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PHOENIX, Sept. 21 /PRNewswire/ -- Mayor Phil Gordon and Phoenix community leaders, including Fire Chief Bob Khan and Chicanos por la Causa President and CEO Edmundo Hidalgo, are partnering with the American Lung Association of Arizona on its Faces of Influenza educational initiative to kick off the 2009-2010 influenza season in Phoenix.
In support of the program, Mayor Gordon, Chief Khan and Hidalgo will receive their seasonal influenza vaccinations and participate in a press briefing on Monday, Sept. 21 at 10:00 a.m. at the Phoenix City Hall Atrium.
The press briefing will focus on the importance of preventing the spread of seasonal influenza through vaccination, as public health experts, including the Centers for Disease Control and Prevention (CDC), recommend seasonal influenza vaccination as the first line of defense this year in protecting against the influenza virus.
"It is the responsibility of every Phoenix resident to find out whether they are a 'face' of influenza recommended for annual seasonal influenza vaccination," said Mayor Gordon. "Many people are affected by seasonal influenza every year and don't realize that getting vaccinated is an easy way to protect their health, their family's health and the health of our community."
The American Lung Association's Faces of Influenza campaign encourages local residents to see themselves and their loved ones among the many "faces" of influenza - people who fall into one or more target groups recommended for annual vaccination by the CDC. Despite recommendations by health experts that more than four out of five Americans should be vaccinated against seasonal influenza annually, fewer than half actually do.
Local "faces" of influenza Chief Khan and Edmundo Hidalgo joined the Faces of Influenza campaign to help educate Phoenix-area families about the importance
|SOURCE The American Lung Association of Arizona
Copyright©2009 PR Newswire.
All rights reserved


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Dandy Wellington - Harlem Rhythm
My good friend Dandy Wellington has just released his debut EP "Harlem Rhythm"! The four tracks - three original compositions - are terrific toe tappers. I especially love the title track which transports me to just the best party of my dreams. What I've always loved about Dandy Wellington and His Band is that they create a mood where ever they go. Their jazz seems to make the night a bit more adventurous, the light a bit more dreamy, the dancers a bit more romantic. Now you can create that mood whenever you want - this technology thing is brilliant!
The debut of Harlem Rhythm was also an opportunity for me and Dandy to collaborate on a photo shoot. Even though I've photographed him many times over the five years we've known each other, we hadn't done a "proper" shoot since we first met on the original Dandy Portraits shoot we did for Fine and Dandy Shop in 2010! We did a few looks, but this amazing midnight blue white tie and tails was just perfect for the cover.
I am Dandy in Chicago!
I'm very excited to be visiting Chicago for events surrounding the opening of the exhibit
This remarkable group photography exhibit is the brain child of curator Shantrelle P. Lewis, a brilliant mover and shaker who has always been a supporter of the Dandy Portraits. I first met Shantrelle as she embarked on her dandy journey with the original incarnation of the Dandy Lion exhibit in Harlem in 2010. I've been a fan of her ever since!
The Dandy Lion exhibit has had many incarnations since, most notably in Amsterdam and Washington DC, and it has only grown and become more relevant. I'm very happy to be exhibiting seven portraits and participating in the symposium for this incarnation in Chicago!
Here are the events not to be missed in Chicago:
April 8 - I am Dandy book signing party at Richard's Fabulous Finds 6-9pm - RSVP event page
April 9 - Opening of exhibit at MoCP 5-7pm - RSVP event page
April 10 - Symposium for exhibit at MoCP 2-6pm - RSVP event page
Here I am inspecting one of the prints...of none other than Dr. Andre Churchwell of course!
Here I am inspecting one of the prints...of none other than Dr. Andre Churchwell of course!
Ragged But Right - A Visit From McDermott & McGough and Barima
David McDermott and Peter McGough outside The Back Room, NYC, Nov 2014
Mike Haar (@mikewillcutyou) hosts a monthly DJ night at The Back Room, and this one was extra special because David McDermott was visiting from Ireland and dropped by with his longtime artistic partner Peter McGough. If you aren't familiar with the legendary duo you must look up McDermott and McGough! Their history, philosophy, and work are fascinating. And if that wasn't enough excitement, my friend and I am Dandy book subject Barima Owusu-Nyantekyi was visiting from London. What a great meeting of worlds! I hope you enjoy my documentation of the evening.
Peter McGough and Barima
Michael Haar
Darlene and Laura Kimsey
Mister Burton finally gets to meet David McDermott
Vanessa and Mr. Haar
As Mister Burton puts it, "A Ripper Street Moment"
Kelly Desmond Bray trying to scare us as we leave The Back Room
I am Dandy in Zurich at AP & Co
Thank you to all who could join us last night to celebrate I am Dandy with our hosts AP & Co - Alferano, Pesko & Co - in Zurich on Oct 2, 2014. What a fantastic crowd! Enjoy these photos by Bon Parinya Wongwannawat and me.
Jeroen van Rooijen, Zurich style guru and creator of the Styleride, presents our book to the crowd.
Raul Sanchez, head of design for Jemoli
Photographing Stephan Feierabend and his custom ride
Jeroen, Natty, and Nico Pesko
Reto Caprez of Alferano, and Artan Kasa, head of made to measure for AP & Co
Our journalist friend Kim Dang kicked things off for us in Zurich with his front page feature for NZZ (Zurich's largest daily newspaper) Stil Sunday magazine "Forever Dandy". That's Winston Chesterfield on the cover!
For more photos visit AP & Co facebook page
Alferano, Pesko & Co
Talstrasse 58
8001 Zurich
+41 43 888 64 64
Do You Know Scavini?
Massimilano Mocchia di Coggiola and Julien Scavini, Paris, Sept 2013
On our last visit to Paris - for the I am Dandy book launch - Massimiliano invited Kelly and I to meet his tailor...and irresistible prospect!
I ask him what he thinks of the word "dandy": "The true dandy is a myth, a fictional character. It is an idea, an essence. The dandy embodies the perfection of nothingness, emptiness. It is an art. And like any perfection, it is not of this world, but the world of ideas and concepts. I prefer the term gentleman for men dressed elegantly. That said, for Massimiliano or Mickael Loir, the term gentleman is wrong, while dandy is best ... funny!"
Scavini's passion for tailoring developed after he came to Paris (from his home town of Biarritz) to study architecture. As a lover of classical architecture, towards the end of his studies he grew weary of the over emphasis on removing "old thinking", and using technology to innovate. He always loved menswear, so he decided to do his thesis about a Hackett shop. "It was very funny," He says. After his graduation he started an apprenticeship with master tailor, André Guillerme-Guilson. He spent a year with Guilson at the l'Association de Formation Tailleur learning the art of tailoring, and, although he is not a cutter, he can make a jacket "from A-Z" by hand.
Scavini brings this keen understanding of tailoring, and a very discerning eye for detail not only to his shop, but to his blog Stiff Collar, which he started in 2009 while studying with Guilson. Stiff Collar is a wealth of information and inspiration, and what you will immediately notice - even if you don't read French - are his charming illustrations. Scavini tells me started drawing during his architecture studies, ironically using his ArchiCad software to express the universe that inspired him - 1930s England, Hercule Poirot, classic tailoring, etc. With their thick black outlines, one can see his artwork has been inspired by classic comics like Tintin, Blake and Mortimer, and illustrator Jean-Claude Floch. Small framed prints of a selection of his artwork are on the walls of his shop, and it adds to the ambience - classic, understated, and a little modern in that practical way I like.
Scavini Tailleur
50 Boulevard de la Tour-Maubourg
50 Boulevard de la Tour-Maubourg
75007 Paris
Apart from visiting Stiff Collar, you can see Scavini's illustrations in Figaro Magazine, and in a soon to be released fully illustrated book on classic wardrobe (title and link to come!). He also will star as a tailoring specialist in an upcoming French TV show called Cousu Main (Hand Sewn), an adaptation of the English reality/game show The Great British Sewing Bee. It premieres Aug 30, 2014 on M6 in France. It appears we will be seeing and hearing a lot from Julien Scavini!
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How is Pirates Backpackers Fremantle rated on other websites?
Hostelz.com Guest Reviews
Nice weekend in Freemantle Popped into town after a month working on the farm and decided to stay a the Pirates backpackers in Freemantle. It was the right call and I had a really enjoyable and relaxing weekend. The owner Vaughan puts in a lot of effort to make his guests' stay enjoyable with lots of organized activities and events throughout the week for people to join in on. The location is also ideal with everything just a few minutes walk away. I'd definitely stay here again if I'm back in Perth. — Noah, age 30, USA (2015-04-28)
Good Nice pleasant staff and guests, clean toilets and bathrooms. — Patrick, age 63, Aus (2014-10-21)
Great little backpackers, always something to do great staff, easy going, like little family -- not baby sitters. little bit of wildlife but not of great concern. a great location. activity board gets everyone in a social group and is fun. — Karl, age 42, aust (2014-02-23)
Your home away from home First thing i ever read about Pirates was the sentence "Your Home away from Home" and i thought thats just one this stupid advertising mottos you hear everywhere. But after altogether 4 month i stayed in this Hostel i realized it's not just a stupid sentence, it is just the truth. Pirates is in the centre of lovely Fremantle and you have everything what you need and more then that. You'll find friends and this awesome Pirates family. It is a small Hostel what makes it easy to meet other people from the Hostel. And especially during all this activities (surfing, snorkeling, rockclimbing, bushwalks, watching sunsets, winetasting tours, barbecue on the beach, dress code partys) which are offered by the Hostel you gonna meet other people easily. Captain Vaughan and his crew making this place to something really special. And even when you staying away for a couple of month and coming back to Pirates, people changing but it's still this good atmosphere. It is still home. It seems to be a place where just awesome people living in. Stay in this Hostel and live the australian dream. i've done it and i'm coming back. I never gonna forget this time, this place, this people. — Anonymous, german (2012-03-22)
Homelike hostel I liked the pirates a lot. it has been like a second home for me. it might not be the cleanest hostel but it is cozy and there are always friendly people around. the staff depends some are nice and some are ignorant. when vaughn was around he was always interested and he would never ignore you. — Posti, german (2011-03-30)
Brilliant little hostel Booked in for just 3 days but ended up staying for 2 weeks. We just loved the place. We had a brilliant time. Everybody has been so friendly and looked after us. Could be a little quiet at times with many people seem to be working. The place was clean, although some people were a bit messy with their clothes on the floor. The courtyard is excellent and we spent most of our time there. We recommend this brilliant little hostel to everyone. — Bek, Canada (2011-03-05)
Pirates Backpackers -- there is no better way to stay! Since 2007 I'm a regular guest. The great location in Fremantle (just footsteps away from the world-famous cappuccino strip, shops, and pubs), the always friendly Pirates Crew, and specially the amazing activities perfect organized by the owner Vaughan (boat trips, camping, surfing lessons, parties) made me stay there almost 1 year instead my planned 3 days. And I wasnt the only long-term guest!! Its never getting boring, it feels like living in 1 big family. Its more than a hostel its like a shared house. Cant wait to go there again!! — Jen, Germany (2010-12-30)
In the top 3 hostels! I spent the best few weeks I've ever had at Pirates. It definitely rates as one of my top 3 hostels of all time! It's small and very relaxed. It made it so easy to meet some amazing people and I had some amazing/funny/interesting/awkward/relaxing/memorable experiences. Cool staff, a very funny cat that loves lying on it's back, and lots to do. Highly recommend this place! — Anonymous, England (2010-12-26)


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Robert Kaplan whom I first encountered when I read his Balkan's Ghost prior to deploying to Kosovo in 2000, is in my opinion one of the big thinkers of our age. Over at Foreign Policy, he has a fascinating article that I urge all to read and think about, entitled Augustine's World.
