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The quiet heroes: The Falklands War medics who had to amputate limbs with a Swiss Army penknife - and no anaesthetic
No one writes about them. Their deeds go unsung. Which is why, for this gripping series, two writers decided to track down the awe-inspiring stories of the Army medics who have saved countless lives in recent British wars.
Yesterday, in the second part of our gripping series about the doctors and nurses who go into the heart of battle to save the wounded and dying, we revealed the exceptional bravery of the RAF nurse in Basra who saved a gunner engulfed in flames after his tank was petrol bombed. Today, in the third instalment, we go back to 1982 and the freezing cold and mud of the Falklands, where courageous Para medics stopped at nothing — including amputating a leg in the middle of a battlefield using nothing but a Swiss Army knife — to save the lives not only of their comrades, but those of the Argentinian enemy, too.
As he advanced with his battalion through a barrage of enemy mortar shells towards the hamlet of Goose Green, Para medical officer Captain Steven Hughes desperately wanted to be on some other enterprise.
'Beam me up, Scotty,' he muttered into the drizzle of the South Atlantic winter, recoiling as a round from a sniper's rifle whistled over his head.
Flying the flag: 2 Para celebrate victory at Goose Green, but the battle saw many of their comrades killed or wounded
'Take me back to the NHS and I'll work 24 hours a day, 365 days a year,' he pledged. 'This is not what I spent all those years at medical school for.'
When appointed as an Army doctor, he had been treated as a waste of space by the macho fighting men of the Paras, as insignificant as a cook or a clerk — until they went to war.
On the British Task Force's long sea journey south to retake the Falkland Islands from their Argentinian occupiers, he watched these tough guys wake up to his importance to them.
As he drilled them in the basics of first aid and what to do if their bellies were split open by a bullet and their guts spilled out, he sensed respect. 'I was no longer considered an idle knacker, but someone who might well save their life,' he said. And now he was in the killing zone with them, doing just that.
He set up his aid post in a gully. It was freezing cold and the smell of cordite was acrid in the air. There was no shelter, not a single tree and scarcely a bush. It was hard to believe how recently he had been working in the warm and dry, in an over-heated London hospital.
The battle was just yards away. Gunfire was too close for comfort. Incoming mortars were as loud as express trains.
Wounded soldiers were being brought in all the time, and Hughes and his small team of medics moved between the rows on the ground, dressing injuries and administering fluids, morphine and antibiotics.
Deciding priorities was heartrending. Every case was urgent, but which ones should he concentrate on?
He knelt over a soldier with multiple cannon fragments in his side and neck, and air leaking through punctures in his throat. His shredded battledress was like blotting paper soaked in his own blood.
Another soldier was half-hidden under a sheet of corrugated metal with part of his brain squeezing out of his head like toothpaste from a tube. He was still conscious.
Surprisingly, he was the least of Hughes's worries. This soldier could wait, he decided. 'If he's survived this long, he isn't in immediate danger.'
In any case, there wasn't a brain specialist at the Task Force's field hospital — a ramshackle warehouse once used for packing mutton — a helicopter ride away at Ajax Bay. The surgeons there would be better off operating on someone with a more easily treatable wound.
Some of his assistants misunderstood and thought Hughes had put the man to one side to die. One was preparing to do the humane thing and give him a fatal dose of morphine.
Hughes intervened just in time. Eventually, the man got his place on a helicopter. He survived. But, on the battlefield and in the fog of war, that was how narrow the line was between life and death.
Lance Corporal Bill Bentley, a medic assisting Hughes, was learning this firsthand as, with a medical pack on his back and Sterling sub-machine gun slung over his shoulder, he went out into the fighting to find casualties.
Fierce: Smoke rises Paras fight to gain victory in the Falklands War
'You're driven on by the thought that your mates are out there, dying and injured,' he said.
One of those he brought back was an old friend, his brains blown out. As he carried a corner of the poncho in which the body was laid, Bentley's knee kept banging against the dead soldier's slumped head. It made a hollow, knocking sound he would never be able to erase from his memory.
On another foray, he leapt into an enemy trench and felt a body beneath a tarpaulin move under his feet. The soldier in him outweighed the medic and he fired into the bundle. This was no time to take chances.
But he did just that with another Argentinian soldier, a youngster shot through the leg and in deep shock.
'He made no aggressive gestures, so I hoisted him over my shoulder and carried him to our own lines,' he said.
Only later did he stop to consider that the enemy dangling down his back could have snatched his bayonet and killed him.
Even braver was his rescue of Private Dave 'Chopsey' Gray, who was bleeding to death in no-man's-land after a mortar shell had practically torn off his right leg, shattered the other and filled him with shrapnel. Bob Cole, one of Gray's mates,
distraught and in tears, managed to reach the aid post to beg for help. 'No problem, I'll go,' said Bentley.
