text:
Press release from Business Wire
CORRECTING and REPLACING COVERGIRL Nails Beauty-with-an-Edge Mantra by Signing Hot Female DJ Duo NERVO

 Trendsetting sisters lend pop-culture prowess to the Brand

 Offering consumers cutting-edge beauty that's always on beat

Wednesday, November 07, 2012
CORRECTING and REPLACING COVERGIRL Nails Beauty-with-an-Edge Mantra by Signing Hot Female DJ Duo NERVO15:14 EST Wednesday, November 07, 2012 HUNT VALLEY, Md. (Business Wire) -- Please replace the release with the following corrected version due to multiple revisions. COVERGIRL nails beauty-with-an-edge mantra by signing hot female DJ duo NERVO - trendsetting sisters lend pop-culture prowess and cutting-edge beauty that's always on beat (Photo: Business Wire) The corrected release reads: COVERGIRL NAILS BEAUTY-WITH-AN-EDGE MANTRA BY SIGNING HOT FEMALE DJ DUO NERVOTrendsetting sisters lend pop-culture prowess to the BrandOffering consumers cutting-edge beauty that's always on beat COVERGIRL announced today the signing of talented DJ duo and sisters NERVO in a bold move that brings some of the most inspired girl power behind electronic dance music (EDM) today to the brand's lineup of talent. Joining an elite fleet of beauties who authentically rock the brand's "doing you" crusade (most recently P!NK and Janelle Monáe), the Australian-born siblings add the title of COVERGIRLs to an already long list of impressive personas, which includes visionary DJs, hit songwriters and trend-setting producers. NERVO will make their public debut as COVERGIRLs doing what they do best – spinning in style at a special COVERGIRL-sponsored event in NYC on November 7. Ensuring all eyes are on the turntables, NERVO will be showcasing extraordinary nail art designs created using the brand's much-anticipated launch of Outlast Stay Brilliant Nail Gloss collection. The event will also celebrate Rolling Stone's "Top 25 DJs" issue hitting newsstands on November 9. "We're both so excited to join COVERGIRL and be part of an iconic brand that shares our beliefs," said Liv Nervo. "It's been really important for us to stay true to ourselves, our style, and our musical choices throughout the years. We believe individuality is a trend that will always be in style and that every woman should rock what makes them unique inside and out." "The NERVO sisters are paving the way for women in a male-dominated industry; their determination is inspiring to all young women working to break barriers and create their own path. With their immense talent and never-give-up mentality, they are the perfect spokesmodels to encourage fans worldwide to dance to their own beat," said Esi Eggleston Bracey, VP and General Manager, COVERGIRL Cosmetics. "We are always looking for fun and fierce young women who can empower the next generation of COVERGIRLs, and NERVO does just that while bringing the heart and energy of EDM music to COVERGIRL." On the front lines of street style trends, NERVO will also serve as Brand correspondents in addition to appearing in promotional and digital COVERGIRL campaigns – sharing with the COVERGIRL community content inspired by their personal styles as well as the different looks they see at shows and on the road. Shared content will also include a range of DIY tips detailing how NERVO incorporates COVERGIRL products into their looks, from Outlast Stay Brilliant Nail Gloss to LashBlast Clump Crusher Mascara and Outlast All-Day Lip Colour. "We like to push our creativity to the limit not only when we spin, but also in the way we style ourselves for shows – from head to toe," said Mim Nervo. "Beyond just clothes and makeup, this also includes our nails, since as DJs, our hands are literally always in the spotlight. That's why COVERGIRL is the right fit for us; we believe beauty is a form of expression, just like music, and when done right, it makes us feel bold, beautiful and free." NERVO, signed to Wilhelmina Artist Management, has quickly become the hottest duo in EDM today, bridging the gap between the underground and mainstream. NERVO has built up a big profile around the world having opened for Britney Spears and Nicki Minaj on the Femme Fatale tour, plus DJ dates at big festivals such as Electric Daisy, Tomorrowland, Electric Zoo, Ultra, Lollapalooza and Winter Music Conference. Recently, NERVO made a historic debut as the only females voted onto the coveted DJ Mag 2012 Top 100 DJ's Poll. As songwriters, NERVO co-wrote David Guetta's Grammy-award-winning hit "When Love Takes Over" featuring Kelly Rowland and penned songs for artists such as Ke$ha, Kylie Minogue, Miley Cyrus and The Pussycat Dolls. As artists, NERVO has released the top 10 Billboard Club Chart single "We're All No One" and top 5 hit "You're Gonna Love Again" plus a slew of collaborations with some of the genre's biggest names including Afrojack, Steve Aoki, Hook N Sling and Dimitri Vegas & Like Mike. They are currently working on their debut album expected to be released by Astralwerks Records in 2013. The girls currently hold a DJ residency in the new Mecca for dance music, Las Vegas, at the Wynn Hotel Group and have a monthly DJ mix show "NERVO Nation" on Sirius XM's BPM Channel 51. Since its introduction in 1961, the COVERGIRL brand has been a pioneer in providing women with the makeup tools needed to rock those everyday cover moments. The long list of famous COVERGIRLs has included Christie Brinkley, Cheryl Tiegs, Rachel Hunter, Tyra Banks, Niki Taylor and Molly Sims. The current roster of COVERGIRLs includes such icons as P!nk, Janelle Monáe, Sofia Vergara, Queen Latifah, Drew Barrymore,Ellen DeGeneres, Taylor Swift and Canadian 2012 Olympian, Mary Spencer (Women's Boxing). Visit www.covergirl.ca for information on COVERGIRL's family of spokeswomen and its extensive lineup of products, including Outlast Stay Brilliant Nail Gloss. To learn more visit facebook.com/covergirl, and follow the brand on Twitter at twitter.com/covergirl. About P&G Beauty & Grooming P&G Beauty & Grooming products help make beauty dreams real for women and help men look, feel and be their best everyday. With 8 billion dollar brands and products available in nearly 130 countries, P&G's beauty and grooming products delivered sales of over $27 billion in fiscal year 2009/10, making it one of the world's largest beauty and grooming companies. P&G Beauty & Grooming offers trusted brands with leading technology to meet the full complement of beauty and grooming needs, including Pantene®, Olay®, Head & Shoulders®, Max Factor®, Cover Girl®, DDF®, Frederic Fekkai®, Wellaflex®, Rejoice®, Sebastian Professional®, Herbal Essences®, Koleston®, Clairol Professional®, Nice 'n Easy®, Venus®, Gillette®, SK-II®, Wella Professionals®, Braun® and a leading Prestige Fragrance division that spans from point of market entry consumers to high end luxury with global brands such as Hugo Boss®, Lacoste®, and Christina Aguilera®. Please visit http://www.pg.com for the latest news and in-depth information about P&G (NYSE: PG) and its brands. Photos/Multimedia Gallery Available: http://www.businesswire.com/cgi-bin/mmg.cgi?eid=50469698&lang=en MSL CanadaAlyssa Haw, email@example.com John, firstname.lastname@example.com


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I am interested in learning about adopting from the Czech Republic. From the research I have done so far, it looks like it would be a long process, maybe 4-5 years. But reading about the Romani children there has pulled at my heartstrings and I want to learn more. If anyone can tell me about adopting from there, adoption agencies that work with American families who want to adopt there, or other info- please share here or PM me.
There is a woman on the transracial board of adoption.com who has adopted Romani children and she talks about it a lot. You may want to check in over there.
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Sydney Spirit
|Sydney Spirit
|West Sydney Razorbacks
Sydney Spirit
|State Sports Centre
|Sydney, New South Wales
The Sydney Spirit (formerly the West Sydney Razorbacks) were an Australian professional basketball team competing in the Australasian National Basketball League. The club was based in Sydney, New South Wales, Australia.
They were the second team to represent Sydney's west in the NBL, several years after the Fosters (West Sydney) Westars were largely absorbed in a merger with the Sydney Supersonics to form the Sydney Kings. After the demise of the Sydney Kings in 2008, the Razorbacks rebranded themselves as the Sydney Spirit to appeal to the greater Sydney community, as they were the sole remaining Sydney-based NBL team prior to the rebirth of the Kings in 2010. The Spirit folded in 2009.
As the West Sydney Razorbacks[edit]
The Razorbacks were formed in 1992 as the West Sydney Slammers in the Continental Basketball Association,[1] and were granted a National Basketball League licence in 1997. The Razorbacks, played their first game in the NBL on 10 October 1998 defeating the Kings 103-97. They qualified for the NBL play-offs in their second season - 1999–2000. Since season 2000–01 the Razorbacks' home venue has been the 5,006 seat State Sports Centre in Homebush. Prior to that their home was the Whitlam Centre in Liverpool.