Syria is the Levant, the geographical core of Late Antiquity. And its disintegration, like the crumbling of Libya, Yemen, and Iraq, along with the chronic unrest in Tunisia and Egypt, signifies not the birth of freedom but the collapse of central authority. Rome could not save North Africa, and the United States will not save the Near East — for as the opinion polls demonstrate, Americans have had enough of foreign military entanglements. Anarchy, perhaps followed by new forms of hegemony, will be the result. – See more at: http://www.foreignpolicy.com/articles/2013/12/03/augustine_s_world_what_antiquity_tells_us_about_syria#sthash.dqoiMYTE.dpuf
His description of the current state of North Africa and the Levant is the best I have seen:
North Africa has since devolved into a borderless world of gangs, militias, tribes, transnational terrorists, anti-terrorist expeditionary forces, and weak regimes gripped in stasis. The adjacent Levant erupted into protracted low-intensity war, with only two strong legal entities left between the easternmost edge of the Mediterranean and the Central Asian plateau: a Jewish state and a Persian one (thus the centrality of Iran arguing for a rapprochement with the United States). – See more at: http://www.foreignpolicy.com/articles/2013/12/03/augustine_s_world_what_antiquity_tells_us_about_syria#sthash.dqoiMYTE.dpuf
He clearly sees what is happening in Europe:
The result is that the European Union itself, so dominant in the first two decades after the fall of the Berlin Wall, has lost some of its geopolitical force in Central and Eastern Europe, just as Russia has re-emerged as authoritarian and powerful, thanks to hydrocarbon revenues. The map of Europe is changing from one uniform color back to divergent shades, with national identities — once presumed to be in retreat — undergoing a resurgence. – See more at: http://www.foreignpolicy.com/articles/2013/12/03/augustine_s_world_what_antiquity_tells_us_about_syria#sthash.dqoiMYTE.dpuf
While we worry about China, Mr. Kaplan notes, that all is not well in the Middle Kingdom– but then again few of our foreign policy elite know anything about the history, culture, and reality of China. Mr. Kaplan notes:
China's economy and military are still growing massively in absolute terms, but the future of the Middle Kingdom is less certain than it was just a decade ago. With ethnic minorities and Han Chinese both pining for more freedom amid fewer opportunities, it is possible that China might one day face a variation on the Soviet Union's fate. – See more at: http://www.foreignpolicy.com/articles/2013/12/03/augustine_s_world_what_antiquity_tells_us_about_syria#sthash.dqoiMYTE.dpuf
He concludes with these I believe prophetic words:
Late Antiquity is useful to the degree that it makes us humble about what awaits us. But whatever comes next, the charmed circle of Western elites is decidedly not in control.– See more at: http://www.foreignpolicy.com/articles/2013/12/02/oppa_gangnam_bubble#sthash.dqoiMYTE.dpuf


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- WND - http://www.wnd.com -
Feds debate whale's fate at Miami Seaquarium
Posted By -NO AUTHOR- On 04/27/2013 @ 12:15 am In Diversions,U.S. | No Comments
(Miami Herald) The federal government has agreed to consider whether to declare Lolita, the Miami Seaquarium's biggest star for more than 40 years, a member of an endangered pack of Northwest Pacific killer whales.
Animal rights activists hailed the decision by the National Marine Fisheries Service as a potential step toward their long-time goal of returning the oldest whale in captivity to its home waters.
"We fully expect them to conclude that Lolita deserves the same protection as her family in the wild," said Jared Goodman, an attorney for People for the Ethical Treatment of Animals, which petitioned the service along with the Animal Legal Defense Fund. "Ideally and realistically, this could ultimately result in her being reunited with her family."
Article printed from WND: http://www.wnd.com
URL to article: http://www.wnd.com/2013/04/feds-debate-whales-fate-at-miami-seaquarium/
Click here to print.
© Copyright 1997-2013. All Rights Reserved. WND.com.


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Associate Shia LaBeouf with the ruining of bot Indiana Jones and Transformers? Think he's some kind of mediocre actor demon spawn? His new movie Horns could be the crutons on your hate salad.
Based on the best-selling book of the same name by Joe Hill, Horns is about a dude named Ig Perrish – whose birth certificate probably got smudged or something – who wakes up from a night of heavy drinking with a pair of horns growing from his head. (I've had worse hangovers.) Now he has to solve the mystery of the horns… oh, and also figure out who killed his girlfriend. Sounds like this could be a USA series too. "He's horny for mysteries."
LaBeouf will star as Ig, Keith Bunin ("In Treatment") is writing the adaptation for Mandalay Pictures. Fun fact: Hill is the son of Stephen King. For reals – if I'm lying, may I grow horns on my head, or something retarded like that. (Variety)
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5 Ways to Instant Inner Peace and Growth
By Joleen Halloran | March 11, 2013
Life hands us opportunities. For some, this means that everything we experience is something that must be "handled', like a spilled glass of milk. When our life experiences feel like something has been taken away from what we assumed was a good day or a good life, this diminishes our peace. For others though, every experience is an opportunity to add something to an already full life. The spilled milk allows us demonstrate a new skill, a new attitude, or to view our world from a different perspective.
In the midst of a troubling event, it's hard for us to conceive that this too is an opportunity for peace, love, or joy. And the reason we feel so disconnected from these (peace, love, joy), is that we have taken our eye off the ball, so to speak and allowed the situation to control us or distract us.
When you start your day and live each moment in fullness, meaning that right where you are this moment is exactly where you must be – then when opportunities arise, they become an addition to an already whole existence. Even in that moment you realize the horrible thing you just said, heard, or saw – when accepted, becomes the jumping off point for the next moments' opportunity.
When we resist something we are experiencing with a, "Oh my – I can't believe I just said that", or "I hate when this happens", or "I can't put up with this" – then we give the experience more power. This immediate resistance to "what is" begins to create powerful feelings, which transpose into emotional reactions of fear, anger, sadness, or grief. But, by allowing the moment to unfold without judgment or reaction, we open ourselves to respond in authenticity, not from inflamed emotion.
Accepting EVERY moment as an opportunity without defining what the moment is (i.e. good, bad, horrible, exciting, scary), is not something we have a great deal of experience with and will take a little bit of practice. By giving this important life skill some attention, we can prepare ourselves towards any situation, no matter how we have defined it in the past. The key is acceptance, and allowing the flow of life to happen, and then observing and responding to the ebbs and flows of whatever it is we experience in that moment. And the beauty, power, and grace of restoring your peace is that you can do it at any moment, no matter how far you feel you feel you have dug your hole, it is always available to you. Here are 5 new practices to add to your day that can increase your capacity for inner peace and growth in every situation you encounter throughout the day.
"Whatever the present moment contains, accept it as if you had chosen it."
—Eckhart Tolle
Set priorities – When setting your days intentions, make sure that growth, peace, joy, and abundance are part of that intention. Do not overcommit on tasks and make sure your spiritual well-being is a priority in every day. Don't get distracted by the busyness of your day – keep your focus on being present and complete the tasks you intend to do. Take frequent breaks throughout the day and always include a few moments in those breaks for rejuvenating your peaceful demeanor by being very still and allowing each moment to lift you up.
Make a commitment – Commit yourself to peace, growth, freedom, and grace. You can only express these natural qualities if you feel them and accept them. While you go about your day, recognize the peace you are experiencing. Recognize the joy or abundance. Expansion in our lives occurs when we appreciate (by being aware of) the qualities of peace and joy in our lives right now! Be a person of peace. Commit to it! Live it!
Practice giving yourself presence – In this case, your gift to yourself is presence in the moment you are in. Frequently check in with yourself throughout the day to ask yourself if you are present. If not, just release to the present moment and relax. The more you practice this – the easier it gets. There is nothing outside of the present moment. The past is a collection of thoughts (it is no longer real) and the future is only thoughts as well – so just stay where you are. Practice, repeat, practice, repeat, and so on.
Do your exercises – Just like exercising our body to strengthen and tone our muscles and bone, we must have a few set of exercises we do each day to strengthen our spirit and expand our soul. Practices like meditation, yoga, or martial arts accomplish both, but some others to consider are deep contemplation, journaling, or doing short exercises to shift your thoughts from one object to another (brain training).
EMERGENCY – Pull the cord – When you want to get off the bus or the train everyone knows that to request this you must pull the emergency cord and the train/bus will stop. To get off the pity, anger, fear, or sorrow train, put something in your life that you can relate to (like pulling the cord) to trigger your intention to get off this bus. The moment can shift immediately if you allow yourself to let go of every single thought you have had about this experience and allow the moment to be new and OK right now. When you feel yourself stepping back onto the train – pull the cord again. Look for ways to pull your cord, like a special prayer or mantra, deep breathing, light a candle, go for a walk. Make your emergency exit something that is special and meaning for you.
- 1 comment • Finding Home • Law of Attraction • Metaphysical • Motivational • New Age • Personal Growth • Practical and Useful • Soulfully Inspiring • Spiritual
- Tagged as: inner growth • inner peace • present moment • spiritual well being
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La Vita E Bella In Miu Miu's Newest Film
This week, Miu Miu will debut a fresh installment of the this series at the Venice Film Festival, ensuring that the voices of women filmmakers are heard. (Huzzah!) Directed by Hiam Abbass, Le Donne della Vucciria is the sixth chapter in the Women's Tales short film project and will premiere August 29. We landed a sneak peek of the teaser, which is set in the beautiful Italian city of Palermo.
Abbass joins an admirable roster of directors who have contributed to the project before her, including Zoe Cassavetes and Ava DuVernay. Check out the trailer below, and see how many polka-dotted pieces you can spot from Miu Miu's dreamy fall '13 collection.


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Smart Repair in Redkey
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Smart repair shops and dealerships near Redkey.