He ducked down and followed Cole up a hill and through a gap in a stone wall. They could see Chopsey 25 yards away on the other side of the slope, lying in a shallow crater in a pool of his own blood and fully exposed to the enemies' guns. Bentley crept down to him.
Para legend has it that when Gray was hit, he shouted: 'I've lost me f***ing leg!' One of his mates behind a nearby tussock replied: 'No, you haven't, Chopsey. It's just landed over here.'
In fact, it was still attached, though not by much. When Bentley reached him, he realised that a desperate situation called for desperate measures. 'I would have to amputate what was left of his right leg.'
He took out his Swiss Army knife, one blade of which he kept razor sharp for emergencies. Lying flat as bullets flew over his head, he sliced away the flesh and sinew, which was all that was keeping the leg attached.
Gray 'just cringed' at this battlefield amputation without anaesthetic — shock must have gone some way to dull the pain.
The medic was tourniqueting the stump when a stretcher party came racing across the hillside towards him. The enemy fire intensified and more bullets peppered into the peat.
'The hill seemed to be exploding. It felt as if the whole Argentinian army was opening up at us,' said Bentley.
The rescue team threw Gray on to the stretcher and, not forgetting the severed leg, ran back up the hill, chased by a line of bullets detonating in the dirt behind them like fireworks.
Back at the aid post, Hughes fought desperately to save the wounded soldier, who had lost so much blood that the doctor couldn't find a vein in his deathly white skin. He had to cut away with a scalpel to make an opening for fluids.
Hughes put him at the top of the queue for immediate evacuation by helicopter. 'But I didn't hold out too much hope for his survival,' he said.
Bentley helped lift the man he had risked his life to save into a casualty pod on the chopper's side. The sawn-off remnant of his leg went, too.
'I don't think it was until that moment that he realised exactly what had happened,' said Bentley. 'He just stared at it.
'I hugged him, gave him a kiss and said: "You'll be all right." Then I closed the top of the pod and they flew him out.'
Defeated: Argentine PoWs being marched from Goose Green after surrendering
Gray was conscious when he was brought into Ajax Bay, breathing hard as if he had just run a marathon and gabbling nonsense.
He had no discernible pulse or blood pressure. When the tourniquet was removed, the leg stump was dry — no blood was getting through. He needed an immediate transfusion.
A medic grabbed some bags of blood — it was kept 'refrigerated' outside in the Antarctic cold, wrapped in wet hessian — and warmed them under his armpits to take off the chill before the blood was dripped into veins that were already beginning to collapse and close up.
Three litres later, on the sixth bag, the stump began to ooze blood. There was colour in Chopsey's cheeks — he was going to make it.
Night was falling at the end of a dreadful day. At Goose Green, the battle slowed and then halted. A desperately tired Hughes could at last take stock. His records, scrawled on notepads smeared with blood, showed he had treated 34 Paras and dozens of Argentinian casualties.
Stretched out in a gully behind a gorse bush were those who had been beyond saving. They looked, he thought, like shop dummies, except for the bullet hole in the middle of one forehead.
'It felt unreal. A few hours ago I had spoken to some of these people, and now they were gone,' he said.
As for all those casualties he had sent to Ajax Bay, he found it impossible to believe any of them could have survived, so basic had been the emergency care he felt he had been able to give them.
The next morning, he awoke to hear that the Argentinians had surrendered.
The battle of Goose Green — the first step in recovering the Falklands — was over.
Hughes did what he could for the enemy wounded, though they were mystified by his compassion.
'Why you treat me?' one asked in faltering English, as the doctor put him on a drip.
The answer was that it was his duty to treat everyone equally, friend or foe. The Geneva Convention demanded it of him, but he also demanded it of himself. All medics did.
The last Argentinian casualty was brought in, more dead than alive, and everyone worked to save him. He had lain on the battlefield for a day-and-a-half before he was discovered at the bottom of a pile of corpses in a waterlogged trench.
With a bullet in one eye, and a broken hand and leg, he was rigid with cold, but also running a fever.
He should have been dead from the gangrene that was eating into him, except that the cold had paralysed the infection and the fever had kept him from freezing. He was warmed up and given blood. When he was operated on, the anaesthetist ventilated him by hand for two hours, squeezing the life back into him breath by breath.
The soldier survived and went home, one-eyed and one-legged.
Others would not be going home. At Ajax Bay, the dozen dead Paras from Goose Green were lined up on stretchers side by side on a concrete strip.
It was a grim, sad business as their clothes were cut away and their personal possessions bagged to be returned to their relatives.
It fell to Surgeon Commander Rick Jolly, the doctor in charge, to examine each body, pronounce on cause of death and complete a field death certificate.
He crouched as he went from man to man and called out his conclusions to a clerk, who scribbled them down — gunshot wounds to the heart, multiofple wounds to chest and abdomen, blast injury.