The Razorbacks made history in season 2001–02 becoming the first Sydney team in the NBL's 24 year history to contest the Grand Final series. The Adelaide 36ers went on to win the championship with a 2-1 result in the best-of-three series.
After breaking club records for most wins in a season, most wins on the road and most wins at home in 2003-04, the Razorbacks made it to their second Grand Final series, this time against the Sydney Kings. In the new best-of-five format, the Razorbacks, who at one stage were up 2-1 in the series, lost 2-3.
During the 2003–04 season, legendary NBL forward, Bruce Bolden, became the first Razorback to have his singlet (#32) retired. In 2005-06 he was joined by #45 Simon Dwight - the NBL's all time top shot-blocker.
After being coached for their first six seasons by Gordie McLeod, a new coach was appointed in 2004–05 – Mark Watkins who previously had been the team's assistant coach.
The club was headed up by basketball Olympian, Robbie Cadee, who launched the franchise in 1998–99. The Razors have had two shocking years in a row. The first of these years started off with Dwight's retirement and promising youngster Steve Markovic walking out on the club to play in Europe just a few weeks before the start of season. The second of the spell started off with import Nick Horvath sustaining a knee injury in the first game (he was out for season).
Cadee axed Watkins and hired Cal Bruton as coach in late 2006 amid a long Razorbacks losing streak. During the 2006-07 season the Razorbacks finally broke a 20 game losing streak against the Wollongong Hawks. Six games after that, the Razorbacks thumped the Kings 106-75 at the Pig Pen, spoiling Brian Goorjian's record of games coached.
In recent years the Razorbacks have been in severe financial difficulty after the original team owner, Canterbury Leagues Club, withdrew its support. Tri Media Group later took over the running of the club, which had almost folded. As a result of their financial troubles, the Razorbacks squad for season 2007–08 was composed mainly of young NBL rookies, headed by first time NBL coach Rob Beveridge.
As the Sydney Spirit[edit]
Shortly after the Kings left the competition, the Razorbacks changed their name to the Sydney Spirit, in an attempt to attract former Kings supporters to the only Sydney team left in the competition. This proved to be a failure as the Spirit could only attract a crowd of 1,500 fans to the opening match of the Spirit's season, and only two months into the 2008–09 NBL season the club's owner, Greg Evans, informed the league's executive that he intended to put the club into administration shortly, resulting in the club's immediate closure.[2] The league stepped in with a rescue package to allow the Spirit to complete the remainder of the season, with the players and coaching staff agreeing to accept the league's minimum salary.[3] The club also established a special financial account to allow interested parties to make contributions to supplement the league's rescue package; the first contribution was A$30,000 by Australian NBA player Andrew Bogut.
Honour roll[edit]
|NBL Championships:
|NBL Finals appearances:
|NBL Grand Final appearances:
|2 (2002, 2004)
|All-NBL First Team:
|John Rillie (2004), Sam Mackinnon (2004)
|NBL Rookie of the Year:
|Steven Markovic (2004)
|NBL Best Defensive Player:
|Simon Dwight (2002)
|NBL Best Sixth Man:
|Bruce Bolden (1999)
|NBL Most Improved Player:
|Liam Rush (2007), Matthew Knight (2009)
Source: www.NBL.com.au
- ^ "From Slammers to Pigs, now they're shooting to be kings". Sydney Morning Herald. 2004. Retrieved 2008-08-22.
- ^ "Statement on Sydney Spirit". www.nbl.com. 2008-11-25. Retrieved 2008-11-26.[dead link]
- ^ "Spirit lives on". Sydney Spirit. 2008-12-03. Retrieved 2009-01-01.


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Genuine Whirlpool part. Whirlpool brands include Whirlpool, Maytag, KitchenAid, Jenn-Air, Amana, Magic Chef, Admiral, Norge, Roper, and others.
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Part Number 67004659 (AP4079487) replaces 1070837, AH2068503, EA2068503, PS2068503.
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We want to ensure that making a return is as easy and hassle-free as possible! If for any reason you decide that your purchase just isn't for you, simply return the item within 30 days of receipt and we'll cover the cost of return shipping. Learn more about our Return Policy.
Puritan Medical PurSwab Foam-Tipped Applicators, Puritan Medical Products 100% Foam Tip, Wide is a reliable and dependable addition to the Puritan Medical Laboratory Consumables family of products. Combining top-notch and uncompromising quality with an affordable price, the Puritan Medical PurSwab Foam-Tipped Applicators, Puritan Medical Products 100% Foam Tip, Wide 1805-PF-RECT/ 10806-032 can fulfill your laboratory needs while still offering a great value for the money.
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These 100 PPI polyurethane shred-resistant swabs are lint-free, nonabrasive, and sized for precision cleaning with solvents such as alcohol or acetone. Select swabs with applicator tips of 100% polyurethane foam or of polyurethane foam over an inner core of USP-grade cotton. Wide style swabs are flat with slightly squared 1.6cm (5/8") wide tips. All tips are securely bonded to the polypropylene shaft.
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We offer only genuine Puritan Medical products, authorized for sale in the U.S. and other markets, so you can be sure that you are buying authentic Laboratory Consumables with a full manufacturer's warranty in addition to our own 100% Satisfaction Guarantee. We are your premier online distributor for Puritan Medical Laboratory Consumables. We are an authorized supplier to all federal and state government agencies, as well as to research and educational institutions, and can facilitate volume orders on many types of laboratory equipment, lab consumables, and other products to help fulfill your specific needs. We offer Free Shipping and FREE USPS shipping to APO and FPO addresses on all orders in our store over $49. Please refer to our APO/FPO Shipping and Export Restrictions for more information on export to APO/FPO addresses.
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Her mousy blond hair swayed as she walked. He looked at her and thought to himself, 'How Lucky am I?' She turned around to him and said "Are you coming or what?". He didn't need to be told twice, as he chased after her. It was Phoebe and Ricks th
Kate's hidden lust
Kate and I had known each other for about a year before deciding to get married. We were both in our early twenties and up for most kinds of fun.
Kate was very good looking blonde with a slim, toned curvy figure. Her firm 34d breasts and smoot
Attraction – The Parking Lot
Our moaning intensified as we kissed, bit and groped each other in the parking lot. The darkness of night, the cold wind and his deep voice added to my excitement. I hoped someone was watching us, the thrill of possibly being seen, of others gettin
The Candy Drop Kisses: Candace and Tiffany's Story
Plain and simple, this was a dream I had a couple weeks ago. Leave comments to let me know how good, bad, horrible it is.
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One night in Germany
I had to go to a business trip to Germany. You know one of those trips where everybody who does not go on business trips are dead jealous, whilst those who do go on these type of trips feel sorry for you. Although hard for a bloke like me I can only
My HEB Girl
Stephanie was the Assistant Manager at the local HEB. She was one of the most beautiful women I had ever seen. I was always trying to not stare. I liked her hair, natural blonde with some black highlights, very cute. Her hair made her gorgeous bl
A Time of Many First
A Time of Many First
Part 1- The Wake Up
It was early. I hate being woken up early unless it is in a special way. Lucky for me this time is indeed very special. Groaning I open my eyes to see the sheets bobbing up and down. As a pull ba
cum corridor
Just a quick blog/fantasy to start a blog here.
This is a fantasy
While leaving the restaurant late the other night, one of waitresses asked me to leave through the kitchen. While walking to the exit, she stopped me and reached into my
For Love of the Game
Alright, just walk down there and do it already. What's the worst that can happen, she tells you to leave? It's your house. Actually… that sounds awful. Okay just do it, one, two, three, now! You didn't move. Damn it I'm a pussy! Okay… GO
catching my wife
it all happened when i was sent out of town for the company i work for.i was supposed to be gone for three days. but plans fell though. and i got back in town early.when i got to the house.ther was no one there.there was a message on the machine.i li
We were both acutely aware of the fact that we should not be here, but it felt so right. We were finally going to have one of those hot & steamy fuck sessions that we talked about on the phone. We meet in Coney Island and head down to the shoreline.
Getting caught in the Shed
This true story happen when I was 19 years old attending my first year of junior college and just started dating my future husband. I come from a small Texas town where everybody knows everybody and my father is pretty well known. Let me describe m
The Work Place (10)
The Work Place
Chapter Ten
Luke Masters was sitting at his desk when his inner warning bell rang, high-pitched and rapid. As the ringing in his head continued, he felt a tingling down below. He sighed and looked down to find his cock sticki
A Day at the Nursery…
A DAY AT THE NURSERY…a true story, even though it sounds like a tale..!!
how well i remember that day….that day at the nursery.
it was a beautiful saturday and i left early to go to my local landscape center to purchase some more
A Day at the Nursery..