4570 W State Road 32, Anderson
AC/Heating, Alignment, Check Engine Light, Classic Car Repair/Restoration, Diagnosis, Mobile Repair, Oil Change & Lube, Roadside Assistance, Scheduled Maintenance, Service & Repair, Timing Belts, Tire Sales/Repair, Transmission Repair
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The US drilling rig count jumped by 24 units during the week ended Mar. 15 reaching a total 1,776 rotary rigs working, Baker Hughes Inc. reported. That compares with 1,984 rigs working in the comparable week last year.
Offshore drilling climbed 2 units to 53 rigs. Inland water drilling, at 22 rigs working, was up by 3 units. Land-based drilling made the largest gain, reaching 1,701 rigs working, 19 units higher than a week ago. Of the rigs drilling offshore, 51 were in the Gulf of Mexico, an increase of 2 units from a week ago.
Rigs targeting natural gas increased 24 units to 431. Oil-targeted rigs were unchanged at 1,341. Four rigs were unclassified, unchanged from a week ago.
Rigs drilling directionally were reported at 209, up 14 units from a week ago but 19 fewer than the comparable week last year. The number of rigs drilling horizontally rose 1 to 1,131. This compared with 1,180 rigs working in the comparable week a year ago.
Of the major oil and gas producing states, Louisiana was up 8 rigs to 114; Oklahoma up 7 to 187; Texas up 3 to 838; New Mexico and Pennsylvania, up 2 rigs each to 80 and 74, respectively; and North Dakota and Wyoming, up 1 rig each to respective counts of 177 and 46. Five states were unchanged this week: Colorado, 57; California, 39; Ohio, 27; Arkansas, 15; and Alaska, 10. West Virginia was down 1 unit to 21 rigs working.
Canada's rig count slumped again this week, down 77 units to 503. This count includes 366 rigs drilling for oil and 137 units drilling for gas. The total was down 14 units from the comparable week last year.


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The short arm of human chromosome 3 is involved in the development of many cancers including lung cancer. Three bona fide lung cancer tumor suppressor genes namely RBSP3 (AP20 region),NPRL2 and RASSF1A (LUCA region) were identified in the 3p21.3 region. We have shown previously that homozygous deletions in AP20 and LUCA sub-regions often occurred in the same tumor (P < 10-6).
We estimated the quantity of RBSP3, NPRL2, RASSF1A, GAPDH, RPN1 mRNA and RBSP3 DNA copy number in 59 primary non-small cell lung cancers, including 41 squamous cell and 18 adenocarcinomas by real-time reverse transcription-polymerase chain reaction based on TaqMan technology and relative quantification.
We evaluated the relationship between mRNA level and clinicopathologic characteristics in non-small cell lung cancer. A significant expression decrease (≥2) was found for all three genes early in tumor development: in 85% of cases for RBSP3; 73% for NPRL2 and 67% for RASSF1A (P < 0.001), more strongly pronounced in squamous cell than in adenocarcinomas. Strong suppression of both, NPRL2 and RBSP3 was seen in 100% of cases already at Stage I of squamous cell carcinomas. Deregulation of RASSF1A correlated with tumor progression of squamous cell (P = 0.196) and adenocarcinomas (P < 0.05). Most likely, genetic and epigenetic mechanisms might be responsible for transcriptional inactivation of RBSP3 in non-small cell lung cancers as promoter methylation of RBSP3 according to NotI microarrays data was detected in 80% of squamous cell and in 38% of adenocarcinomas. With NotI microarrays we tested how often LUCA (NPRL2, RASSF1A) and AP20 (RBSP3) regions were deleted or methylated in the same tumor sample and found that this occured in 39% of all studied samples (P < 0.05).
Our data support the hypothesis that these TSG are involved in tumorigenesis of NSCLC. Both genetic and epigenetic mechanisms contribute to down-regulation of these three genes representing two tumor suppressor clusters in 3p21.3. Most importantly expression of RBSP3, NPRL2 and RASSF1A was simultaneously decreased in the same sample of primary NSCLC: in 39% of cases all these three genes showed reduced expression (P < 0.05).
The short arm of human chromosome 3 (region 3p21.3) contains clusters of tumor suppressor genes (TSG) involved in multiple cancer types including lung, kidney, breast, cervical, nasopharyngeal and other carcinomas [1-5]. We performed a comprehensive deletion survey of 3p in more than 400 of major epithelial cancer samples and identified two most frequently affected regions - LUCA at the centromeric and AP20 at the telomeric border of 3p21.3 [6-10]. Aberrations in these loci were detected in more than 90% of studied tumors. Homozygous deletions (HD) were frequently detected in all tumors in both the LUCA and AP20 regions. The frequent chromosome losses in these regions suggested that they harbor several multiple TSG [9,10]. More than 20 genes were localized in these two regions and among them at least three TSG were identified: RBSP3 (RB protein serine phosphatase from chromosome 3 gene or HYA22 or CTDSPL; CTD small phosphatase family), NPRL2 (nitrogen permease regulator-like 2 gene or G21 or TUSC4; NPR family) and RASSF1A (Ras association domain family member 1 gene). RBSP3 was mapped to AP20 and the others two to the LUCA region [1,11-13].
The RBSP3 gene occupies more than 120 kb and contains at least 8 exons coding for a 4.8 kb mRNA that is ubiquitously expressed in normal tissues including lung. By sequence analysis RBSP3 belongs to a gene family of small C-terminal domain phosphatases that may control the RNA polymerase II transcription machinery [14]. Two sequence splice variants of RBSP3 (A and B) were identified and an initial analysis of RBSP3 was performed in lung and other cancers [12]. The expression of the gene was greatly decreased in several small cell lung cancer (SCLC) and NSCLC cell lines. RBSP3 showed growth suppression with regulated transgenes in cell culture and suppression of tumor formation in SCID mice. It was demonstrated that transient expression of variant A and B resulted in drastic reduction of the phosphorylated form of RB protein presumably leading to a block of the cell cycle at the G1/S boundary. In addition, frameshift, nonsense and missense mutations in RBSP3 have been reported [15]. All these features are consistent with classical characteristics of a TSG.
The NPRL2/G21 gene covers 3.3 kb and contains 11 exons coding for the main 1.8 kb transcript with multiple splice isoforms that are expressed in all tested normal tissues including lung. By sequence analysis, the main product of NPRL2/G21 encodes a soluble protein that has a bipartite nuclear localization signal, a protein-binding domain, similarity to MutS core domain, and a newly identified nitrogen permease regulator 2 domain with unknown function. This information suggests that the nuclear protein NPRL2/G21 may be involved in DNA mismatch repair, cell cycle checkpoint signaling, and regulation of the apoptotic pathway. NPRL2 plays an important role in cisplatin-induced resistance in human non-small-cell lung cancer cells [16,17]. Previously obtained results indicated that NPRL2/G21 is a multiple tumor suppressor gene [16,18,19].
The RASSF1 gene occupies 7.6 kb and contains 5 exons coding for 2 kb alternatively spliced mRNAs [6,11,20]. One of the major splicing forms is RASSF1A. Several studies have shown that loss of RASSF1A expression occurs in many different cancers because of tumor acquired promoter DNA methylation and the gene is able to suppress growth of lung cancer cells in culture and tumor formation in mice [13,21-24]. For example, RASSF1A is silenced by promoter hypermethylation in 100% of SCLCs and in 63% of NSCLCs cell lines and in 50-100% SCLC and 21-58% NSCLC primary tumors [25-28]. As in the case of RBSP3, frameshift, nonsense and missense mutations in RASSF1A have been discovered [15,29]. The amino acid sequence of RASSF1A (340 amino acids) contains a predicted diacylglycerol (DAG) binding domain and a Ras association domain. Association of human proteins RASSF1C and RASSF1A with Ras protein was demonstrated [30,31]. RASSF1A can induce cell-cycle arrest by engaging the Rb-family cell cycle checkpoint [32]. RASSF1A is involved in several growth regulating and apoptotic pathways and regulates cell proliferation, cellular integrity and cell death [24,27]. These and other results strongly suggest that RASSF1A is an important human TSG involved in the development or progression of many epithelial tumors.
Previously only few studies were performed to compare expression of several 3p TSG in the same tumor sample [33,34]. To investigate this further we chose RBSP3, NPRL2 and RASSF1A and analyzed their expression by qPCR in primary tumors: non-small cell lung cancer (NSCLC) - adenocarcinoma (AC) and squamous cell lung cancer (SCC).
For the first time we found that expression of all three genes was significantly decreased in 67-85% of tested NSCLC cases. Moreover, the simultaneous down-regulation of RBSP3, NPRL2 and RASSF1A in the same tumor sample was observed in 39% of all cases. Both genetic and epigenetic mechanisms contributed to deregulation of these three genes representing two TSG clusters in 3p21.3.
Tissue specimens
Paired specimens of non-small cell lung cancer (NSCLC) tissues including 41 squamous cell carcinomas (SCC), 18 adenocarcinomas AC) and adjacent morphologically normal tissues (conventional "normal" matched control samples) were obtained after surgical resection of primary lung cancer prior radiation or chemotherapy and stored in liquid nitrogen. "Normal" matched controls were obtained minimum at 2 cm distance from the tumor and confirmed histologically as normal lung epithelial cells. The diagnosis was verified by histopathology and only samples containing 70% or more tumor cells were used in the study. The samples were collected in accordance to the guidelines issued by the Ethics Committee of Blokhin Cancer Research Center, Russian Academy of Medical Sciences (Moscow). All patients gave written informed consent that is available upon request. The study was done in accordance with the principles outlined in the Declaration of Helsinki. All tumor specimens were characterized according to the International System of Clinico-Morphological Classification of Tumors (TNM), based on the tumor-node-metastasis and staging classification of 1989 [35] and WHO criteria classification of 1999 [36]. Relevant clinical and pathological characteristics of the patients with NSCLC included in this study are summarized in Table 1. Normal lung tissues (autopsy material) were obtained post mortally from ten healthy individuals (age 23-49 lacking cancer history with absence of chronic diseases).
Table 1. Clinical and pathological characteristics of patients with NSCLC
DNA and total RNA extraction and reverse transcription reaction
DNA was extracted using the Dneasy Tissue kit (Qiagen, USA) and total RNA was isolated with Rneasy mini kit (Qiagen, USA) according to the manufacturer's recommendation. RNA quality was assessed with spectrophotometer NanoDrop ND-1000 (NanoDrop Technologies Inc. USA) and by gel electrophoresis. All RNA samples were treated with RNAse free DNase I (Fermentas, Lithuania) and cDNA was synthesized using MMLV reverse transcriptase and random hexamers according to standard manufacturer's protocol (Fermentas, Lithuania).