On the face of the dead Colonel 'H' Jones, the 2 Para commander whose heroic but fatal dash had played a large part in breaking the Argentinian resistance, Jolly saw a quizzical smile. His eyes were open, but he looked peaceful. A single bullet had killed him. 'I closed his eyes gently. Then we lifted him, placed his body into a plastic shroud and then into a body bag.'
Shortly afterwards, Steven Hughes, his face grey with fatigue, flew in for the funerals.
He avoided Jolly's eyes, which surprised the surgeon. He knew and liked the Para doctor, and had heard nothing but praise for him from the casualties who had passed through his hands.
Jolly opened his mouth to speak, but was silenced by an outburst from Hughes, apologising, absurdly, for not having done enough in the middle of the battlefield and for sending back so many casualties in poor condition.
Jolly interrupted this flow of desperate remorse. Hughes had nothing to blame himself for. Every one of the wounded paratroopers ferried from Goose Green to the hospital was alive.
Hughes was incredulous. All the way in on the helicopter, he had been dreading discovering how many more his men had died from their injuries. Jolly gripped his arms and, almost shouting to get home his point, declared: 'No, Steve, no! They are all still alive.'
Hughes shook his head. It could not be so. He pulled out his field notebook and picked a name. 'What about this one? Private Gray?'
Jolly jumped in: 'Do you mean Chopsey? I looked after him myself. Virtually ex-sanguinated when he got here, but he's fine now.'
Hughes stared in amazement as the truth sank in. Then the colour returned to his cheeks, just as it had to Chopsey's. He was transfused with relief and joy.
All those battered young bodies he had administered to on the edge of a raging battle, patched up as best he could and then sent back down the line, were alive! But his sense of achievement did not last.
'We had been through an experience none of us had ever expected,' he would recall. He felt himself shutting down emotionally, blanking out the awful pictures of suffering. Later, years after he had left the Army, they would come back to haunt and overwhelm him.
He learned what few other people, soldiers or civilians, realise: that medics are steeped in the horrors of war in a way that no one else ever can be. All the carnage comes funnelling through them. They can't look the other way.
Other soldiers may see mates wounded or be injured themselves. But only the medics see the non-stop pain and misery of war. They run their eye over the butcher's bill and many of them, like Hughes, pay the price with their peace of mind.
- Extracted from Medic: Saving Lives From Dunkirk To Afghanistan by John Nichol and Tony Rennell, to be published by Viking on October 29 at £20. © John Nichol and Tony Rennell 2009. To order a copy at £18 (P&P free), call 0845 155 0720.
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subset: commoncrawl


text:
Dancer is one of the sub-types of the Fairest seeming. Whether entertainer, courtesan, artist or murderer, the Dancers are happiest when moving to the sound of their inner rhythms. As creatures of particular agility and grace for whom motion is itself beauty and art, theirs is the blessing of the Fae Grace.
One aspect of beauty is grace. Dancers embody this aspect in ways that many Fairest emulate, but few can reach. Lightfooted, elegant and swift, a Dancer is the very archetype of the faerie as something more graceful than a human can hope to be. Though Dancers' durances were no more pleasant or fondly remembered than those of any other Lost, Dancers are unable to fully resent the gifts of agility and poise they received in Arcadia.
The process of attaining these gifts may have been tortuous and best forgotten, but the grace lingers with them every minute of every day. Therefore, a Dancer is often among the camp of Lost that tries to see the best of what they've become, making the most of their blessings even if they regret having to go to Arcadia to receive them. Of course, for those not as beautiful and elegant as a Dancer, it's a hard line to swallow.
The most distinguishing feature of a Dancer is her agility. Every physical motion is imbued with added grace, no matter how small. This is a quality that cannot be entirely disguised by the Mask — Dancers stand out in a crowd, if anyone is watching. Even the way they neatly place their feet when walking is an indication of their instinctive agility and precision. Beyond that shared preternatural grace, though, Dancers vary widely in appearance. Some are pale and faintly luminescent, almost ethereal; others have pale skin marked with whorls of color.
Many Dancers were literally taken to dance. They spent endless nights in glass ballrooms, waltzing with the Fae until the Dancers' feet bled. They capered on stages, tied to invisible strings. They were placed on red-hot bronze so that their Keepers could watch their cavorting. Sometimes Dancers were taken to act as partners, sometimes just to be display objects.
Other Dancers were given the blessing of great agility for other purposes, perhaps salacious. A human taken as a sexual slave may not have been sufficiently… limber to entertain a Keeper without some modifications. The castle of a Keeper might have been built of narrow swords laid across deep chasms, where the servants were required to learn exquisite balance simply to keep their lives. Whatever the reason a Dancer was taken, his preternatural grace may have been the result of arduous effort, odd elixirs or bizarre surgeries.
