oncogenic transformation of breast cancer cells leads to chronic activation of NF-κB required to upregulate Lin-28B and downregulate the negative microRNA regulator of IL6, Let-7a [56]. As a result, IL6 provides an inflammatory feedback loop that further activates NF-κB as well as the STAT3 signaling pathway [56], [58]. Interestingly, this pro-inflammatory feedback loop also exists in some prostate and hepatocellular carcinomas, but only a subset of lung cancers showed increased IL6 expression [56]. In addition to the autocrine feedback mechanism, IL6 signaling pathways downregulate mir200c in a chemically-induced transformed breast cancer cell line. Loss of mir200c subsequently results in constitutively activated NF-κB through an inflammatory feedforward signaling circuit [59]. In these papers [55], [56], [59], IL6 was found to be required for the maintenance of breast CICs.

Additional work is needed to determine the importance of IL8 and IL6 as feedforward mediators of NF-κB activation in mesenchymal NSCLC cell lines. As shown in Figure 5B, IL8 is highly upregulated and maintained in mesenchymal A549 cultures; however, IL6 transcripts do not significantly change between untreated 3D and cytokine-treated 3D cultures (Figure S2C). Thus, in agreement with IIiopoulos and colleagues [59], IL6 may not be a common requirement for CICs in lung cancer. However, since CXCR1 is highly expressed in A549 cells following exposure to DNA methytransferase inhibitors [60], inflammatory circuits that regulate promoter demethylation, as observed for IL6 signaling [59], may play an important role for controlling the IL8CXCR1 responsiveness in lung cancers. Future work is needed to explore the importance of IL8CXCR1 in the maintenance of constitutive NF-κB activation and development of NSCLC CICs.

Supporting Information Figure S1.

Cytokine-treated 3D A549 cells show increased fibroid and mesenchymal morphology. Monolayer (2D) and 3D A549 cultures were left alone or treated with TNF and TGFβ for ninety-six hours. Cells were subsequently disaggregated, replated on glass coverslips, and cultured for an additional eighteen hours in 2% FBS. The cells were then fixed in methanol, and indirect immunofluorescence was used to detect the presence of junctional E-cadherin. Images are a representative field from three independent experiments.

doi:10.1371journal.pone.0068597.s001

(TIF)

Figure S2.

TNF and TGFβ-treated 3D A549 cells show increased RelA phosphorylation and nuclear DNA binding activity. (A) Immunoblot analysis of 3D A549 cells indicates that cells display constitutive RelA phosphorylation upon co-stimulation with both TNF and TGFβ over the three day period. (B) Nuclear extracts from cytokine-treated 3D control A549.V cells show elevated NF-κB binding activity by EMSA, compared to unstimulated cell extracts. The NF-κB DNA-protein complex is composed of both RelA and p50 proteins as detected by antibody super shift (SS) assays. (C) In contrast to IL8 expression shown in Figure 5B, cytokine-treated 3D cultures fail to upregulate IL6 transcripts as measured by QRT-PCR.

doi:10.1371journal.pone.0068597.s002

(TIF)

Table S1.

QRT-PCR Primers.

doi:10.1371journal.pone.0068597.s003

(DOC)

Contributions

Conceived and designed the experiments: MWM MK DFA NNB JJW. Performed the experiments: MK DFA NNB JJW. Analyzed the data: MWM MK DFA NNB JJW. Contributed reagentsmaterialsanalysis tools: AJK SB DRJ. Wrote the paper: MWM MK DFA JJW.

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