Analysis of mRNA and DNA copy number by qPCR
The sequences of primers and probes are shown in Table 2. All reactions were performed using ABI 7000 PRISM™ SDS (Applied Biosystems) with RQ software (PCR program: 10 min at 95°C, then 40 two-step cycles 15 s at 95°C and 60 s at 60°C) in total volume 25 μl in triplicate. All probes contained the dye FAM at 5'-end and RTQ1 at 3'-end. Final concentrations of primers and probes for target and reference genes were: RBSP3 cDNA primers- 350 nM, probe - 150 nM; RBSP3 DNA primers - 150 nM, probe - 100 nM; ACTB DNA primers- 200 nM, probe - 100 nM, NPRL2 cDNA primers - 500 nM, probe 300 nM; RASSF1A cDNA primers - 300 nM, probe 300 nM;GAPDH cDNA primers - 300 nM, probe - 150 nM; RPN1 cDNA primers - 350 nM, probe - 200 nM;RPN1 DNA primers - 200 nM, probe - 100 nM; GUSB cDNA primers - 350 nM, probe - 250 nM; GUSB DNA primers - 200 nM, probe - 200 nM. PCR products were analyzed in 1.8% agarose gels and nucleotide sequences of the amplicons were verified by sequencing with 3730 DNA Analyzer automated sequencer (Applied Biosystems).
Table 2. Primers and probes for target and reference genes for expression levels and copy number studies
qPCR data were analyzed using the relative quantification or ΔΔCt-method [37,38] based on mRNA (or DNA) copy number ratio (R) of a target gene versus reference gene in a given tumor sample relative to matched normal control sample (see above Tissue specimens section) according to the formula:
where E - efficiency of reaction, CT - threshold cycle, ref - reference gene, tar - target gene.
All preliminary validation steps have been done: standardization of all assays, reproducibility of the qPCRs in parallel and in independent runs, selection of reference samples and testing of reference genes http://www.gene-quantification.info/ webcite.
NotI-microarray analysis
Microarrays were constructed essentially as previously described [39,40]. In brief, two oligonucleotides:
NotX: 5'-AAAAGAATGTCAGTGTGTCACGTATGGACGAATTCGC-3'
and NotY: 5'-GGCCGCGAATTCGTCGGTATGCACTGTGTGTGACATTCAAA-3"
were used to create the NotI linker. Annealing was carried out in a final volume of 100 μl containing 20 μl of 100 μM NotX, 20 μl of 100 μM NotY, 10 μl of 10×M buffer (Roche Molecular Biochemicals) and 50 μl of H2O. Two micrograms of tumor and normal control DNA (50 μg/ml) were digested with 20 U of Sau3A (Roche Molecular Biochemicals) at 37°C for 5 h and then 0.4 μg of the digested DNAs were circularized overnight with the T4 DNA ligase (Roche Molecular Biochemicals) in the appropriate buffer in 1 ml reaction mixture. Then DNA was concentrated with ethanol, partially filled in and digested with 10 U of NotI at 37°C for 3 h. Following digestion, NotI was heat inactivated and DNAs were ligated overnight in the presence of a 50 M excess of NotI linker at room temperature. NotI- representation (NR) probes were labeled in a PCR reaction with NotX primer. The majority of products of the DNA amplification step were in the 0.2-1.0 kb range. Repeated PCR was conducted for labeling NR with fluorophores.
Hybridization of coupled normal/tumor NotI samples was carried out at 42°C for 15 h in a Lucidea Base device (Amersham Pharmacia Biotech) according to manufacturer's recommendations. Automatic washing of the microarrays was performed in the same device using manufacturer's protocol. The following solutions were sequentially used for the washing: 1) 0.2% SDS+1 SSC; 2) 0.2% SDS+0.1 SSC; 3) 0.1 SSC; 4) de-ionized water; 5) isopropyl alcohol. Then microarrays were scanned in the GenePix 4000 A and results were processed with GenePix Pro 6.0 software (Amersham Pharmacia Biotech).
Statistical analysis
Nonparametric Wilcoxon test was used to compare mRNA expression differences of target and reference genes for the same NSCLC sample. Then groups of samples were compared in respect to average level of mRNA decrease (LDav) and the frequency of decrease (FD). The LD was calculated as 1/R and reflects the n-fold factor by which the mRNA content decreased in the tumor compared to normal tissue. Nonparametric Kruskal-Wallis and Mann-Whitney rank-sum tests were used to test mRNA differences (both LDav and FD) for each target gene in NSCLC (AC, SCC) and with and without metastases. Nonparametric Spearmen's criterion was used to calculate the coefficient of correlation between the levels of mRNA decrease (LDav) for each set of pairs of target genes. P-values < 0.05 were considered statistically significant. All statistical procedures were performed using the BioStat software [41].
Expression of RBSP3, NPRL2 and RASSF1A genes in lung tissues of healthy donors
Genomic structure and location of primers and probes is shown in Figure 1.
Figure 1. Schematic representation of the RBSP3 (A), NPRL2/G21/NPR2L/TUSC4 (B), and RASSF1A (C) genes. Exon-intron structure of known mRNAs is shown. Coding and non-coding mRNA regions are depicted by dark and light grey boxes, respectively. Primers and probes designed for qPCR are shown by arrows and boxes above mRNAs. A. RBSP3 (3p21.3, AP20 region). Forward primer crossed the 1st/2nd exons boundary, reverse primer and probe were located in the 2nd exon. B. NPRL2 (3p21.3, LUCA region): Forward primer crossed the 5th/6th exons boundary, reverse primer crossed the 6th/7th exons boundary, probe was located in the 6th exon. C. RASSF1A (3p21.3, LUCA region): Forward primer crossed the 1st/2nd exons boundary that provides specificity to isoforms A and E. Reverse primer crossed the 2nd/3rd exons boundary that provides specificity to isoforms A, B and D. Combination of forward and reverse primers provided specificity to isoform A only. Probe was located in the 2nd exon.
It is known that normally looking cells surrounding tumors can already harbor genetic and epigenetic changes compared to the cells isolated from the same tissue from healthy individuals. Therefore we decided to check expression of these 3 genes in samples obtained from healthy donors and in biopsies of "normal" (matched) control samples obtained from patients with NSCLC. The mRNA levels were measured in lung tissues from healthy donors using different reference samples. RNA pool isolated from lung tissues of 10 healthy donors was used in one set of experiments and in another test the same samples were examined against RNA pool isolated from adjacent to tumor "normal" tissues of 30 NSCLC patients. The GAPDH was used as a reference gene and its expression did not vary more than 2-fold in all samples examined. Very similar results were obtained for target genes in both experiments. We concluded that expression of all three genes did not significantly vary (R = 1.5 ± 0.5) in normal lung tissues from healthy donors and patients with NSCLC ("normal" matched controls).
Expression of three TSGs in primary NSCLC - AC and SCC
The results of mRNA level quantification for three genes and statistical analysis are shown in Figures 2 and 3 and Table 3. The variability of GAPDH and RPN1 mRNA was less than 2-fold in studied samples, therefore in this study we considered significant greater than or equal to 2-fold decrease of target genes expression.
Figure 2. The relative expression levels of RBSP3, NPRL2/G21 and RASSF1A genes in AC and SCC. The mRNA level of each target gene was normalized by that of the reference gene GAPDH. Data for ADC and SCC were presented according to cancer progression stages and to the presence of metastases.
Figure 3. Simultaneous down-regulation of tumor suppressor genes RBSP3/CTDSPL, NPRL2/G2,1 and RASSF1A in primary non-small cell lung cancer (AC and SCC). The data were for the same 36 primary tumor samples as the data presented in Figure 2. The mRNA level of each target gene was normalized by those of two reference genes GAPDH and RPN1.
Table 3. The frequency of mRNA decrease (FD) and the average level of mRNA decrease (LDav)
Significant (from 2 to 94-fold) decrease of the mRNA level (LD) was observed in 85% (50 of 59, P < 0.001) of all NSCLC cases. Frequency of mRNA decrease (FD) was high in both AC (78%) and SCC (88%). Interestingly at Stage I of the SCC cases expression of RBSP3 was down-regulated already in 100% cases and only in 71% of AC samples (Table 3). On the other hand the tendency of more severe deregulation of RBSP3 transcription during tumor progression was more evident for AC cases: in average a 3-fold declined expression in 70% of cases without metastases and a 6-fold in 88% of samples with metastases. The same was correct for cases at Stage I (LDav = 3, FD = 71%) and Stage III (LDav = 6, FD = 100%). Spearmen's coefficient of reverse correlation (rs) between tumor Stages (I, II, III) and expression R values was 0.36 (P = 0.2, i.e. non-significant).
Significant decrease of LD (from 2 to 100-fold) was seen in 73% (43 of 59, P < 0.001) of all NSCLC cases. Down-regulation of NPRL2 was more significant in SCC compared to AC: 9-fold and 85% vs. 4-fold and 44%, respectively, P = 0.002 (see Table 3). No expression was found in 12% (5 of 59) of SCC cases. The FD was comparable in SCC cases with metastases and without (82% and 88%, respectively). However FD in AC samples without metastases was less than with metastases (20% against 75%; P = 0.08). Again at Stage I of the SCC cases expression of NPRL2 was down-regulated already in 100% cases and in AC only 14% samples showed decreased expression. The tendency of more frequent decrease of NPRL2 mRNA level with tumor progression was seen only for AC cases, where Spearmen's coefficient of reverse correlation (rs) between tumor Stages (I, II, III) and expression R values was 0.47 (P = 0.06). Eighty three percent of AC cases had decreased expression at Stage III as compared to 14% at Stage I.
Significant decrease of LD (from 2-fold to 57-fold) was detected in 67% (24 of 36, P < 0.01) of all NSCLC cases, including 64% of AC and 68% of SCC samples (Figure 2, Table 3). More severe deregulation of RASSF1A expression was observed during tumor progression both in AC and SCC. Spearmen's coefficient of reverse correlation (rs) between tumor Stages (I, II, III) and R values was 0.54 (P = 0.05) for AC. Statistically significant decrease of LDav and increase of FD was observed in AC for patients with metastases and without (7-fold and 100% vs. 4-fold and 29%, P < 0.05). The same tendency was seen for LDav and FD in SCC cases with and without metastases: namely, 6-fold and 81% vs. 4-fold and 33%, respectively; P = 0.196. Moreover an average decrease of RASSF1A mRNA (LDav) in SCC at Stage I was 4-fold and at Stage III it was already 7-fold.
FD increased for both SCC (33% vs. 82%) and AC (29% vs. 100%) during tumor progression from Stage I to Stage III.
Surprisingly a 3-to-4 fold increase of mRNA level of two genes - NPRL2 and RASSF1A was found for several AC samples at Stage I (Figure 2, samples 1, 3, 5). These tumor samples were highly differentiated in contrast to samples 2, 4, 9, 10 and others that displayed low degree of differentiation and significant decrease of NPRL2 and RASSF1A. Some of them showed so high degree of anaplasia that the epithelial phenotype was almost undetectable. These undifferentiated cells were highly malignant and showed the strongest decrease of NPRL2 and RASSF1A expression.