Dancing is a common element in faerie folklore. Traditional fairy-tale elements may make interesting components when recast in the shadowed language of the Gentry — red shoes, rings of white mushrooms, secret dancing halls deep under the earth. Many faeries are described as dancing rather than walking when seen, and a Dancer may have had to become what she is simply to keep up.
May only wear cloth shoes, cannot wear gold jewelry, enraptured by harpsichord music, may not eat beef or mutton, must laugh when hears laughter, must keep a feather on her person at all times
- Changeling: The Lost (Rulebook), p. 114
- Winter Masques, p. 81


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Paarl (South Africa): India's Sachin Tendulkar added another feather to his glittering cap when he became the leading scorer in World Cup history at the Boland Park on Wednesday. The 29-year-old crossed Pakistani Javed Miandad's tally of 1,083 runs when he reached 25 against the Netherlands in the group 'A' league match.
Tendulkar, playing his 304th One-dayer, began the tournament with more runs (11,546) and more centuries (33) than any batsman in Limited Overs cricket. He has also scored 8,811 runs from 105 Tests with 31 centuries.


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Living Colors Moss Bathroom Towels Collection
Keep your bathroom fully stocked with style and comfort when you outfit it with Living Colors™ towels in Moss. Crisp color provides an airy and fresh feel to a space to inspire relaxation and tranquility. With everything to keep you clean and dry from head to toe these towels are a must have addition to any home. These versatile towels can stand alone or be paired with other colors in the Living Colors collection to create a look all your own.
Set up the look of your bathroom décor with this earthy green bath towel. This 100% cotton bath towel dries quickly to stay fresh for the ultimate comfort and convenience.
The natural color of this green hand towel is a great way to freshen up your bathroom décor! 100% cotton and 100% perfect for your fun style. Wash, rinse, dry and repeat!
Grab a natural, green wash cloth and create a light, clean theme for your bathroom décor. Its soft absorbency will ensure it's more than just a good looking piece of décor!


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The Pelican Egg is a item that first appears in Grand Mario Land U. This item transforms Mario into Pelican Mario.
The Pelican Egg is a egg with eyes. The Pelican Egg also has two tiny wings on both of its sides.
Grand Mario Land U
Pelican Eggs first appear here. These eggs allow Mario and his friends to become Pelican Mario.


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|Arrival at the Sand Planet
|Episode Data
|Episode #
|Animation Director
|Episode 019
|Episode 021
Arrival at the Sand Planet!! (砂の星にやってきた!!, Suna no Hoshi ni Yattekita!!) is the twentieth episode of the Inazuma Eleven GO Galaxy series.
Just as Galaxy Nauts Gou was about to start the warp, Tetsukado heard some weird sounds, and the team decided to see what was lurking in the spaceship with them. Unlike what they thought, the weird sounds came from Shinsuke, who had secretly went abroad along with them! At this point, Kuroiwa decided to let Shinsuke join Earth Eleven, as Shinsuke also had Soul. The train then went into a warped tunnel and arrived Sandorius. After they have arrived, Ishigashi Gorham, a co-ordinator of Ginga Renpou Hyougikai, welcomed the team and allowed them to explore the neighbourings before the match with Sandorius Eleven started.
Meanwhile, in Faram Obius, a princess named Lalaya Obies was watching the videoes of Inazuma Japan in the preliminaries of Grand Celesta Galaxy along with her servant, Minel Eiba. Lalaya was immediately attracted to Tsurugi Kyousuke, watching him performing Bicycle Sword. Despite Minel's protest of Tsurugi being a "Earthling", Lalaya requested him to bring Tsurugi right in front of her...
As the team explored Sandorius, several members got lost and Tenma, Tetsukado, Kusaka, Matatagi and Shinsuke were challenged by several Sandoriusians to a soccer-battle. The Sandorius players played in a violent manner, charging at every single member, damaging them so that they can play in the match. Just as Matatagi was about to get hurt as well, Kazerma Woorg, the captain of Sandorius Eleven, halted the match and greeted Tenma and the others, telling them they will play fair despite of the consequences.
As Minel returned to Lalaya to tell her about Tsurugi's abduction plan, Lalaya recited a legend she heard about Earth: "When danger befalls our world, our future will be in the hands of those from the blue planet that lies beyond the seventh light." Minel, at this point. informed her that Shitennou will make sure Earth will lose...
Major EventsEdit
- The team heard some weird sounds and went off to investigate.
- The team discovered the strange sound was Shinsuke.
- Tetsukado and Shinsuke became friends quickly.
- Nishizono Shinsuke was finally accepted to be in the Earth Eleven.
- Galaxy Notes Gou travelled into a warped tunnel.
- Earth Eleven arrived at Sandorius and met some aliens.