A DAY AT THE NURSERY…a true story, even though it sounds like a tale..!!
how well i remember that day….that day at the nursery.
it was a beautiful saturday and i left early to go to my local landscape center to purchase some more
Watching The Daughter
Juliet was on her way home from another long day. She had to get the kids and her husband out of bed since everyone seemed to forget to set the alarms again. Her husband was the first to leave without eating breakfast but that was something she was u
Her best friend 2
We admit, it is a bit sick to have a thing for your best friend. And yet both of you still be in a relationship, but to go through every day wanting the other so bad it hurts. You want both of them. Your heart aches for them.
But life isn't fa
Jacob's Seduction Pt 2
Jacob followed Mandy inside of her home like an obedient dog. He stared at her heart-shaped ass as she walked, it swayed left and right and the only thing he could think about was screwing her brains out. Her home was a two-story house in the suburbs
The BBW's twin joins us for the rest of the night
Standing there in front of me was another woman who looked like another version of Chloe. Except she was not quite as big. About twenty or thirty pounds lighter, long blonde hair in a pony tail, and an eyebrow ring. Other than that it was Chloe


text:
Peter Grant has accepted his departure from Norwich was the right move for all concerned after his 12-month tenure ended on Tuesday.
The 42-year-old agreed to leave by mutual consent after Monday's 1-0 defeat at bottom club QPR left the Canaries in the relegation zone with just eight points from their opening 10 matches.
"It was a very, very difficult decision for us to make but it was right for myself and for the football club to go forward," Grant told the club's official website.
"I have had fantastic support from the board from minute one right up until the time of leaving but we are in a results-driven business and I don't like being second best at anything."
"Unfortunately for me I feel that the balls haven't bounced the right way for me over the last 12 months," he added.
"But the most important things for me have always been the supporters and the club itself.
"They are the two constants while managers and players come and go.
"For the first time in 27 years, I've got a day off away from football. But it's the best job in the world - and I want to be back in it by tonight.
"There's no better feeling in the world than getting up of a morning and going on to a football pitch and I hope to be back as soon as possible."
Grant has lost no self-belief during his chastening experience.
"My results weren't good enough - and I accept that," he said.
"But I believe I have the abilities to be a good manager and hope that I can learn from the experience of this year."


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Columbia attractions are listed below. Find things to do in or near Columbia, MO for your upcoming individual or group travel for Columbia visitors. We also offer the great discounts on Columbia Missouri hotel and motel rooms.
Reserving group travel? Save with Columbia Group Rates or Get hotel bids for your Columbia Meeting
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Virgin Gorda 2007
by Wes Kennedy
Trek across the hidden beaches and giant granite maze known as the Baths and marvel at the panoramic vistas
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poetry 1998 - 2001
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A group of bouncers from Jay-Z's 40/40 Club in Atlantic City have been fired.
This follows news that security for the club beat and kicked patrons Tyrell Durant and Leonard Clark on November 28. Video of the incident was posted online.
Couri Glen, who was the security supervisor, told TMZ.com that he and the other bouncers were "just doing [their] jobs" by removing the men from the club. He claims the two men were actually grabbing and touching women inappropriately on the night in question.
The bouncers have since been fired for misconduct, and police are mulling whether to press assault charges against them.
— Sonya Eskridge
Here's more:
Jay-Z to 'Decode' rhymes in book
Jay-Z's 40th birthday bash


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Thomas Robinson is 'pissed off'
Oct 4, 2013, 11:15 AM EDT
Thomas Robinson, when the season begins, will be playing for his third team in two years. He was drafted by the Kings, traded mid-season to the Rockets and traded again in the offseason to the Trail Blazers.
The last top-five pick to play for so many teams in his first two years was drafted in 1997, and Robinson isn't happy he's experienced so much more upheaval than other recent top picks.
Robinson, via Chris Haynes of CSN Northwest:
"I'm just pissed off," he told CSNNW.com.
"I feel disrespected," Robinson went on to say. "A lot of people don't know the ins and outs of this business. That's how it works, man. There's no point in even talking about it. That's the way things work. I'm just ready to play."
"I think last year was the first time thinking I was the one being hunted, as far as people coming for me or feeling worried. That was too much pressure," Robinson said. "I rather be the one chasing somebody down."
That last top-five pick to play for three teams in his first two years was Chauncey Billups, who went on to a pretty remarkable career that included an NBA Finals MVP. After being traded from the Celtics to the Raptors to the Nuggets, Billups kept working on improving his game. He wasn't stubborn about remaining a scoring guard and refined his passing skills. Most importantly, he was driven to get better.
It sounds like Robinson is emulating that drive. The question is whether he changes his game. Robinson doesn't have the size to bang inside like he did at Kansas, but he can still be an effective rebounder and hustle player. The key step is choosing to fill that role, and that very well could determine whether Robinson sticks in Portland.
- cmehustle - Oct 4, 2013 at 5:13 PM
I really wonder why so many teams gave up on this guy so quick? I thought he was excellent a KU, and would go on to a very productive career. Lets hope it works out in Portland.
- whereyaat - Oct 4, 2013 at 9:08 PM
I think the Kings dumped him because they wanted Patterson, and the Rockets legitimately wanted Robinson. But then when Dwight came around, the Rockets had to dump players/salary fast, and they had some limitations with who they could dump. So basically, they just dumped him in the effort to get Dwight.
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Andie Graph is an Android port of the AlmostTI emulator by Marat Fayzullin - http://fms.komkon.org/
*** ROM not included. Why? http://dgmltn.com/roms ***
The TI-8X series of calculators work great as standalone calculators, showing 8 lines of history, using standard mathematical notation. Each model was designed with more specific purposes in mind:
The TI-82 is designed for algebra and trigonometry. Graph and compare functions, perform variable substitution, and solve single variable equations.
The TI-83 is designed for statistics and trigonometry. Graph and compare functions, and perform data plotting and analysis.
The TI-85 is great for engineering and computer science. Graph and compare functions, perform bit manipulation and convert between numeric bases.
The TI-86 is great for engineering and calculus. Graph and compare functions, perform bit manipulation and convert between numeric bases. Solve differential equations, plot vector fields.
All models allow you to write programs in TI-BASIC and perform basic scientific calculator operations.
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What's New
Wabbitemu, like all emulators, requires a ROM image. Wabbitemu includes an easy to use setup wizard that will help you create a ROM image for your TI.
Frequently Asked Questions:
1. How do I bring up the menu?
You can bring up the menu by swiping from the left edge of the screen, or pressing the menu key (if your device has one).
2. How do I disable vibration when I tap on a button?
Open the menu and click on settings. Under the 'Skin' category unselect 'Vibrate on keypress'
3. How do I load apps like PlySmlt2 or Inequalz?
To load any file to your calculator you need to first download it to your SD card or internal storage on your device. You can find the official TI applications for the 84 Plus (non color) available here: http://education.ti.com/en/us/software/search/ti-84-plus-family-ti-83-plus-family#view=math-apps. Once you have downloaded the app to your device open Wabbitemu. Swipe from the left edeg of the screen to bring up the menu, and select 'Load file'. Find the file you just downloaded in the list and select it. The file will be sent to your calculator. Apps for the 83+ and 84+ calcs will end in *.8xk. Apps for the 84+CSE will end in *.8ck or *.8xk.
4. I'm missing fraction options when I press Alpha+F1! How do I get these fraction options?
Fraction options (and the rest of the Alpha+FN menus) are only available on OS 2.55 MP. Wabbitemu allows you to download both 2.43 and 2.55 MP. You can check your OS version by hitting '2nd' then '+'. Then press enter to go to the about screen. If it not 2.55 MP or 2.53 MP you will need to upgrade.
To upgrade you will need to recreate your ROM by going through the setup wizard again. The setup wizard is runnable if you bring up the menu by swiping from the left edge of the screen. Then select 'Re-run setup wizard'. As you go through the setup make sure you have selected OS 2.55 MP on the OS selection screen.
More documentation can be found on the official website, http://wabbit.codeplex.com
Graph 89 Free
The ROM image is not included. You need to extract this from you own calculator. Make sure you have the ROM ready before downloading this emulator.
Graphing Calculator TI-84 Man.
• This manual shows all important functions of the graphing calculator TI 84 Plus, which are very useful for high school (also suited for TI 83 Plus and TI 84 Plus C).
• The app shows the exact key press sequence for every topic!
• You can swipe through screenshots of the original graphing calculator which show every step you have to do.
• There are helpful descriptions in the app which give some additional info.