Simultaneous down-regulated expression of three genes in the same tumor samples
As expression of all three target genes was frequently decreased in the same NSCLC samples we tested the probability that this decrease was not random. To do this we used 36 NSCLC samples and two control reference genes GAPDH and RPN1 (Figure 3, Table 4). Results demonstrated that this simultaneous down-regulation was very frequent and statistically valid: all three genes had reduced expression (≥2) in 39% (P < 0.001) of NSCLC specimens (36% in AC and 41% in SCC). Simultaneous decreased expression of RBSP3 and NPRL2 was observed in 61% of cases and for RBSP3 and RASSF1A this occurred in 50%. In the case of NPRL2 and RASSF1A simultaneous down-regulation was detected in 44% of tumors (Table 4). Spearmen's coefficient values rs for RBSP3, NPRL2/G21, RASSF1A gene pairs were high (0.63 - 1.0, P < 0.001) in different groups especially in AC and SCC with metastases.
Table 4. The frequencies of simultaneous mRNA level decreases (FD) for the combination of genes RBSP3, NPRL2/G21 and RASSF1A in different groups
Decreased expression of RBSP3, NPRL2 and RASSF1A in primary NSCLC can be caused by genetic and epigenetic factors
To understand the mechanism underlying the observed down-regulation of RBSP3 in NSCLC samples we used qPCR to test DNA copy number changes, i.e. genetic factors [9,10] and NotI microarrays to examine methylation i.e. epigenetic factors [39]. Methylation was detected in 38% of AC (3 of 8) and in 80% of SCC (8 of 10) cases with decreased expression of RBSP3. Deletions were detected in 25% of AC samples and in 30% of SCC tumors (Table 5). Both methylation and hemizygous deletions were observed in two SCC specimens. Methylation or/and deletions of RBSP3 were detected in 63% of AC and in 90% of SCC cases. The data suggested that both genetic and epigenetic mechanisms are important for transcriptional inactivation of RBSP3 in NSCLC.
Table 5. Comparison of qPCR data and NotI-microarrays for RBSP3 gene
Using NotI microarrays we cannot test methylation of NPRL2 and RASSF1A promoters because they don't contain NotI sites. However we can check genes SEMA3F and GNAI2 that are located in the same LUCA sub-region as NPRL2 and RASSF1A. We tested how often deletions and methylations occurred in the same NSCLC sample at both LUCA and AP20 sub-regions and found that this occurred in 58% of all studied cases (15 of 26). Thus, most likely both genetic and epigenetic mechanisms are responsible for simultaneous down-regulation of expression of these three genes.
Several candidate TSG from the 3p21.3 AP20 and LUCA sub-regions were examined in the gene inactivation test, GIT [2,12,13,16,34,42,43]. The test is based on the functional inactivation of analyzed genes that can be achieved in different ways: by mutation, deletion, methylation etc. According to these results, at least three genes can now be considered as bona fide lung TSG: NPRL2 and RASSF1A from LUCA and RBSP3 from AP20 sub-regions [12,13,16].
Earlier the decrease of RBSP3 expression was shown in SCLC, NSCLC, cervical, renal, breast, ovary, leukemia cell lines and primary tumors by Northern blot analysis, RT-PCR and qPCR [12,44-46]. The decrease or absence of NPRL2/G21 expression was detected in some SCLC, NSCLC and renal cancer cell lines using Northern blot analysis [6,16]. The decrease or absence of RASSF1A expression was found in SCLC, NSCLC and many other tumors and cancer cell lines [see [24,27,28]].
It was reported that promoter methylation was the main mechanism of RASSF1A loss of expression in lung cancer (see Introduction). Homozygous deletion of 3'-part of NPRL2 gene and rare mutations were found in NSCLC and SCLC cell lines [6,16]. There are no methylation data explaining the loss of RBSP3 expression in lung cancer. However frequent deletions and mutations were reported [10,12,15]. In some leukemia cell lines (up to 98%) and acute leukemia lymphoma blood samples (24%) methylation of the promoter region of RBSP3 was reported [44]. Methylation (up to 26%), deletions and decreased expression of RBSP3 were significantly associated with poor prognosis of cervical cancer [45]. Thus, inactivation of RBSP3 might be one of the early events in cervical carcinogenesis.
Loss of heterozygosity and quantitative real-time PCR demonstrated that aberrations in both LUCA and AP20 sub-regions occurred simultaneously in the same tumor with high probability. Thus, it was suggested that aberrations in both LUCA and AP20 sub-regions could be linked [9,10]. Indeed, homozygous deletions in both regions often occur in the same tumor (P < 3 × 10-7). The estimation of possible interdependency between all aberrations in the loci NLJ-003 (AP20) and NL3-001 (LUCA) as different events was carried out using a permutation test for four types of cancers: lung, renal, breast and ovarian. This test also revealed a significant correlation between different aberrations in these two loci (P < 10-6). The same results were obtained using Pearson correlation for numeric values of copy number changes of these loci. Indeed, proteins RBSP3 and RASSF1A could collaborate in cell cycle arrest: RASSF1A by inhibiting cyclin D1 [32] and RBSP3 by dephosphorylating pRB [12]. Thus functional collaboration of these two genes could result in activation of the RB1 gene.
In this study we tested the hypothesis that TSG in AP20 and LUCA regions were not only deleted but their expression could also be simultaneously down-regulated in NSCLC. This suggestion was indirectly supported by other studies that showed that genes over large chromosomal regions could be regulated in a coordinated fashion [33,47-49].
First we found that expression of all three genes is rather uniform in lung samples isolated from healthy donors and from normally looking lung samples obtained from NSCLC patients ("normal" matched control samples). Thus adjacent morphologically normal tissues from the patients can be used as paired reference controls to tumor samples. In the study two parameters were analyzed - the level of mRNA decrease and frequency of mRNA decrease in two major NSCLC histological subtypes (AC and SCC) and their subgroups with different characteristics such as clinical Stage, grade, tumor localization, presence of metastases and others. Although both parameters reflect deregulation of gene expression, they are not randomly but rather functionally related.
Expression analysis of the three genes revealed the following main features.
1. Expression of the three studied TSG was significantly decreased in NSCLC: 85% for RBSP3, 67% for RASSF1A and 73% for NPRL2 (P < 0.001). It was statistically valid both for SCC and AC.
2. Down-regulation of the three genes was already evident at Stage I of NSCLC samples. Statistically significant down-regulation of both NPRL2 and RBSP3 was seen in 100% cases at Stage I of SCC.
3. The degree and frequency of the expression decrease for all three genes was more strongly pronounced in SCC than in AC samples (see Table 3). This difference was statistically valid in the case of NPRL2 (P = 0.002).
4. All studied genes were involved in progression of AC. The tendency of more severe expression down-regulation of the RBSP3 was evident during tumor progression of AC with respect to FD and LD (70% and 3-fold in cases without metastases in contrast to 88% and 6-fold decrease in cases with metastases, P = 0.13). For NPRL2, this tendency was also seen only for AC - 83% of cases had decreased expression at Stage III compared to 14% at Stage I and in 75% of AC cases with metastases vs. 20% of cases with metastases, P = 0.08, see Table 3). Expression of RASSF1A revealed the most strongly pronounced correlation between decrease of expression (FD and LD) and tumor progression both in SCC and AC. For example, difference in FD values was obvious between cases with and without metastases. For SCC cases this difference was 33% vs. 81% (P = 0.196) and for AC it was even more sharp, 29% vs. 100% (P < 0.05).
5. Expression of RBSP3 and RASSF1A was most seriously affected in respect to FD. For RBSPS3 it was detected in 85% of all NSCLC cases and for RASSF1A in 67%. However, regarding LD, the expression of NPRL2 was most strongly inhibited (LDav = 8), while RBSP3 showed weaker inhibition (LDav = 5).
6. Preliminary data suggested that no statistically significant difference was observed in cases with relation to age, smoking history and other cytological and pathological characteristics.
7. NotI microarrays and qPCR on genomic DNA we tested for possible mechanisms of the declined expression of RBSP3 in NSCLC. The data suggested that both genetic and epigenetic mechanisms were important for transcriptional inactivation of RBSP3 in NSCLC. Altogether deletions were detected in 25% of AC samples and in 30% of SCC patients. Methylation of RBSP3 was detected in 38% of AC and in 80% of SCC cases. With NotI microarrays we also tested how often LUCA (NPRL2 and RASSF1A) and AP20 (RBSP3) regions were deleted or methylated in the same tumor and found that this occurred in 58% of all studied cases (18 of 26). Thus, most likely both genetic and epigenetic mechanisms are responsible for simultaneous down-regulation of expression of these three TSG.
The detailed analysis of mRNA expression levels of three 3p TSG was performed in two histological subtypes of NSCLC - AC and SCC respectively. The most important finding was that expression of RBSP3, NPRL2 and RASSF1A decreased in the same samples of primary NSCLC: all 3 genes have reduced expression in 39% of cases (P < 0.05). Declined expression of RBSP3 and NPRL2 was observed in 61% of samples (P < 0.05), RBSP3 and RASSF1A in 50% (P < 0.05), and NPRL2 and RASSF1A in 44% (P < 0.05) of cases. RASSF1A and NPRL2 are located in the same locus and if close localization is the reason for simultaneous decrease of expression then such decrease should be significantly less frequent when genes from the LUCA region are compared with RBSP3. However this was not the case and thus close localization is not the main factor for simultaneous decrease of expression. At present expression of many thousands of genes in NSCLC is investigated using cDNA microarrays [50,51]. These experiments already produced very valuable data. However, results of different studies varied significantly. They should be proved by independent methods and qPCR is one of the methods of choice that allows more detailed analysis of particular genes. This work is one of such studies. All presented data confirm and extend our previous results for lung, renal, breast, cervical cancers [9,10] and support the hypothesis that two TSG clusters (in AP20 and LUCA) are very likely co-regulated by common mechanisms.
qPCR: quantitative real-time reverse transcription-polymerase chain reaction; NSCLC: non-small cell lung cancer; AC: adenocarcinoma; SCC: squamous cell carcinoma; RBSP3: RB1 serine phosphatase from human chromosome 3; NPRL2: nitrogen permease regulator-like 2 gene; RASSF1A: Ras association domain family 1A; ACTB: beta actin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; RPN1: Ribophorin 1; GUSB: Glucuronidase, beta.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
VNS designed the experiments, carried out the qPCR analysis, and wrote the manuscript; EAA collected and classified all lung tumors, performed the qPCR data; TTK - contributed with clinical information for these patients and drafting the manuscript; GSK - was responsible for primers and probes design and analyzed the data; AAD -was responsible for calculations and statistical analysis. VIZ, TVP, VIK performed the experiments and analyzed the NotI-data; MIL helped in the evaluation of the results and revised the manuscript critically for important intellectual content; ERZ was responsible for design study and coordination, wrote the manuscript. All authors read and approved the final manuscript.