- Lalaya Obies was shown to be watching the match of Inazuma Japan.
- She was shown to be particularly interested in Tsurugi Kyousuke after watching him using Bicycle Sword.
- She told Minel Eiba to bring Tsurugi to her.
- Earth Eleven played a five-people soccer battle against those aliens, with Shinsuke as the goalkeeper.
- The alien players were shown to be playing in a violent manner.
- Kazerma Woorg came and halted the match.
- Lalaya's servant, Minel Eiba told her about Shitennou, that Shitennou will make sure Earth will lose.
- Lalaya also recited a legend she heard about Earth: "When danger befalls our world, our future will be in the hands of those from the blue planet that lies beyond the seventh light."
- However, Lalaya and Minel interpreted the legend saying that Earth will threaten Faram Obius.
[Nishizono Shinsuke] I have chosen to come here! I have to do all that I can!
- Jii-san from the manga Dangerous Jii-san made a cameo appearance.


text:
RepairPal is your advocate for car care advice and guidance
Q: I have a 1998 Chrysler Sebring Convertible. 6 cyl. The car will not start. on 1998 Chrysler Sebring
Called AAA for a jump start and nothing. No "rrrr" or anything. Lights, etc were not left on. Starter seemed to be okay before. The car ran fine. I parked it and went to start it and nothing. Battery tests fine for cold crank, etc. Now I have to have it towed! Any ideas why it wont start?
it is a fuse. ignition fuse, under the hood in the fuse box compartment
| |
could be your battery is just shorted internally and will not take a jump
Thank you for the response. I was told it was a fuse that blew the starter. I am not sure. I used to dabble in working on cars, but everything is too different now. Thanks again for the help.
I aggree with globalhelper. Bad cell inside the battery.
Thanks to you too! I see you both agree. I am told to replace the starter. It will run into more money than I thought. I did manage to replace the battery - too hard! I posted another question. I appreciate your response.
Find Out Why Your Car Won't Start
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text:
Learn more about SURREY LITHO LIMITED. Check the company's details for free and view the Companies House information, company documents and list of directors.
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|Tangible Fixed Assets
|£ 214,770
|Current Assets
|£ 32,183
|Tangible Fixed Assets Depreciation
|£ 284,849
|Provisions For Liabilities Charges
|£ 6,169
|£ 28,033
|Shareholder Funds
|£ 162,663
|Profit Loss Account Reserve
|£ 55,833
|Revaluation Reserve
|£ 106,730
|Called Up Share Capital
|£ 100
|Net Assets Liabilities Including Pension Asset Liability
|£ 162,663
|Total Assets Less Current Liabilities
|£ 168,832
|Net Current Assets Liabilities
|£ 45,938
|Creditors Due Within One Year
|£ 78,121
|Cash Bank In Hand
|£ 23,413
|Stocks Inventory
|£ 4,150
|Share Capital Allotted Called Up Paid
|£ 100
|Number Shares Allotted
|£ 100
|Tangible Fixed Assets Disposals
|£ 137,455
|Tangible Fixed Assets Additions
|£ 13,143
|Tangible Fixed Assets Cost Or Valuation
|£ 403,410
|Tangible Fixed Assets Depreciation Charged In Period
|£ 13,918
|Tangible Fixed Assets Depreciation Decrease Increase On Disposals
|£ 42,021
Information about the Private Limited Company SURREY LITHO LIMITED has been prepared for information purposes only. It is not intended to be nor does it constitute legal advice. This is public information provided by the official company register.


text:
Timestamp: 2012-01-04
Location: Future Technology, Paris branch
- Ardant: You called me all the way to Paris for this madness?
- Gramática: Listen, it's been months and we are no closer to getting the Shroud to work. The microscopic technology in its fibers is similar to what we found in other Pieces of Eden. An alloy we've dubbed [Bathoricka]. This alloy seems to react specifically to thought. But the artifact itself remains inert.
- Ardant: That's still no reason for this course of action. What if the Shroud doesn't save you?
- Gramática: Do you know just how much of Abstergo's money we've embezzled into our secret project here? Because I'm sure Mr. Rikkin noticed by now. And if we have nothing to show for it... so I'm going to wrap myself in the Shroud. And you're going to do what needs to be done. One way or another, our current dilemma will be resolved.
- Ardant: Fine.
- Ardant: Oh, shit!
[Sound of the Shroud activating]
- Consus: I am Consus, the Erudite God.
[Isabelle Ardant screaming]


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Do you want to make Ohio.com your homepage?