• More than 60 math problems plus quiz to train your skills!
Not yet convinced? Here's the detailed description:
•Every topic shows a detailed instruction, through Screenshots of the original graphing calculator TI-84 Plus, how to solve the exercise. You only have to swipe to get the next Screenshot which looks similar with your calculator!
•To "become warm" you have the possibility to insert a sample function in your calculator which is used in the App. The App shows an example for every topic. After that, press the first key of the respective key press sequence on the graphing calculator. Then swipe once again to get the next Screenshot. These will look like the display of your calculator. The App contains for each step a picture, so it's easier to solve the exercise.
•The app is also useful for preparing your examination, because you often don't remember all instructions of the TI 84 Plus.
•Your teacher will be grateful as well, because he doesn't have to walk around the classroom until everyone's calculator shows the right solution. You only have to take a look at the app and know how to do!
•The app contains the following topics:
Differential Calculus, Integral Calculus, Solve Equations, Matrices, Tips and Tricks,
The TI 84 Plus is a trademark of Texas Instruments.
All trademarks are the property of their respective owners.
Graphing Calculator by Mathlab
Furthermore, Graphing Calculator by Mathlab displays calculations as it performs them on the high-quality display of the Android device, making it easier for the user to understand the calculations and see them clearly. This app has two great strengths. First, it acts as a fine scientific calculator, but, more than that, it displays the intermediate steps of the calculations as you type. It allows the students to both watch and learn how the calculations are made and how to find the final answer. Second, the graphing ability is absolutely stunning! Not only does the calculator beautifully display the graphs, but it automatically generates the x- and y- values and displays them as well.
Free version requires internet connection and contains ads! Upgrade to PRO!
Help site with instructions and examples: http://help.mathlab.us
If you have a question, send email to firstname.lastname@example.com
* Arithmetic expressions +,-,*,/,÷
* Square root, cube and higher roots (hold '√' key)
* Exponent, logarithms (ln, log)
* Trigonometric functions sin π/2, cos 30°, ...
* Hyperbolic functions sinh, cosh, tanh, ... (hold "e" key to switch)
* Inverse functions (hold direct function key)
* Complex numbers, all functions support complex arguments
* Derivatives sin x' = cos x, ... (hold x^n key)
* Scientific notation (enable in menu)
* Percent mode
* Save/load history
* Multiple functions graphing
* Implicit functions up to 2nd degree (ellipse 2x^2+3y^2=1, etc.)
* Polar graphs (r=cos2θ)
* Parametric functions, enter each on new line (x=cos t, y=sin t)
* Function roots and critical points on a graph. Select the checkbox on the left from the function to show their coordinates on the graph, click the graph-button on the upper menu to display their coordinates as a list.
* Graph intersections (x^2=x+1)
* Tracing function values and slopes
* Scrollable and resizable graphs
* Pinch to zoom
* Fullscreen graphs in landscape orientation
* Function tables
* Save graphs as images
* Save tables as csv
* Simple and complex fractions 1/2 + 1/3 = 5/6
* Mixed numbers, use space to enter values 3 1/2
* Linear equations x+1=2 -> x=1
* Quadratic equations x^2-1=0 -> x=-1,1
* Approximate roots of higher polynomials
* Systems of linear equations, write one equation per line, x1+x2=1, x1-x2=2
* Polynomial long division
* Polynomial expansion, factoring
* Matrix and vector operations
* Dot product (hold *), cross product
* Determinant, inverse, norm, transpose, trace
* User defined constants and functions (PRO)
* Save/load expressions
Free Graphing Calculator
Does far more than most of the paid calculators out there . . . let alone the free ones.
1) Scientific Calculator. Simple to grasp and easy to use, but powerful features are available when you need them. Available functions include the following:
• the usual arithmetic functions and exponentiation.
• square root, cube root, nth root, natural log, log base 10, log of arbitrary base, absolute value, factorial, permutations (nPr), combinations (nCr), modulus, random integer, bell curve, cumulative normal distribution, decimal to fraction.
2) Graphing. Capabilities:
• Graph up to four equations at once.
• Graphs are labeled.
• You can drag the graph or pinch to zoom in or out.
• Calculator can find roots and intersections.
• Graph in polar coordinates.
• Graph parametric equations
• Can graph implicit functions, such as x^2+y^2-4=0. Most calculator apps can't do this!
3) A unit converter. With a tap, you can enter the result of your conversion into the calculator. Currently converts different units of the following: acceleration, angle, area, density, distance, energy, force, mass, power, pressure, speed, temperature, time, and volume. Great for doing physics homework!
4) Constants for scientific calculations -- speed of light, strength of gravity at Earth's surface, etc. etc. etc. Tapping on a constant will insert it into your calculation -- i.e, you don't have to key in the value. Again, great for doing physics homework!
5) It can make a table of the values of a function you enter. You can choose the starting x value of the table, as well as how much x increases for each successive row.
6) Help screens linked directly to many of the available functions and constants. Tap the disclosure arrow to see the definition.
7) Forgot the quadratic formula? Or the double-angle formulas for sine and cosine? The math/science reference hits the high points of various subjects. Currently includes algebra, differential and integral calculus, geometry, trigonometry, vectors, vector calculus, and classical mechanics.
8) Statistics -- enter data and make a histogram, box and whisker plot, or scatter plot with optional regression line.
I'd love to hear your comments or suggestions. Because of spammers, my email address is not in this description, but it is inside the app, and it's great to hear from real users.
Desmos Graphing Calculator
To achieve this vision, we've started by building the next generation of the graphing calculator. Using our powerful and blazingly-fast math engine, the calculator can instantly plot any equation, from lines and parabolas up through derivatives and Fourier series. Sliders make it a breeze to demonstrate function transformations. It's intuitive, beautiful math. And best of all: it's completely free.
Graphing: Plot polar, cartesian, or parametric graphs. There's no limit to how many expressions you can graph at one time - and you don't even need to enter expressions in y= form!
Sliders: Adjust values interactively to build intuition, or animate any parameter to visualize its effect on the graph
Tables: Input and plot data, or create an input-output table for any function
Statistics: Find best-fit lines, parabolas, and more.
Zooming: Scale the axes independently or at the same time with the pinch of two fingers, or edit the window size manually to get the perfect window.
Points of Interest: Touch a curve to show maximums, minimums, and points of intersection. Tap the gray points of interest to see their coordinates. Hold and drag along a curve to see the coordinates change under your finger.
Scientific Calculator: Just type in any equation you want to solve and Desmos will show you the answer. It can handle square roots, logs, absolute value, and more.
Inequalities: Plot cartesian and polar inequalities.
Offline: No internet access required.
Visit www.desmos.com to learn more and to see the free online version of our calculator.
Note: the app doesn't yet support saving & sharing of graphs. If you need to save and share, we recommend visiting www.desmos.com on your device.
Graph 89 - (with TI84 support)
*Please remember to read the ROM section below before downloading this application!
It will turn your phone or tablet into an exact replica of your calculator. The emulator will provide the same functionality and generate the same results as your real calculator. Being ported to Android means that it will always fit in your pocket, have a backlight, be rechargeable and also run faster.
You would be able to install applications by copying the App file to the internal memory of your phone, pressing the 'Back' button and selecting 'Install Application/Send File'.
Graph89 would be great tool for math, science and engineering courses in high school, college and beyond. Some of these calculators feature Computer Algebra System (CAS) having the capability to simplify and symbolically solve mathematical expressions.
Graph89 combines two powerful emulation engines which make it the only app in the Android Play Store to support the full range of TI graphing calculators.
1) TiEmu - http://lpg.ticalc.org/prj_tiemu providing support for the Motorola 68K family: TI89, TI89 Titanium, TI92 Plus, Voyage 200
2) TilEm - http://lpg.ticalc.org/prj_tilem providing support for the Z80 family: TI 83, TI 83+, TI 83+ SE, TI 84+, TI 84+ SE
Emulators are computer software which simulate a specific hardware. In order for the emulator to do anything useful it needs some software to run. The software that runs in your calculator (ROM) is copyrighted by TI, and as such, it can not be distributed by Graph89 or any other emulator for that fact.
This means that you will have to provide the ROM file yourself by extracting it from your own calculator. Transfer it to your phone, and then tell Graph89 where to find it. To extract the ROM you can follow the instructions from http://www.ticalc.org/programming/emulators/romdump.html and by using TiLPII from http://sourceforge.net/projects/tilp/files Google and youtube are also great sources of tutorials and help.