The authors are grateful to Ms. Leah V. Lerman, esq. for the help in preparation of the manuscript. This study was financially supported by the Russian Foundation for Basic Research (project no 05-04-49408), The Agency of Science and Innovations (State Contracts no 02.512.11.2241, Agreement 1-2008/07/07) and by research grants from the Swedish Cancer Society, the Swedish Research Council, the Swedish Foundation for International Cooperation in Research and Higher Education (STINT), the Swedish Institute, and Karolinska Institute.
Zabarovsky ER, Lerman MI, Minna JD: Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers.
Oncogene 2002, 21:6915-6935. PubMed Abstract | Publisher Full Text
Imreh S, Klein G, Zabarovsky ER: Search for unknown tumor-antagonizing genes.
Genes Chromosomes Cancer 2003, 38:307-321. PubMed Abstract | Publisher Full Text
Ji L, Minna JD, Roth JA: 3p21.3 tumor suppressor cluster: prospects for translational applications.
Future Oncol 2005, 1:79-92. PubMed Abstract | Publisher Full Text
Klein G, Imreh S, Zabarovsky ER: Why do we not all die of cancer at an early age?
Adv Cancer Res 2007, 98:1-16. PubMed Abstract | Publisher Full Text
Angeloni D: Molecular analysis of deletions in human chromosome 3p21 and the role of resident cancer genes in disease.
Brief Funct Genomic Proteomic 2007, 6:19-39. PubMed Abstract | Publisher Full Text
Lerman MI, Minna JD: The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium.
Cancer Res 2000, 60:6116-6133. PubMed Abstract | Publisher Full Text
Braga E, Senchenko V, Bazov I, Loginov W, Liu J, Ermilova V, Kazubskaya T, Garkavtseva R, Mazurenko N, Kisseljov F, Lerman MI, Klein G, Kisselev L, Zabarovsky ER: Critical tumor suppressor gene regions on chromosome 3 regions in major human epithelial malignancies: allelotyping and quantitative real time PCR.
Int J Cancer 2002, 100:534-541. PubMed Abstract | Publisher Full Text
Protopopov A, Kashuba V, Zabarovska VI, Muravenko OV, Lerman MI, Klein G, Zabarovsky ER: An integrated physical and gene map of the 3.5-Mb chromosome 3p21.3 (AP20) region implicated in major human epithelial malignancies.
Cancer Res 2003, 63:404-412. PubMed Abstract | Publisher Full Text
Senchenko V, Liu J, Braga E, Mazurenko N, Loginov W, Seryogin Y, Bazov I, Protopopov A, Kisseljov FL, Kashuba V, Lerman MI, Klein G, Zabarovsky ER: Deletion mapping of cervical carcinomas using quantitative real time PCR identifies two frequently affected regions in 3p21.3.
Oncogene 2003, 22:2984-2992. PubMed Abstract | Publisher Full Text
Senchenko VN, Liu J, Loginov W, Bazov I, Angeloni D, Seryogin Y, Ermilova V, Kazubskaya T, Garkavtseva R, Zabarovska VI, Kashuba VI, Kisselev LL, Minna JD, Lerman MI, Klein G, Braga EA, Zabarovsky ER: Discovery of frequent homozygous deletions in chromosome 3p21.3 LUCA and AP20 regions in renal, lung and breast carcinomas.
Oncogene 2004, 23:5719-5728. PubMed Abstract | Publisher Full Text
Dammann R, Li C, Yoon JH, Chin PL, Bates S, Pfeifer GP: Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3.
Nat Genet 2000, 25:315-319. PubMed Abstract | Publisher Full Text
Kashuba VI, Li J, Wang F, Senchenko VN, Protopopov A, Malyukova A, Kutsenko AS, Kadyrova E, Zabarovska VI, Muravenko OV, Zelenin AV, Kisselev LL, Kuzmin I, Minna JD, Winberg G, Ernberg I, Braga E, Lerman MI, Klein G, Zabarovsky ER: RBSP3 (HYA22) is a tumor suppressor gene implicated in major epithelial malignancies.
Proc Natl Acad Sci USA 2004, 101:4906-4911. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Li J, Wang F, Protopopov A, Malyukova A, Kashuba V, Minna JD, Lerman MI, Klein G, Zabarovsky E: Inactivation of RASSF1C during in vivo tumor growth identifies it as a tumor suppressor gene.
Oncogene 2004, 23:5941-5949. PubMed Abstract | Publisher Full Text
Yeo M, Lin PS, Dahmus ME, Gill GN: A novel RNA polymerase II C-terminal domain phosphatase that preferentially dephosphorylates serine 5.
J Biol Chem 2003, 278:26078-26085. PubMed Abstract | Publisher Full Text
Kashuba VI, Pavlova TV, Grigorieva EV, Kutsenko A, Yenamandra SP, Li J, Wang F, Protopopov AI, Zabarovska VI, Senchenko V, Haraldson K, Eshchenko T, Kobliakova J, Vorontsova O, Kuzmin I, Braga E, Blinov VM, Kisselev LL, Zeng YX, Ernberg I, Lerman MI, Klein G, Zabarovsky ER: High mutability of the tumor suppressor genes RASSF1 and RBSP3 (CTDSPL) in cancer.
PLoS ONE 2009, 4:e5231. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Li J, Wang F, Haraldson K, Protopopov A, Duh FM, Geil L, Kuzmin I, Minna JD, Stanbridge E, Braga E, Kashuba VI, Klein G, Lerman MI, Zabarovsky ER: Functional characterization of the candidate tumor suppressor gene NPRL2/G21 located in 3p21.3C.
Cancer Res 2004, 64:6438-6443. PubMed Abstract | Publisher Full Text
Ueda K, Kawashima H, Ohtani S, Deng WG, Ravoori M, Bankson J, Gao B, Girard L, Minna JD, Roth JA, Kundra V, Ji L: The 3p21.3 tumor suppressor NPRL2 plays an important role in cisplatin-induced resistance in human non-small-cell lung cancer cells.
Cancer Res 2006, 66:9682-9690. PubMed Abstract | Publisher Full Text
Ji L, Nishizaki M, Gao B, Burbee D, Kondo M, Kamibayashi C, Xu K, Yen N, Atkinson EN, Fang B, Lerman MI, Roth JA, Minna JD: Expression of several genes in the human chromosome 3p21.3 homozygous deletion region by an adenovirus vector results in tumor suppressor activities in vitro and in vivo.
Cancer Res 2002, 62:2715-2720. PubMed Abstract | Publisher Full Text
Otani S, Takeda S, Yamada S, Sakakima Y, Sugimoto H, Nomoto S, Kasuya H, Kanazumi N, Nagasaka T, Nakao A: The tumor suppressor NPRL2 in hepatocellular carcinoma plays an important role in progression and can be served as an independent prognostic factor.
J Surg Oncol 2009, 100:358-363. PubMed Abstract | Publisher Full Text
Wei MN, Latif F, Bader S, Kashuba V, Chen JY, Duh FM, Sedico Y, Lee CC, Geil L, Kuzmin I, Zabarovsky E, Klein G, Zbar B, Minna JD, Lerman MI: Construction of a 600-kilobase cosmid clone contig and generation of a transcriptional map surrounding the lung cancer tumor suppressor gene (TSG) locus on human chromosome 3p21.3: progress toward the isolation of a lung cancer TSG.
Cancer Res 1996, 56:1487-1492. PubMed Abstract | Publisher Full Text
Dreijerink K, Braga E, Kuzmin I, Geil L, Duh FM, Angeloni D, Zbar B, Lerman MI, Stanbridge EJ, Minna JD, Protopopov A, Li J, Kashuba V, Klein G, Zabarovsky ER: The candidate tumor suppressor gene RASSF1A from human chromosome 3p21.3 is involved in kidney tumorigenesis.
Proc Natl Acad Sci USA 2001, 98:7504-7509. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Kuzmin I, Gillespie JW, Protopopov A, Geil L, Dreijerink K, Yang Y, Vocke CD, Duh FM, Zabarovsky E, Minna JD, Rhim JS, Emmert-Buck MR, Linehan WM, Lerman MI: The RASSF1A tumor suppressor gene is inactivated in prostate tumors and suppresses growth of prostate carcinoma cells.
Cancer Res 2002, 62:3498-3502. PubMed Abstract | Publisher Full Text
Inda MM, Castresana JS: RASSF1A promoter is highly methylated in primitive neuroectodermal tumors of the central nervous system.
Neuropathology 2007, 27:341-346. PubMed Abstract | Publisher Full Text
Hesson LB, Cooper WN, Latif F: Evaluation of the 3p21.3 tumour-suppressor gene cluster.
Oncogene 2007, 26:7283-301. PubMed Abstract | Publisher Full Text
Burbee DG, Forgacs E, Zöchbauer-Müller S, Shivakumar L, Fong K, Gao B, Randle D, Kondo M, Virmani A, Bader S, Sekido Y, Latif F, Milchgrub S, Toyooka S, Gazdar AF, Lerman MI, Zabarovsky E, White M, Minna JD: Epigenetic inactivation of RASSFA1 in lung and breast cancers and malignant.
J Natl Cancer Inst 2001, 93:691-699. PubMed Abstract | Publisher Full Text
Honorio S, Agathanggelou A, Schuermann M, Pankow W, Viacava P, Maher ER, Latif F: Detection of RASSF1A aberrant promoter hypermethylation in sputum from chronic smokers and ductal carcinoma in situ from breast cancer patients.
Oncogene 2003, 22:147-150. PubMed Abstract | Publisher Full Text
Dammann R, Schagdarsurengin U, Seidel C, Strunnikova M, Rastetter M, Baier K, Pfeifer GP: The tumor suppressor RASSF1A in human carcinogenesis: an update.
Histol Histopathol 2005, 20:645-663. PubMed Abstract | Publisher Full Text
Agathanggelou A, Cooper WN, Latif F: Role of the Ras-association domain family 1 tumor suppressor gene in human cancers.
Cancer Res 2005, 65:3497-3508. PubMed Abstract | Publisher Full Text
Pan ZG, Kashuba VI, Liu XQ, Shao JY, Zhang RH, Jiang JH, Guo C, Zabarovsky E, Ernberg I, Zeng YX: High frequency somatic mutations in RASSF1A in nasopharyngeal carcinoma.
Cancer Biol Ther 2005, 4:1116-1122. PubMed Abstract | Publisher Full Text
Vos MD, Ellis CA, Bell A, Birrer MJ, Clark GJ: Ras uses the novel tumor suppressor RASSF1 as an effector to mediate apoptosis.