Gap closes in Brown-Mandel senate race
Republican candidate for U.S. Senate, Ohio state Treasurer Josh Mandel, (left) is closing, but still trails incumbent and U.S. Senator Sherrod Brown, D-Ohio in the race for the U.S. Senate. (AP Photos)
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Crazy Horse was the first of the semi-custom Sllim 37 IRC Cruiser/Racer and is a development of Mark Mills' highly successful IRC 39 Mariners Cove. The boat was originally targeted at the middle boat class in the 2008 Commodores Cup but was entered as the small boat with much reduced sail area. Extending the philosophy of true dual-purpose Cruiser/Racers, the design has been optimised to be raced as efficiently as possible, without compromising dual-use features like the secure and comfortable cockpit layout that was refined over many designs, or the smart seamanlike interior, which can sleep 8. Although originally a custom project, due to the level of interest it evolved to become a semi-production build to allow the talented New Zealand builder Davie Norris to reduce costs for subsequent builds. The S glass/foam core/epoxy construction was engineered by High Modulus NZ, and the project was managed by Chris Main to ensure a high quality package on her Key West race Week 2008 debut. After her change of ownership, the yacht was been sailed in the Irish Sea, participating solely in Irish regattas. The yacht has undergone a refit in the early part of 2015, but due to a change in circumstances, is now seriously for sale. Crazy Horse would be an ideal yacht for the 2016 Commodores Cup
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Sandra Bullock New Movie
December 27, 2011 by staff
Sandra Bullock New Movie, Sandra Bullock, an Oscar-winning actress who weathered a divorce and a cheating scandal, feels empowered by a single word – "Mama." Her son Louis, who she adopted just before her much-publicized 2010 split from husband Jesse James, turns 2 years old in January and is learning how to talk.
"He calls me 'Mama' and it gives you a sense of power," the 46-year-old actress told OnTheRedCarpet.com. "It breaks your heart and opens it up in the best way. It is astounding. It's like something I just can't even explain. But I think everyone who has a child knows what it feels like. It makes you a better person."
Bullock and James, a motorcycle customizer, married in 2005. He was her first husband. The two split in March 2010 amid reports James had cheated on Bullock, which he later confirmed, and after she won her first Oscar, given for her performance in the movie "The Blind Side." The actress filed for divorce in April 2010 and it was finalized that June.
Bullock maintains a home in New Orleans with her son, who is her only child. She and James had adopted him in January 2010. After their split, she announced she would raise him as a single parent.
James, who has children from a previous relationship, said in May, in interviews to promote his memoir, that he misses Louis.
Bullock made her comments about her son to OnTheRedCarpet.com an interview to promote her new film "Extremely Loud and Incredibly Close," which sees her playing a mother to 9-year-old Oskar, portrayed by Thomas Horn. Her son searches New York City for a lock that matches a key left behind by his father, who died in the Sept. 11, 2001 attacks on the city's World Trade Center.
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Genomics and proteomics approaches to the study of cancer-stroma interactions
The development and progression of cancer depend on its genetic characteristics as well as on the interactions with its microenvironment. Understanding these interactions may contribute to diagnostic and prognostic evaluations and to the development of new cancer therapies. Aiming to investigate potential mechanisms by which the tumor microenvironment might contribute to a cancer phenotype, we evaluated soluble paracrine factors produced by stromal and neoplastic cells which may influence proliferation and gene and protein expression.
The study was carried out on the epithelial cancer cell line (Hep-2) and fibroblasts isolated from a primary oral cancer. We combined a conditioned-medium technique with subtraction hybridization approach, quantitative PCR and proteomics, in order to evaluate gene and protein expression influenced by soluble paracrine factors produced by stromal and neoplastic cells.
We observed that conditioned medium from fibroblast cultures (FCM) inhibited proliferation and induced apoptosis in Hep-2 cells. In neoplastic cells, 41 genes and 5 proteins exhibited changes in expression levels in response to FCM and, in fibroblasts, 17 genes and 2 proteins showed down-regulation in response to conditioned medium from Hep-2 cells (HCM). Nine genes were selected and the expression results of 6 down-regulated genes (
A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein.