Wabbitemu http://wabbit.codeplex.com/ is also a great tool for extracting the ROM from your TI83/TI84
Supported ROM files:
TI89, TI89 Titanium, TI92 Plus and Voyage 200:
*.rom, *.89u, *.v2u, *.9xu, *.tib
TI83 Plus, TI83 Plus SE, TI84 Plus and TI84 Plus SE:
*.rom, *.8Xu
Firmware updates (*.89u, 9xu, *.v2u, *.8Xu) which are normally used to restore the operating system of your calculator can also be used as a ROM image.
Needless to say, you will be very disappointed if you purchase Graph89 without having the ROM file ready. You will just see some instructions and a blank screen.
Graph89 needs permission to look at your Android Account in order to generate a unique ID shown under F1/About. This works only for TI89/V200. Note that there is no internet connection required for this App.
TiEmu, TilEm and Graph89 have been developed independently of Texas Instruments and are not affiliated with TI.
Texas Instruments and TI are trademarks of Texas Instruments Incorporated.
Revision History:
An 8Xu (firmware update) file can now be used as a ROM for TI84+, TI84+SE, TI83+ and TI83+SE
Added support for: TI-83, TI-83 Plus, TI-83 Plus SE, TI-84 Plus and TI-84 Plus Second Edition using the TilEm2.00 engine.
Bug fixes on 'State Save' and 'Out of Memory' errors on some older phones.
Backup Manager
Dot Matrix LCD simulation
Click Screen to Zoom
Reset RAM
Landscape mode for TI89, TI89T
TI92+ skin
Bug fixes
Emulates Voyage 200 and TI92 Plus
Multiple calculator instances
Take screenshots
Generate an ID under F1/About
Sync clock
Acoustic feedback on keypress
Automatic overclock
Grayscale support
Send group files (*.89g, *.tig)
Receive files, (var-link/F4/F3/send)
Performance improvements
Customizable LCD colors
AlmostTI - TI Calc Emulator
To run AlmostTI, you will need at least one calculator system ROM. AlmostTI does not include any calculator ROMs on its own, as they are intellectual property of Texas Instruments. You should place your own ROM, preferably dumped from the calculator you own, onto your internal storage. AlmostTI recognizes following ROM files (in upper or lower case):
For information on dumping a ROM from your own calculator, see here:
For general information about AlmostTI, see its homepage:
For Android-specific help, please see this web page:
And to discuss your problems with the author and other users, use this discussion group:
Graphing Calculator MathlabPRO
Furthermore, Graphing Calculator by Mathlab displays calculations as it performs them on the high-quality display of the Android device, making it easier for the user to understand the calculations and see them clearly. This app has two great strengths. First, it acts as a fine scientific calculator, but, more than that, it displays the intermediate steps of the calculations as you type. It allows the students to both watch and learn how the calculations are made and how to find the final answer. Second, the graphing ability is absolutely stunning! Not only does the calculator beautifully display the graphs, but it automatically generates the x- and y- values and displays them as well.
Help site with instructions and examples: http://help.mathlab.us
If you have a question, send email to firstname.lastname@example.com
* 3D graphs
* Save constants, functions and expressions in the library
* Internet is not required
* No advertisements
* Arithmetic expressions +, -, *, /, ÷
* Square root, cube and higher roots (hold '√' key)
* Exponent, logarithms (ln, log)
* Trigonometric functions sin π/2, cos 30°, ...
* Hyperbolic functions sinh, cosh, tanh, ... (hold "e" key to switch)
* Inverse functions (hold direct function key)
* Complex numbers, all functions support complex arguments
* Derivatives sin x' = cos x, ... (hold x^n key)
* Scientific notation (enable in menu)
* Percent mode
* Save/load history
* Multiple functions graphing
* Implicit functions up to 2nd degree (ellipse 2x^2+3y^2=1, etc.)
* Plar graphs (r=cos2θ)
* Parametric functions, enter each on new line (x=cos t, y=sin t)
* Function roots and critical points on a graph. Select the checkbox on the left from the function to show their coordinates on the graph, click the graph-button on the upper menu to display their coordinates as a list.
* Graph intersections (x^2=x+1)
* Tracing function values and slopes
* Dcrollable and resizable graphs
* Pinch to zoom
* Fullscreen graphs in landscape orientation
* Function tables
* Dave graphs as images
* Save tables as csv
* Simple and complex fractions 1/2 + 1/3 = 5/6
* Mixed numbers, use space to enter values 3 1/2
* Linear equations x+1=2 -> x=1
* Quadratic equations x^2-1=0 -> x=-1,1
* Approximate roots of higher polynomials
* Systems of linear equations, write one equation per line, x1+x2=1, x1-x2=2
* Polynomial long division
* Polynomial expansion, factoring
* Matrix and vector operations
* Dot product (hold *), cross product
* Determinant, inverse, norm, transpose, trace
* User defined constants and functions
* Save/load expressions
Simple Graph Drawer Full
The perfect tool for students, teachers and anyone involved in math:
- supports standard graphs in Cartesian coordinate system, parametric graphs, graphs in polar coordinates and parametric graphs in polar coordinates, point graphs;
- simple and easy to use intuitive interface;
- 46 mathematical functions, built-in constants;
- any number of graphs on one screen;
- save graphs as images with various options;
- work in real time, gesture support, two virtual keyboard to choose, examples, help section, history, settings and more.
ACT/SAT Math Booster (lite)
Link to FULL VERSION: https://market.android.com/details?id=com.actfull
Boost your math score on the ACT test and SAT test by programming your TI 83/84 with these totally allowed (see ACT/SAT rules and regs on their website) sneaky, super easy-to-program calculator programs. We give you step-by-step directions and show you how to use them on real ACT and SAT test questions.
MathsApp Scientific Calculator
Looking for a graphing calculator, matrix support, or simply want to support the cause of creating the greatest scientific calculator app on Android? Get MathsApp Graphing Calculator: https://play.google.com/store/apps/details?id=com.mathsapp.graphing
MathsApp Scientific Calculator includes:
-Landscape mode
-Easily adjust previous calculations or insert previous results
-Binary, octal and hexadecimal number support
-Scientific, engineering and regular number formatting
-Physical constants
-User-friendly interface
-No advertisements
-Advanced calculations
-Trigonometric functions
-Advanced statistical distribution functions
-List support
-Complex number support
-Percent support
Graph It!
The integration of functions is facilitated by self-completion of statements, also expressions of the interpreter is very flexible and allows the inclusion of any function: even the most complex are recognized and interpreted correctly.
The main functions recognized are: abs(x), acos(x), acosh(x), asin(x), asinh(x), atan(x), avg(x1,x2,...,xN), ceil(x), combin(x,y), cos(x), cosh(x), e(), exp(x), fact(x), floor(x), lg(x), ln(x), log(x,y), max(x1,x2,...,mN), min(x1,x2,...,xN), mod(x,y), pi(), pow(x,y), rand(), sign(x), sin(x), sinh(x), sqrt(x), sum(x1,x2,...,xN), tan(x), tanh(x), toDeg(x), toRad(x).
The graph allows an accurate view of the trend of function at any point in the plane. You can zoom and pan through the classical gestures.
There is also a tool which can calculate the value of the functions included, given the value of x (via a number or expression).
Beebdroid (BBC Micro emulator)
See that Android phone in your hand? That's not Intel inside - it's powered by an ARM processor. The 'A' in 'ARM' stood for Acorn, a visionary British company that produced the seminal BBC Micro, and thirty years later - coming full circle - your phone hosts its venerable ancestor today.
Have fun reliving classic games from the 1980s, hosted by BBC Micro preservation website stairwaytohell.com - any questions, please email firstname.lastname@example.com
Works at the full 50 frames per second on most recent powerful Android phones and tablets. This means anything significantly better than an HTC Desire or Nexus One (except, annoyingly, the brand new Galaxy Nexus, because it has the archaic GPU from 2010's Galaxy S pushing 240% of the pixels), or any Android 3.x or 4.x tablet. If you try to run this thing on something like a Wildfire or a ZTE Blade, please don't whinge to us that it runs like a dog - just go and buy a Galaxy S II already ;) It's an extremely faithful emulation, way moreso than other machines' emulators, but if you think you can make it quicker, get involved - it's open-source - code it!
Works with the specialised controls of the Sony Ericsson Xperia Play (but doesn't run at 50 frames per second).
This FREE and OPEN SOURCE application - the source code is published on Github at https://github.com/littlefluffytoys/Beebdroid - is based on B-Em for Linux by Tom Walker and licensed under GNU General Public License v2.0 as detailed at http://www.gnu.org/licenses/gpl-2.0.html - we encourage others to assist us in developing Beebdroid further. This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the license for more details.