J Biol Chem 2000, 275:35669-35672. PubMed Abstract | Publisher Full Text
Ortiz-Vega S, Khokhlatchev A, Nedwidek M, Zhang XF, Dammann R, Pfeifer GP, Avruch J: The putative tumor suppressor RASSF1A homodimerizes and heterodimerizes with the Ras-GTP binding protein Nore1.
Oncogene 2002, 21:1381-1390. PubMed Abstract | Publisher Full Text
Shivakumar L, Minna JD, Sakamaki T, Pestell RG, White MA: The RASSF1A tumor suppressor blocks cell cycle progression and inhibits cyclin D1 accumulation.
Mol Cell Biol 2002, 22:4309-4318. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Sinha S, Singh RK, Alam N, Roy A, Roychoudhury S, Panda CK: Frequent alterations of hMLH1 and RBSP3/HYA22 at chromosomal 3p22.3 region in early and late-onset breast carcinoma: clinical and prognostic significance.
Cancer Sci 2008, 99:1984-1991. PubMed Abstract | Publisher Full Text
Wang F, Grigorieva EV, Li J, Senchenko VN, Pavlova TV, Anedchenko EA, Kudryavtseva AV, Tsimanis A, Angeloni D, Lerman MI, Kashuba VI, Klein G, Zabarovsky ER: HYAL1 and HYAL2 inhibit tumour growth in vivo but not in vitro.
PLoS ONE 2008, 3:e3031. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Speissel B, Beahrs OH, Hermanek P, Hutter RVP, Scheibe O: TNM atlas: illustrated guide to the TNM/pTNM classification of malignant tumours. 3rd edition. Springer-Verlag Berlin New York; 1989.
Travis WD, Coby TV, Corrin B, Shimosato Y, Brambilla E: World Health Organization International Histological Classification of Tumours; Histological typing of lung and pleural tumours. 3rd edition. Springer Berlin; 1999.
Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-ΔΔCT) method.
Methods 2001, 25:402-408. PubMed Abstract | Publisher Full Text
Relative Quantitation of Gene Expression [http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf] webcite
Li J, Protopopov A, Wang F, Senchenko V, Petushkov V, Vorontsova O, Petrenko L, Zabarovska V, Muravenko O, Braga E, Kisselev L, Lerman MI, Kashuba V, Klein G, Ernberg I, Wahlestedt C, Zabarovsky ER: NotI subtraction and NotI-specific microarrays to detect copy number and methylation changes in whole genomes.
Proc Natl Acad Sci USA 2002, 99:10724-10729. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Dunwell TL, Hesson LB, Pavlova T, Zabarovska V, Kashuba V, Catchpoole D, Chiaramonte R, Brini AT, Griffiths M, Maher ER, Zabarovsky E, Latif F: Epigenetic analysis of childhood acute lymphoblastic leukemia.
Epigenetics 2009, 4:185-193. PubMed Abstract | Publisher Full Text
Glantz SA: Primer of biostatistics. 4th edition. McGraw-Hill New York; 1994.
Li J, Protopopov AI, Gizatullin RZ, Kiss C, Kashuba VI, Winberg G, Klein G, Zabarovsky ER: Identification of new tumor suppressor genes based on in vivo functional inactivation of a candidate gene.
FEBS Lett 1999, 451:289-294. PubMed Abstract | Publisher Full Text
Protopopov AI, Li J, Winberg G, Gizatullin RZ, Kashuba VI, Klein G, Zabarovsky ER: Human cell lines engineered for tetracycline-regulated expression of tumor suppressor candidate genes from a frequently affected chromosomal region, 3p21.
J Gene Med 2002, 4:397-406. PubMed Abstract | Publisher Full Text
Shu J, Jelinek J, Chang H, Shen L, Qin T, Chung W, Oki Y, Issa JP: Silencing of bidirectional promoters by DNA methylation in tumorogenesis.
Cancer Res 2006, 66:5077-5084. PubMed Abstract | Publisher Full Text
Mitra S, Mazumder Indra D, Bhattacharya N, Singh RK, Basu PS, Mondal RK, Roy A, Zabarovsky ER, Roychoudhury S, Panda CK: RBSP3 is frequently altered in premalignant cervical lesions: Clinical and prognostic significance.
Genes Chromosomes Cancer 2010, 49:155-170. PubMed Abstract | Publisher Full Text
Anedchenko EA, Kiseleva NP, Dmitriev AA, Kiselev FL, Zabarovsky ER, Senchenko VN: Tumor suppressor gene RBSP3 in cervical carcinoma: copy number and transcriptional level.
Frigola J, Song J, Stirzaker C, Hinshelwood RA, Peinado MA, Clark SJ: Epigenetic remodeling in colorectal cancer results in coordinate gene suppression across an entire chromosome band.
Nat Genet 2006, 38:540-549. PubMed Abstract | Publisher Full Text
Gao C, Furge K, Koeman J, Dykema K, Su Y, Cutler ML, Werts A, Haak P, Woude GF: Chromosome instability, chromosome transcriptome, and clonal evolution of tumor cell populations.
Proc Natl Acad Sci USA 2007, 104:8995-9000. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Smith IM, Glazer CA, Mithani SK, Ochs MF, Sun W, Bhan S, Vostrov A, Abdullaev Z, Lobanenkov V, Gray A, Liu C, Chang SS, Ostrow KL, Westra WH, Begum S, Dhara M, Califano J: Coordinated activation of candidate proto-oncogenes and cancer testes antigens via promoter demethylation in head and neck cancer and lung cancer.
PLoS ONE 2009, 4:e4961. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Oyama T, Osaki T, Baba T, Nagata Y, Mizukami M, So T, Nakata S, Ichiki Y, Uramoto H, Sugaya M, Yoshimatsu T, Morita M, Hanagiri T, Sugio K, Kawamoto T, Yasumoto K: Molecular genetic tumor markers in non-small cell lung cancer.
Anticancer Res 2005, 25:1193-1196. PubMed Abstract | Publisher Full Text
Petty RD, Nicolson MC, Kerr KM, Collie-Duguid E, Murray GI: Gene expression profiling in non-small cell lung cancer: from molecular mechanisms to clinical application.
Clin Cancer Res 2004, 10:3237-3248. PubMed Abstract | Publisher Full Text
Pre-publication history
The pre-publication history for this paper can be accessed here:


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Can Kate Hardcastle save the Bradford Bulls in Super League?
Award-winning business woman, founder of a charity, semi-professional singer, guest speaker, "mum of the year" to a three-year-old daughter - and now the only female director in the Super League.
Kate Hardcastle has achieved many things in her life but could she be the person to propel Bradford Bulls into the future?
A little under a year ago, the four-time Super League champions were on the brink of extinction and were forced to appeal to fans to raise £1m to keep the club afloat.
In June they went into administration and were hanging on to their future in the Super League .
A life-long fan, Hardcastle admits that she thought her team might cease to exist.
Hardcastle's varied ventures
- At 18-years-old, Kate Hardcastle was the lead singer of The Fabulous BB Kings and also their manager.
- She was in other bands - Bare Faced Cheek and Kiss Me Kate - and still sings semi-professionally.
- Hardcastle is a founding partner of Insight with Passion which evaluates and improves businesses with a focus on branding and marketing.
- In 2007 she founded Charity Dreamgirls which helps promote and support smaller charities across the UK.
- In 2012 her charity launched 'Positive Image' - a campaign to inspire confidence.
- Hardcastle has won awards such as Innovator of the Year and Outstanding Business Woman.
- She has a three-year-old daughter and in January 2013 she was named Tesco Mum of Year.
- Hardcastle is a non-executive director of the Bradford Bulls.
She says: "I rushed my daughter there last year, actually thinking 'my goodness me, I've got to take her because this genuinely might be it, this might be the end' - and that filled me with dread."
Hardcastle explains that she was first taken to Odsal as a two-year-old, by her father, Martin, and her grandfather.
"I was taken in at half time because it was free then so there was no concern that if I didn't enjoy it they would have wasted the money," she says.
"But I did enjoy it. I loved the buzz of it."
And so her love affair with the club began - a love which has seen her step in to become a non-executive director on the board, alongside owner Omar Khan, who bought the club in August to secure its future.
She says her father will be keeping a close eye on the job she is doing.
"He said "don't mess it up"," she says.
"I have got a guy, with Bradford Bulls running through him, who will be sat with me at Sunday dinner poking me saying 'right what are you doing next? What are you going to do about this?'
"I get emails at three o clock in the morning from him. I'm like 'dad for goodness sake, we are going as fast as we can'."
Hardcastle, 36, was the lead singer in a successful band The Fabulous BB Kings, and still sings semi-professionally. She has her own company - "Insight with Passion" - which helps revive businesses and she has also set-up Charity Dreamgirls , which helps promote smaller charities across the UK. This woman of many talents has also recently been named Tesco Mum of the Year for 2013.
She explains her main roles at Bradford Bulls will include looking at the branding of the club, attracting more families and appealing to women to get involved in the sport.
She says: "I am trying to understand what the future of rugby league actually is and it's about inclusivity for me.
"We have a 40% female fan base, the biggest in the Super League, so I thought there was huge potential to understand how we could get more female fans, and make sure that the female fans we have are enjoying it.
"We want to make sure that all of the fans understand the brand and are getting excited about the brand again and that we were taking that message through to grassroots, because those are the children who are going to be the fans of the future."
A BBC Inside Out report in January revealed that Super League clubs are facing combined debts of £68.5m, and - like the Bulls - Salford City Reds were battling financial problems until they were bought out on the eve of the new season .
But is this a sport which can be saved?
She says: "It's only going to happen if it understands that the future is about evolution.
"It's got to understand that the future isn't just about doing what we've always done, and cross our fingers and tighten our belts a bit and it's going to be OK.
"And I think the Bradford Bulls are starting to show some indication and some signs that that's going to be the case."
She explains some of her ideas to bring about her vision of a real family day out to see the Bulls - something which she has seen is possible whilst working in Australia and attending cricket matches and rugby matches in the country.
She says the club will offer childcare facilities on match days so families with young children can still attend the game, she will look at food options ("not just Bovril and a pie") and she has already asked the fans about what merchandise they would like to see available.
On the subject of evolution, Hardcastle says that it is "crazy" that she is the only woman on a Super League board.
Kate Hardcastle on comparisons with Karren Brady, Vice-Chairman of West Ham United
"Karren Brady is on the same speaking circuit as me, we both do seminars and speeches, and it was actually very different for her.
"I find the comparison quite challenging sometimes because she was brought in at a very young age to a football club, without having gone and proven herself as a business person and then went on to prove herself brilliantly. I have gone out and been a business woman and I'm being brought in to help a board with my expertise.