Solid tumors are characterized by the presence of two major components: neoplastic cells and a specialized nonmalignant stroma in which they are immersed and are essential for their survival and proliferation. In carcinomas, a basement membrane is usually present between these components
The tumor stroma is distinguished by an enrichment of microvessel density, abundance of endothelial cells and precursors, inflammatory cells including lymphocytes, neutrophils, macrophages, dendritic and mast cells, and a connective tissue with fibroblasts, myofibroblasts and histiocytes responsible for remodeling and deposition of extracellular matrix (ECM) components - fibronectin, collagens, elastin, and glycosaminoglycans
Substantial evidence indicates that the development and the progression of cancer not only depend on its genetic characteristics but also on interactions with its microenvironment
In this context, inflammation and infection have gained special attention. Well known examples connecting infection-related or -unrelated chronic inflammation and increased risk for cancer development are described in the literature
Macrophages represent one of the main inflammatory regulators in tumor stroma and are responsible for proliferation, invasion and immunosuppressive signaling, with the production of angiogenic and growth factors, chemokines, cytokines and matrix metalloproteinases
Regardless of the fact that they are easily identified by their morphology, specific cellular markers for fibroblasts remain unknown, presumably because of their large diversity
Many studies have analyzed the role of fibroblasts in cancer initiation and progression. To address this issue, several approaches have been used, as co-culture of cancer cells and fibroblasts and cultures with conditioned medium, combined or not with
Much of the answer to the question of tumor-stroma interactions lies in the identity of ligands, receptors and effectors of signaling patterns expressed by stroma and tumor cells. Numerous growth factors, cytokines, chemokines, hormones, enzymes and cells responsible for their expression have been characterized but the cross-signaling between pathways in this complex network is far from solved
An important perspective in the study of tumor stroma is the potential use of the gene expression pattern of their cells for diagnostic or prognostic evaluation and as a target for therapy. Supporting this idea are the results from studies on outcome prediction and molecular marker analysis of the stroma
In order to investigate potential mechanisms by which the tumor microenvironment might contribute to cancer phenotype, we asked whether soluble paracrine factors produced by stromal and neoplastic cells
Primary tumor samples
For conditioned medium experiments, a primary epidermoid (squamous cell) carcinoma of the retromolar area was obtained from a 49-year-old male patient, prior to radiation and/or chemotherapy. Twenty-four laryngeal and 23 oral tongue squamous cell carcinoma (SCC) samples from patients undergoing tumor resection were used for gene expression analysis. All carcinoma samples were reviewed by senior pathologists and exhibited the presence of at least 70% tumor cells; the corresponding surgical margins were classified to be free of tumor cells.
The study protocol was approved by the National Committee of Ethics in Research (CONEP 1763/05, 18/05/2005), and informed consent was obtained from all patients enrolled.
Epithelial cancer cell line and primary tumor cell cultures
The Hep-2 cell line, originally established from an epidermoid carcinoma of the larynx (ATCC, Rockville, Maryland, USA), was seeded at a density of 1 × 106 cells/mL per 75 cm2 culture flask (Corning, NY, USA) in medium MEM-Earle (Cultilab, Campinas, SP, Brazil), pH 7.5, supplemented with 20% fetal calf serum (Cultilab), 1% non-essential amino acids, 0.1% antibiotic/antimycotic (Invitrogen Corporation, Carlsbad, CA, USA), and cultured at 37°C in a humid atmosphere of 5% CO2.
A primary carcinoma of retromolar area sample showing epithelium and adjacent connective tissues was rinsed multiple times with 100× antibiotic and antimycotic solutions (Invitrogen) and minced into 2-4 mm fragments. Single-cell suspensions were obtained by digestion at 37°C for 1 hour with 40 mg/mL collagenase type I (Sigma Chemical, St Louis, USA). After centrifugation, the cells were washed with PBS, resuspended in DMEM medium supplemented with 20% fetal calf serum (Cultilab), 2 mM glutamine (Invitrogen), 1% non-essential amino acids (Invitrogen), and 0.1% antibiotic/antimycotic (Invitrogen). The cells were seeded at a density of 1 × 106 cells/mL per 75 cm2 culture flasks (Corning) and cultured at 37°C in a humid atmosphere of 5% CO2. Cell medium was changed at 72 h intervals until the cells became confluent. Since fibroblasts were mixed with the epithelial tumor cells at the time of initial plating, fibroblasts were selected by plating the cells growing in medium supplemented with 20% serum for at least 3 weeks
Preparation of conditioned medium
Conditioned medium (CM) was prepared from Hep-2 cell or tumor stromal fibroblast cultures showing 80% confluence. Twenty-four, 48 and 72 hours after medium replacement, the supernatant or conditioned medium (CM24, CM48 and CM72, respectively) from three replicas was aspirated and filtered through a 0.22 μm membrane (Millipore) to remove any cell debris and stored at -80°C. Before using, the CM was diluted 1:1 in complete medium. The dilution 1:1 and CM72 were chosen to maximize the chance of detecting a cell response to soluble factors. Optimization experiments showed that dilutions lower than 1:1 resulted in higher numbers of dead cells.
Hep-2 cell-conditioned medium is referred to as HCM and fibroblast-conditioned medium is referred to as FCM.
Growth curve
Hep-2 cells were seeded at a density of 5 × 104 cells in plastic 6-well plates in two sets of quadruplicates. Twenty-four hours later, when cells had already adhered, Hep-2 cultures were incubated with FCMs. One replica in each set was treated with self-conditioned medium and one replica was treated with complete medium.
Medium was replaced on day 4 and cell morphology was observed every day. After 1, 3, 5 and 7 days, cells were harvested and counted using a Neubauer hemocytometer. The same experiment was repeated twice.