Beebdroid does not ship with any third party programs such as games - these are accessed via third party websites which have hosted such programs for years. Should any licence holders object to third party websites providing such programs, please contact the third party websites in question. We are furthermore also happy on a best-effort basis to block the visibility of specific programs within Beebdroid upon written request from verified product licence holders. We are also able to offer an in-app payment solution for your programs in such circumstances - please contact us for details.
The rights to the BBC Micro ROMs are believed by the emulator community to have effectively fallen into the public domain, having lain unclaimed for many years - Acorn itself is of course long bankrupt, and the documentation for the rights is rumoured to have gone down in the World Trade Center on 9/11. If you believe that you have a claim to the ROMs that has never been disclosed to the emulator community, or indeed has never been proven under law, remember that this is a labour of love, a free and open source preservation effort, and so please talk to us in the first instance, cheers!
8085 Emulator
★★ Trainer Kit
The first ever 8085 Emulator on Mobile devices. Now Perform 8 bit μP operations on your ANDROID device. All logical, arithmetic and register direct, indirect operations are added.
✰ Simple and elegant design
★★ PRO In built default programs - basic programs that this 8085 emulator supports. One touch to execute these programs
✰ Drag down button to get last execution results easily
✰ Stack Memory from 8000 - 9FFF
★★ Assembler
✰ All new assembler of 8085 first time on android device
✰ Now use both trainer kit and assembler on your android device
★★ PRO Save instructions as file on your device and access them any time
✰ More default programs
The app is not intended for low end devices with slower processors, you might face a lot of lag.
More from developer
Display the list of states in a grid view, list view, or CoverFlow! Filter by only seen, only unseen, or both.
Have any feature requests? Ask!
Boxee Remote by supware.net
This branch adds GESTURE MODE, new icons, a new settings screen, and improved Now Playing screen.
TIP: use volume keys to adjust boxee volume.
TIP: to use keyboard (on this or any app), press and hold the menu button.
Instructions and code at http://boxeeremote.googlecode.com/
* One time morphs into the next as time ticks by.
* Background changes to match the sun's position where you live.
* Large, easy-to-read time, appropriate to any device.
* Subtle animations don't distract from work or sleep.
* Your coarse location is used to calculate the sunrise and sunset times.
* That's it.
This app is based on open source libraries:
* Android Number Morphing, by Davy Leggieri https://github.com/bydavy/android-number-morphing
* Solar Positioning library, by Klaus Brunner https://github.com/KlausBrunner/solarpositioning
UPnP Browser
Source code available here:


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Great for Dogs with Derma Issues
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Pros: Easy to use
Cons: none
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text:
Currently available vaccines against leptospirosis are of low efficacy, have an unacceptable side-effect profile, do not induce long-term protection, and provide no cross-protection against the different serovars of pathogenic leptospira. The current major focus in leptospirosis research is to discover conserved protective antigens that may elicit longer-term protection against a broad range of Leptospira. There is a need to screen vaccine candidate genes in the genome of Leptospira interrogans.
Bioinformatics, comparative genomic hybridization (CGH) analysis and transcriptional analysis were used to identify vaccine candidates in the genome of L. interrogans serovar Lai strain #56601. Of a total of 4727 open reading frames (ORFs), 616 genes were predicted to encode surface-exposed proteins by P-CLASSIFIER combined with signal peptide prediction, α-helix transmembrane topology prediction, integral β-barrel outer membrane protein and lipoprotein prediction, as well as by retaining the genes shared by the two sequenced L. interrogans genomes and by subtracting genes with human homologues. A DNA microarray of L. interrogans strain #56601 was constructed for CGH analysis and transcriptome analysis in vitro. Three hundred and seven differential genes were identified in ten pathogenic serovars by CGH; 1427 genes had high transcriptional levels (Cy3 signal ≥ 342 and Cy5 signal ≥ 363.5, respectively). There were 565 genes in the intersection between the set encoding surface-exposed proteins and the set of 307 differential genes. The number of genes in the intersection between this set of 565 and the set of 1427 highly transcriptionally active genes was 226. These 226 genes were thus identified as putative vaccine candidates. The proteins encoded by these genes are not only potentially surface-exposed in the bacterium, but also conserved in two sequenced L. interrogans. Moreover, these genes are conserved among ten epidemic serovars in China and have high transcriptional levels in vitro.
Of the 4727 ORFs in the genome of L. interrogans, 226 genes were identified as vaccine candidates by bioinformatics, CGH and transcriptional analysis on the basis of the theory of reverse vaccinology. The proteins encoded by these genes might be useful as vaccine candidates as well as for diagnosis of leptospirosis.
Leptospirosis is a globally important zoonotic disease caused by pathogenic Leptospira species[1]. Leptospires are thin, helically coiled, motile bacteria, classified into 17 genomospecies (including the saprophyte Leptospira biflexa and the pathogen Leptospira interrogans) on the basis of DNA-DNA hybridization studies, or serologically classified into more than two hundred pathogenic serovars on the basis of structural heterogeneity in the carbohydrate component of the lipopolysaccharide[2,3]. Currently available vaccines, based on inactivated whole bacteria or membrane preparations from pathogenic leptospires, are of low efficacy, have an unacceptable side-effect profile, require annual booster immunizations and do not confer cross-protective immunity against different serovars [4-6]. Because of these concerns, the current major focus in leptospirosis research is to discover cross-species-conserved or cross-serovar-conserved protective antigens that may elicit longer-term protection against a broad range of Leptospira[5,7]. New vaccine development strategies are thus needed for preventing this zoonosis. Reverse vaccinology, which based on the genomic approach, has been applied to some bacteria, and novel vaccine candidate sequences have been identified [8-11]. The genome projects of two Leptospira strains give us intensive knowledge on the whole genome level [12-14]. Although many efforts have been made to identify the surface-exposed proteins of leptospires, finding perfect vaccine candidate antigens that provide cross-protection against different serovars of pathogenic L. interrogans still requires further work[7,15-17].
In our current study, we identified 226 potential candidate vaccine genes against L. interrogans using in silico analysis, comparative genomic hybridization (CGH) and transcriptional analysis, based on a genome-wide DNA microarray comprising 3528 open reading frames (ORFs) derived from the original annotation of L. interrogans strain #56601. These candidate genes not only encode surface-exposed proteins of L. interrogans strain #56601, but also have high transcription levels in vitro. Moreover, the proteins encoded by these genes are conserved in two sequenced L. interrogans and ten epidemic pathogenic serovars in China.
In silico analysis for identification of genes encoding surface-exposed proteins
In 4727 ORFs of L. interrogans strain #56601, 1282 proteins were predicted to be surface-exposed using P-CLASSIFIER, 654 proteins had signal peptides, 813 were predicted to have no more than four α-helices with transmembrane topology, 96 were predicted to have β-barrel topology implying that they are integral β-barrel outer membrane proteins, and 158 were predicted have a lipoprotein signal peptide using SpLiP. The number of genes in the intersection between the set of surface-exposed proteins identified by P-CLASSIFIER and the set of proteins characterized by at least one of the four characteristic topologies is 688. We calculated the similarity of proteins between serovar Lai and serovar Copenhageni as well as between serovar Lai and human (cut-off value: similarity >70% and E value = 1e-10 for two serovars, E value = 1e-10 for serovar Lai and human) using BLASTP. We found 3672 orthologs between the two serovars, and 605 proteins that are similar in serovar Lai and human. Finally, 616 genes were yielded by the bioinformatics study by retaining the orthologs between the two serovars and subtracting the genes that were similar in serovar Lai and human.
Comparative genomic hybridization
We prepared a gene chip microarray corresponding to the complete genome sequence of L. interrogans strain #56601. The chips were hybridized to labelled total DNA extracted from strain Fiocruz L1–130 and ten pathogenic serovars. On the basis of test hybridizations of strain Fiocruz L1–130 vs. the reference sample, we considered genes that gave hybridization ratios between 1.0 and 3.0 to be present in both strains and greater than 10.0 to be absent from the test strain. Ambiguous values between 3.0 and 10.0 may have been due to highly divergent genes or hybridization to paralogous genes. The CGH results revealed that 307 genes of L. interrogans strain #56601 were absent or highly divergent in at least one strain tested. After subtracting these 307 differential genes, we were left with 565 genes, which not only encode presumably surface-exposed proteins but also are conserved in the ten pathogenic serovars.