"So what the comparison basically is, is that we are business women in sport and that's really sad actually because that's about as far as it goes. I think that just proves there are not enough women in there and we've got to make a change there. "
She says: "If those teams are going to start to attract a different audience - and women are a fantastic audience to attract because they are not necessarily being appealed to through the other sports as well - then you need to understand women.
"You need that balance on the board.
"The right person has to do the job, regardless of sex, but if women never actually get the chance to get through, because something is deemed to be male dominated or a man's job, how are you going to be able to prove that you are the right person to do the job anyway?"
Although Hardcastle is not the first woman to grace a Super League boardroom - Kath Hetherington was chairperson of Hull FC for 12 years between 1999 and 2011 - she knows it will be a challenge.
"The fans have been very supportive in the main so far, as have the players and of course the board," she says.
"I imagine, if you go digging for it, there would be the odd comment on a forum or whatever about the fact that I'm a woman or the fact that I look a certain way - I don't know.
"No one is going to push me out of this sport.
"It's been my sport since I was two so I will be delivering on my job and when I feel I've succeeded at that job, and I've had the time to do that, then we can have a discussion about how I've got on."


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Get the most out of your car at the pumps and on the road with smartphone apps that measure fuel economy and performance. We put five of the best of both to the test. Whether you're concerned about the cost of filling up, or just want to impress your mates with your car's performance figures, there's almost certainly a smartphone app that can help.
There's a host of data loggers that can be used to record your car's acceleration and calculate fuel economy, but which is best for each task? We downloaded a mix of free and paid-for apps to find the ideal driving companions. How did we test them, you ask? Well, to rate ease of use, we scrolled through the menus and looked for clear interfaces and legible text.
Our MPG calculators had to give accurate reports of our mileage that were easy to understand. Bonus points went to those that could be used to track maintenance costs. All apps scored highly if you could share data via E-mail or social networking sites, and value for money was our final consideration.
1. g-tac free
Price: Free
Available for: iOS
The superb g-tac free is hard to beat. While there are two paid for apps in the g-tac stable – a £6.99version adds a horsepower calculator, while the £13.99 pro version gets rid of adverts – this free app is more than enough for the average motorist. It's easy enough to get started, with clear instructions, and it provides comprehensive data from your maximum speed, brake distances and acceleration times, all accurate to 5mph.
2. Dynolicious
Price: £5.99
Available for: iOS
Unlike g-tac, there's no free version of Dynolicious, but it's worth paying for. You can export your results via E-mail, Facebook or Twitter, and you can save runs from more than one car as well. The display looks great, and it's simple to use, plus was accurate to within 1mph of our VBOX. Track-day enthusiasts will also like the skidpan measurements, which show g-forces pulled in corners.
3. PocketDyno
Price: Free
Available for: iOS
PocketDyno put in a strong challenge to our top two, as it's extremely easy to understand, and you get clear instructions about how to start – either tap to get going, or double tap for a traffic light countdown. However, you can't store the results of more than one car, and there are far too many adverts. It was accurate to within 5mph of our VBOX, and provided plenty of data, but you can't export any of it.
Price: £2.49
Available for: iOS
For this price, we expected more than just a speedometer. The display is clearer than Speedo HD Free, and the interface is really slick. We also love the fact you can put your iPhone on the dash and the app will act as a head-up display. But it doesn't provide as much data as the other apps, and you can't export any data. Almost as accurate as the Dynolicious, though.
Price: Free
Available for: iOS
We hoped that revisiting this app after a year would have given the developer time to address our previous concerns, but sadly that's not the case. The app only shows your speed, and worse still, it didn't reflect what was on our VBOX– the app was out by about 7mph. There's no head-up display, and you can't record any acceleration or top speed, so this app could never hope to challenge for top honours.
MPG Calculators
1. Gas Manager
Price: Free
Available for: Android, iOS
A recent update propels Gas Manager to victory. The interface looks great, and we love how it displays the uploaded picture of your car. There are plenty of graphs to help track your mpg, and inputting info is simple, as is exporting reports. Unlike our recommended choice Road Trip (right), it can only track your fuel consumption, but it does show nearby fuel stations and car parks. Plus it's free, and is available for Android and iOS.
2. Road Trip
Price: £2.99
Available for: iOS
A great app. Inputting data is simple, as is exporting your reports. You can upload a picture of your car, and use the app to record info on more than one vehicle. The summary crams all the info on to one page, and you can tilt your iPhone to view each graph in landscape mode. It's the most expensive of the mpg calculators, but you can use it to track garage costs, too.
3. Car Care
Price: £1.99
Available for: iOS
Looks as good as Gas Manager, and lets you input maintenance costs plus fill-ups. It's not free like our Best Buy, but it's slightly cheaper than Road Trip. The data entry process is a little more complicated, and we don't like the fact you have to search for costs – mpg is the main focus on this app. But the pitstop feature, which times your fill-up, is fun, and we like the way your car's picture becomes your background.
4. Gas Cubby
Price: £1.49
Available for: iOS
Gas Cubby differs from our top two as you have to enter your car's make and model before getting started – and our 2012 Peugeot wasn't on the list, so we had to input the manufacturer's claimed figures manually. Still, it's very easy to understand, and the menus are clear – if a little dull compared to rivals'. And while you can export reports, it's slightly irritating that you have to enter the time of each fill-up.
5. AccuFuel
Price: 69p
Available for: iOS
We were disappointed with AccuFuel. The display is clear, but you have to scroll individual wheels (rather than use the keypad) to enter your mileage, which takes too much time. We'd also prefer it if you could enter the total cost, rather than the pence per litre price, and the graphs are rather simplistic. You can't assign pictures of your cars to their reports, but at least you can export data.
I presume this article is a joke?
Someone clearly forgot they were supposed to be checking iOS and Android and it seems the author only remembered at the last min to do both and not carry on making love to his/her iphone.
Where is the Torque Pro app? Constantly voted as one of the best on most other motoring websites yet AE doesn't seem to know it exists. That does all you have wanted above and more!
I guess we shouldn't expect anything less from AE, I mean I'm still waiting to hear when we can get the mag in Google Magazines, only been asking since January and was told the summer, well that's been and gone and still don't have the decency to reply to me about it.
Andy your comment is spot on, the Torque Pro app is probably the best car related app in the world, unless like the author you must have a photo of your car uploaded.
On a lighter note, the car in the picture is a Gen1 Mazda 6 (could be any trim above the S). Plus it's in left hand drive trim.
GhosTrack is the perfect application for speed calculation
in Android Platform. Not only speed actually GhosTrack is a multipurpose
application. You can track anyone simply using it. You can track location,
speed, distance even you can spy over someone by just simply activating spy
mode. Its available on google play try it now.


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Tag: Bonnaroo
2012 Music Festival Survival Guide – Bonnaroo
By Nathan Erb on May 30, 2012 at 11:55 AM
In just a day or two, a 700-acre farm in Manchester, Tenn., will draw up to 80,000 people from all over the world for four days of music, art, film and entertainment. The name "Bonnaroo" is slang for "a really good time", and was chosen both for its literal meaning and to honor the rich […]
Posted in: Odds & Ends
Outdoor jammin', part 2: Bonnaroo Music and Arts Festival
By Nathan Erb on June 7, 2010 at 9:00 AM
June 10-13, 2010: Manchester, TN Four days of not showering, sleeping in tents in a field and walking miles in the Tennessee sun in June may not be for everyone. But for me and up to 80,000 music lovers, Bonnaroo is a unique experience that makes you eager to come back every year. Each June, […]
Posted in: On the Web


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Mortazavi is an assistant professor of developmental & cell biology in the School of Biological Sciences. His project will explore how DNA codes the precise activities of genes involved with development. His lab will create methods to measure how this gene expression is affected by changes in the organization of DNA in embryonic stem cells as they differentiate into neurons and cardiac muscle cells. Mortazavi believes his research will identify fundamental principles of gene regulation as well as the specific DNA elements critical to stem cell differentiation.
The New Innovator Award highlights two important goals of the NIH by stimulating highly original research and supporting promising new investigators. In 2013, under its High Risk-High Reward program, the NIH is awarding 12 Pioneer Awards, 41 New Innovator Awards, 10 Transformative Research Awards and 15 Early Independence Awards. The total funding, which represents contributions from the NIH Common Fund and multiple NIH institutes and centers, is approximately $123 million.
|Contact: Cathy Lawhon
University of California - Irvine


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Incumbents continued to hold strong in the Senate as lawmakers retained their seats and candidates battled to fill the seats of term-limited legislators.
Oklahoma elections: Senate races
Three incumbents kept their seats and primary elections decided the outcome in six races in the Senate. Only one Republican incumbent will face a Democrat challenger in November.
Republican Sen. Anthony Sykes, of Moore, beat his opponent Linda Molsbee with 5,060 votes, or 77 percent of the vote, with all precincts reporting, according to the state Election Board.
In Senate District 28, Sen. Harry Coates, R-Seminole, beat Tim Clem by getting 2,898 votes, or 54 percent of the vote, with all precincts reporting. Clem received 2,434 votes, or 46 percent of the vote.
Democrat Sen. Connie Johnson held her northeastern Oklahoma City seat and avoided a runoff election by receiving 4,293 votes, or 68 percent of the vote, in a race that included three other opponents.
Sen. Cliff Aldridge, R-Midwest City, beat James Lane with 4,213 votes, or 76 percent of the vote, with all precincts reporting. Aldridge will face Democrat Mike Kelly in the fall.
In another Oklahoma City district, David Holt, chief of staff to the Oklahoma City mayor, got 5,125 votes, or 64 percent of the vote, in the race to fill the Senate District 30 seat vacated by term-limited Senate President Pro Tem Glenn Coffee. Holt, a Republican, wins the seat since there is not Democrat challenger.
The primary also determined the winner of the Senate District 22 race where Rep. Rob Johnson, R-Kingfisher, received 5,214 votes, or 54 percent of the vote, in a field of three candidates to fill his father Sen. Mike Johnson's Senate seat. The elder Johnson is prohibited from running again because of term limits.
Across the state there were several battles among Republican and Democrat candidates seeking their party's nominations to fill seats vacated by term-limited lawmakers and legislators seeking a higher office.
In the Tulsa area, Republican Rick Brinkley beat Tim Coager with 65 percent of the vote to fill the Senate District 34 seat vacated by Sen. Randy Brogdon, R-Owasso. Brogdon lost the Republican nomination for governor to U.S. Rep. Mary Fallin on Tuesday.
Continue reading this story on the...


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How Safe is Your Hospital?
Updated: 07/31/2015 - We trust hospitals to help make us well. What we don't expect is to get sick in a hospital. But every year about 648,000 hospital patients develop infections during their stay and about 75,000 die. Consumer Reports latest research finds that while some hospitals have been successful at cutting their infection rates, many have not.