Transcriptome analysis
Microarray analysis of the mRNA extracted from in vitro grown leptospires revealed that the fluorescence signals of Cy3 and Cy5 ranged from 10.5 to 51,707 (see Figure 1); 1427 genes were expressed above the median level (Cy3 signal ≥ 342 and Cy5 signal ≥ 363.5) in the microarray and therefore as genes with high transcriptional levels. The intersection between the sets of 565 and 1427 genes contained 226 genes. Among them, 8.0% (18/226) were located extracellularly, 53.1% (120/226) in the outer membrane, 16.4% (37/226) in the periplasmic space and 22.6% (51/226) in the inner membrane according to predictions. These vaccine candidates were classified further according their gene names and clusters of orthologous groups (COGs) [18,19](Table 1, 2, 3, 4); 60.6% (137/226) of the candidates had COG annotations.
Figure 1. Identification of highly expressed genes in L. interrogans by microarray. Bacteria were grown in EMJH medium at 37°C and were collected when the culture reached mid-exponential-phase. RNA was purified and labelled with either Cy3 or Cy5 and hybridized with the microarray of L. interrogans strain #56601 (3528 genes). Transcription analysis revealed that 1427 genes were highly expressed (cy3 signal ≥ 342 and cy5 signal ≥ 363.5).
Table 1. The result of vaccine candidates according to localization sites: extracellular
Table 2. The result of vaccine candidates according to localization sites: outermembrane
Table 3. The result of vaccine candidates according to localization sites: periplasmic
Table 4. The result of vaccine candidates according to localization sites: innermembrane
Vaccines composed of whole cells or outer membrane envelope are available in some countries to prevent human leptospirosis, and clinical trials have been reported [20-23]. In view of their disadvantages, especially their inability to elicit longer-term protection against different serovars of pathogenic leptospires, efforts have been focused on developing subunit vaccines[24]. During recent years, Hap1[25] (also known as LipL32[26]), LipL41, OmpL1[27] and Lig[28,29] proteins have been identified as promising vaccine candidates for preclinical trials.
The availability of complete genome sequence information for many pathogens and the development of sophisticated computer programs have led to a new paradigm in vaccine development. Now it is possible to screen potential vaccine candidate genes in a reverse manner starting from the genome. This reverse vaccinology was first applied to MenB[30] and is now applied routinely in vaccine development, as in the search for vaccines against S. pneumoniae, Streptococcus agalactiae, Staphylococcus aureus, Porphyromonas gingivalis, Chlamydia pneumoniae and other microorganisms[10]. Bioinformatics analysis is the first important strategy of reverse vaccinology. Gram-negative bacteria have five subcellular location sites: cytoplasm, inner membrane, outer membrane, periplasm and extracellular space. The surface-exposed proteins, i.e. those located in sites other than the cytoplasm, are the most suitable vaccine candidates because they are more susceptible to antibody recognition and can therefore elicit protective immune responses. Many sophisticated computer programs have been developed to predict the subcellular locations of putative proteins in the whole genome [31-33]. Analyzing the gene transcription profile using DNA microarrays provides a second vaccine candidate selection strategy in reverse vaccinology. A gene having a fluorescent signal above the median value corresponds to an expression level higher than 5–10 mRNA copies per genome[34]. Those highly expressed genes could be potential vaccine candidates[34]. Finally, other approaches such as proteomic technology can be used to screen vaccine candidates. Using combined these strategies, genes encoding potential vaccine antigens can eventually be identified.
In our preliminary selection, all genes in L. interrogans strain #56601 were searched using P-CLASSIFIER, a system for predicting the subcellular locations of proteins on the basis of amino acid subalphabets and a combination of multiple support vector machines[33]. Moreover, four topologies were predicted by the corresponding programs. Proteins predicted to be surface-exposed and having any of these four topologies were screened as preliminary vaccine candidates. All proteins with more than four predicted transmembrane spanning regions were removed from the list of candidates, not only because they are likely to be completely embedded in the cell membrane and therefore inaccessible to antibodies, but also because they are difficult to express in E. coli[34]. We retained the genes shared by the two sequenced serovars and subtracted genes that had human homologues. The reason we subtracted human homologues is they are likely to cause problems of autoimmunity[35]. Finally, we narrowed the list of vaccine candidates to 616 genes in the genome of L. interrogans strain #56601.
In order to explore vaccine candidates that could generate cross-protection against the diverse serovars of leptospires, we applied CGH to identify genes that are conserved among the ten pathogenic strains involved in most infections[36]. This approach allowed us to refine the vaccine candidate shortlist further by eliminating antigens that were not conserved among these serovars. The 565 vaccine candidates not only presumably surface-exposed but also conserved among the ten prevalent serovars in China were identified as the result of this approach.
Transcriptome analysis was performed using DNA microarrays of L. interrogans in order to assess the transcription levels of all genes in the genome. A graph of the signal obtained for each gene gave a diagonal distribution reflecting the expression level of that gene. After subtracting genes with transcriptional levels below the median, we were left with 226 genes as vaccine candidates.
Applying the theory of reverse vaccinology, 226 genes had been identified as potential vaccine candidates against L. interrogans combined bioinformatics, CGH and transcriptional analysis. Among them, 60.6% (137/226) have COG annotations; thus, nearly 40% either have an unknown function or have no COG annotation. This group of gene products offers great promise as it comprises a pool of previously unexploited vaccine targets. To evaluate our results, we compared our candidates with those identified by others. Gamberini et al. (2005) found approximately 20% potential surface proteins using in silico approach, and sixteen proteins were recognized by antibodies present in human sera[15]. However, only three of them (LA0222, LA2637 and LA2741) appear in our final set. This is not unexpected, since 206 genes encoding hypothetical or unknown proteins were selected from approximately 20% of the genome for cloning and expression. Nally et al. (2005) characterized 32 proteins in outer membrane vesicles of L. interrogans serovar Copenhageni by two-dimensional gel electrophoresis, including previously-described outer membrane proteins (OMPs); in addition, unknown, hypothetical and putative OMPs were also identified[17]. Interestingly, only two proteins (LA0222 and LA2637) are represented among the sixteen proteins found by Gamberini and co-workers. There is an overlap of eight genes between our result and that of Nally et al. (2005) (LA0222, LA0505, LA0616, LA1495, LA2024, LA2295, LA2637 and LA3091). The reasons responsible for the discrepancies among the results may be due to differing methodologies. Genomics, transcriptional profiling and proteomics have emerged in the post genomic era with potential to speed up the vaccine discovery research process. It should be pointed out that those methods have their respective advantages and limitations, and can be complementally utilized in the development of the novel vaccines. Genomics involves the use of various softwares to predict sublocalization of proteins. However, some algorithms have limited accuracy. Although transcriptome analysis uses gene chip array to measure gene expression but suffers from the fact that mRNA levels may not reflect protein levels. Expression of a transcribed gene may be regulated at the level of translation. It is believed that the proteome maps of microorganisms are important to understand cellular status at the protein level, which cannot be deciphered from genome or transcriptome analysis[37]. Proteomics of outer membrane can rapidly identify almost all proteins in outer membrane. However, some of the proteins identified in membrane preparations are in fact typical cytoplasmic proteins[10,38]. Moreover, one of the major disadvantages of subproteomic studies by 2-D gel electrophoresis and mass spectrometry is the potential for contamination via leaky fractionation or lysis[39]. Nally et al. (2005) also revealed that outer membrane vesicles contain small amounts of inner membrane or cytoplasmic proteins in their proteomic study[17]. It is worth mentioning here that mainly surface-exposed proteins such as LipL32 (LA2637)[26,40], LipL41 (LA0616)[27,40], LipL45 (LA2295)[41] and LipL21 (LA0011)[42] have higher transcriptional levels in our results; this suggests that the genes with higher transcriptional levels identified in our current research may be preferable for development as vaccine candidates.
This is the first time that CGH and transcription analysis have been used to identify potential candidates for vaccines against L. interrogans. Our present work corroborates previous studies, showing the advantages of reverse vaccinology[8,11]. The next step following our present research is to verify whether the selected vaccine candidates are surface-exposed and to evaluate the protective activities of these proteins. Such studies will lead to the development of safe and effective new vaccines against leptospirosis in the future.
We have performed high-throughput in silico and microarray-based processes that are useful for determining potential vaccine candidates against leptospirosis. In total, 226 genes were identified in the genome of L. interrogans serovar Lai type strain #56601 using bioinformatics, CGH and transcriptional analysis. The proteins encoded by these genes are not only potentially surface-exposed in the bacterium, but also conserved in two sequenced L. interrogans. Moreover, these genes are conserved among ten epidemic serovars in China and have high transcriptional levels in vitro. These proteins might therefore be useful for vaccine candidates as well as for the diagnosis of leptospirosis. Further research, including verification that these vaccine candidates are surface-exposed and evaluation their protective activities, will aid in the study of vaccines against leptospirosis in the future.
Bacteria strains and growth condition
Ten strains of L. interrogans were used in this study (Table 5). All the strains were obtained from the Institute for Infectious Disease Control and Prevention (IIDC), Beijing, China. Leptospires were maintained by serial passages in guinea pigs for preservation of virulence and were cultured in liquid Ellinghausen-McCullough-Johnson-Harris (EMJH) medium at 28°C or 37°C with shaking under aerobic conditions. Culture conditions were then developed to ensure that only mid-exponential-phase bacterial cultures at a mean density of 106/ml were used in further experimentation. The cells were harvested by centrifugation at 10,000 g for 10 min at 4°C.
Table 5. Bacterial strains used in the study
The L. interrogans serogroup Icterohaemorrhagiae serovar Lai type strain #56601 (strain Lai) was used to construct the DNA microarray. The genomic DNA of strain Fiocruz L1–130 was kindly provided by the Centro de Pesquisas Goncalo Moniz.
In silico analysis
Genes and protein data for human and for the sequenced L. interrogans (serovar Lai and serovar Copenhageni) were downloaded from NCBI. P-CLASSIFIER[33] was applied to predict the subcellular locations of proteins in L. interrogans strain #56601. Signal peptide prediction was carried out using SignalP 3.0[43]. α-Helix transmembrane topology prediction was carried out using TMHMM[44]. BOMP was used to predict β-barrel outer membrane proteins[45]. Putative lipoproteins were predicted by SpLiP[46]. To identify proteins orthologous between serovar Lai and serovar Copenhageni as well as between serovar Lai and human, all predicted proteins were searched against each other locally using BLASTP[47].
Comparative genomic hybridization
DNA microarrays of L. interrogans strain #56601 consisting of 3528 annotated ORFs longer than 250bp were prepared as previously described [48]. The genomic DNA of L. interrogans strain #56601 was used for reference in the double-fluorescence hybridization, and the genomic DNA of strain Fiocruz L1–130 was used as a control. A CGH microarray analysis of strain Lai and strain Fiocruz L1–130 was performed first. The qualified threshold determined in this control experiment was used to identify gene deletions in other strains. Reference or test DNA was fluorescently labelled through direct incorporation of Cy3-dCTP or Cy5-dCTP (Amersham Pharmacia Biotech) respectively by a randomly primed polymerization reaction. Unincorporated nucleotides and random primers were removed using QIAquick Nucleotide Removal columns (QIAGEN) according to the manufacturer's instructions.
Hybridizations were conducted in a hybridization chamber at 42°C overnight. Slides were washed at 55°C with 1 × SSC containing 0.2% SDS for 10 min and then at 55°C with 0.1 × SSC containing 0.2% SDS for 20 min and finally at room temperature with 0.1 × SSC for 3 min. Competitive hybridization was performed twice for each strain. In the first experiment, L. interrogans strain #56601 reference DNA and the sample DNA were labelled with Cy3 and Cy5, respectively. In the second hybridization, the dyes for labelling were interchanged.
Microarrays were scanned using a Chipreader laser scanner GenePix 4000B AXON (Axon Instruments, Union City, CA) according to the manufacturer's recommendations. Spot quantification, signal normalization and data visualization were performed using the programs GeneSpring 5.0.2 (Silicon Genetics) and Microsoft Excel.
Transcriptome analysis
L. interrogans was grown in EMJH medium at 37°C under aerobic conditions for transcriptome analysis. Only mid-log-phase cultures at a mean density of 106/ml in 100 ml were used in transcriptional experiments.
Total RNA was isolated from leptospires using Trizol reagent (Invitrogen) according to the manufacturer's protocol. Contaminating DNA was digested with RQ1 RNase-free DNase (Promega Corp.). The treated RNA was purified with a QIAGEN RNeasy Kit (QIAGEN).
RNA (10 μg) was labelled with Cy3 by reverse transcription using Superscript α (Invitrogen). Unincorporated dye was removed using a QIAquick Nucleotide Removal Kit (QIAGEN) as specified in the manufacturer's protocol. Samples were hybridized under cover slides to the microarray slides overnight at 42°C, and then washed as usual. The hybridization slides were processed by Tiffsplit (Agilent) and data were further analyzed using Genespring software 5.0.2 and normalized using mean values combined with Microsoft Excel software. Microarrays were used to assay relative RNA abundance. Flagged spots or SN<2 spots were excluded for intrachip and interchip reproducibility analysis. We calculated the coefficients of three spots in same chip for each gene to estimate intrachip reproducibility using Microsoft Excel. The signal values from the experiments represent average mRNA abundances. As in the CGH experiments, the dyes for labelling Cy3 and Cy5 were interchanged in the second hybridization.
Figure 2 is a scheme of the procedure we used to identify the vaccine candidates as described above (the numbers in parentheses are the results after the corresponding procedure step).
Figure 2. Schematic representation of general procedure to identify the vaccine candidates in the genome of L. interrogans (the numbers in parentheses are the results after the corresponding procedure step).
Authors' contributions
HLY and XKG designed the research project. HLY and YZZ carried out the bioinformatics analysis. PH and HLY completed the CGH. JHQ and HLY coordinated the transcriptome analysis. HLY and XKG drafted the manuscript. XCJ and GPZ participated in the design of the study and helped to draft the manuscript. All authors contributed to the writing and preparation of the manuscript. All authors read and approved the final manuscript.
We thank Bao-Yu Hu and Yang Yang (Department of Microbiology and Parasitology, Shanghai Jiao Tong University School of Medicine) for help in bacterial culture preparation. This work was supported in part by grants from the National Natural Science Foundation of China (No.30370071 & 30670102), the National High Technology Research and Development Program of China and Shanghai Leading Academic Discipline Project (T0206). Moreover, we are grateful to the editors and anonymous reviewers for thoughtful comments on the manuscript.
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Fans will soon pack MetLife Stadium for Super Bowl XLVII. The National Football League's 2013-2014 season championship game will be held on February 2 at 6:30 PM (Eastern). Following the power outage during last year's Super Bowl, organizers of this year's event are taking extra precautions to avoid disruptions to the East Rutherford, New Jersey stadium's electricity supply. At the same time, organizers are promoting the environmental aspects of the power supply for this year's game.
During Super Bowl XLVII at the Mercedes-Benz Superdome in New Orleans, Louisiana, a power outage moments after the beginning of the second half of play caused many of the stadium lights and systems to go dark. Between restoring power supply, rebooting computer systems, and letting stadium lighting cool and return to full power, it took 34 minutes for play to resume. Subsequent investigation revealed that the outage occurred when load-monitoring equipment had opened a breaker after detecting an abnormality in the system.
Organizers hope this year's championship is free from such disruption. MetLife Stadium and the rest of the Meadowlands Sports Complex is served by utility Public Service Electric & Gas Co. or PSE&G. Two power lines feed into the substation serving the complex, and on-site generators add additional capacity. PSE&G has been reported as expecting the game to draw as much as 20 megawatts of power, and the utility, NFL, and stadium owner have collaborated on measures and testing to ensure continuity of service during the big game.
Meanwhile, PSE&G's parent PSEG has partnered with the NFL Environmental Program to source renewable energy for the game. PSEG has agreed to purchase and retire a renewable energy credit, or REC, for every megawatt-hour of electricity used at the stadium, the AFC and NFC team hotels, and Super Bowl in Times Square. 240 solar RECs are slated to come from PSE&G's nearby 3-megawatt Kearny solar farm, as well as 5,700 additional RECs from the 7.5-megawatt Jersey Atlantic Wind Farm near Atlantic City.
Beyond electricity, event organizers have committed that all the waste oil generated from food production during the game will be processed into biodiesel fuel by Tri-State Biodiesel, and that all other food waste will be composted.
Presumably, most fans' attention will be focused on the game. Will the organizers' measures prevent power outages in an environmentally friendly manner?
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EDM Social Media InternshipBookmark This
DescriptionOneBeat is a music media company that specializes in Electronic Dance Music. We work with the top EDM artists, venues, and festivals from around the world creating original content, through compelling articles, photo, and video. OneBeat is looking for passionate EDM fans to contribute to our team.
Social Media Intern:
Assist in managing and creating content for OneBeat's social media platforms.
ResponsibilitiesAssist with or create posts for all social media platforms
Help maintain OneBeat's social media calender (I.e. Hootsuite)
Help build a social media following: Twitter, Instagram, Vine
RequirementsPassion for Electronic Dance Music
Proficient with: Hootsuite, Facebook, Twitter, Instagram, Vine
Excellent writing skills
Excellent communication skills
A creative and forward thinking mind
Excellent time management and organization skills
A self starter
The ability to meet multiple deadlines in a timely manner