Immunofluorescence analysis
The Hep-2 cell line or tumor stromal fibroblasts were grown in culture chambers (Nunc, Naperville, IL, USA) and, after 3 days, the chambers were carefully removed, and the slides with adherent cells were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde, 0.1 mol/L sodium phosphate buffer, pH 7.4, for 2 hours at 4°C. The slides were washed in the same buffer and incubated with 0.1% albumin bovine and 3% normal serum in PBS (PBSA) to block nonspecific binding. The cells were immunostained with primary mouse monoclonal antibodies (Ab) anti-vimentin (NCL-VIM-V9, Novocastra, Benton Lane, Newcastle, UK) or anti-cytokeratin (M3515, antibodies to all types of cytokeratins; AE1-AE3; Dako, Carpinteria, CA, USA) diluted at 1:200 in 1% PBSA, followed by overnight incubation at 4°C. For negative controls, the cells were incubated with nonimmune mouse serum (1:200 working dilution; Sigma-Aldrich). After repeated washings in 1% PBS, a goat anti-mouse IgG (Fc fragment-specific, Dako, Glostrup, Denmark) antibody conjugated to FITC (1:50; British BioCell International, Cardiff, UK) was added, followed by 1 hour incubation at room temperature. Thus, the cells were washed thoroughly in PBS. Analysis was conducted using an Axioskop 2 light microscope (Zeiss, GR) equipped with a digital camera. Digital images were captured by using software AxioVision (Zeiss, GR).
Immunohistochemical analysis
Apoptosis was assayed using AnxA5 staining as described
RNA extraction for Rapid Subtraction Hybridization (RaSH) and real time PCR experiments
Hep-2 cells and stromal fibroblasts were seeded at a density of 1 × 106 cells/mL per 75 cm2 culture flasks in complete medium (controls) and in conditioned medium. Hep-2 cells and fibroblasts were cultured for 5 and 3 days, respectively, and harvested by addition of TRIzol Reagent, following treatment with DNase (Invitrogen). Total RNA from primary tumor samples was also extracted using TRIzol Reagent and treated with DNase. cDNA synthesis was performed using a High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA) as described by the manufacturer.
RaSH technique was performed as described by Jiang et al. (2000)
The cDNA was digested with MboI (Invitrogen) at 37°C for 3 h followed by phenol/chloroform extraction and ethanol precipitation. The digested cDNAs were mixed with the adaptors XPDN-14 5'-CTGATCACTCGAGA and XPDN-12 5'-GATCTCTCGAGT (Sigma Chemical, final concentration 20 μM) in 30 μl of 1× ligation buffer (Gibco BRL), heated at 55°C for 1 min, and cooled down to 14°C within 1 h. After adding 3 μl of T4 DNA ligase (5 U/μl) (Gibco, BRL), ligation was carried out overnight at 14°C. After phenol/chloroform extraction and ethanol/glycogen precipitation, the mixtures were diluted to 100 μl with TE buffer (10 mM Tris/1 mM EDTA); 40 μl of the mixtures were used for PCR amplification.
The PCR mixtures were set up using 10 μM XPDN-18 5'-CTGATCACTCGAGAGATC, 0.4 mM dNTPs, 10 × PCR buffer, 1.5 mM MgCl2 and 1U Taq DNA polymerase (Invitrogen). Thermocycler conditions were one cycle at 72°C for 5 min, followed by 25 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, ending in a final extension at 72°C for 3 min. Ten μg of purified PCR product (tester) was digested with 20U XhoI (Invitrogen) followed by phenol/chloroform extraction and ethanol precipitation.
One-hundred nanograms of the tester cDNA were mixed with 5 μg of the driver cDNA in hybridization solution (0.5 M Nacl, 50 mM Tris/HCl, SDS2% and 40% formamide) and, after heating at 95°C, incubated at 42°C for 48 h. After extraction and precipitation, the hybridization mixture (1 μg) was ligated with XhoI-digested pZero plasmid and transformed into competent bacteria. Bacterial colonies were picked and used as DNA template for PCR. Clones were sequenced using an automated DNA sequencer and sequence homologies were searched using the BLAST program
Quantitative PCR
For validation experiments, cells were seeded at a density of 1 × 106 cells/mL per 75 cm2 culture flasks in two sets of quadruplicates. Twenty-four hours later, when cells had already adhered, Hep-2 culture replicas were treated with FCMs and fibroblast cultures were treated with HCMs. One replica in each set (control) was treated with self-conditioned medium. Hep-2 cells and fibroblasts were harvested after 5 and 3 days, respectively, and RNA was extracted as described above.
Nine differentially expressed genes were selected for validation by quantitative real time PCR experiments according to their direct or indirect involvement in tumorigenesis. Their expression was checked in treated samples relative to matched non-treated samples. One of these genes (
The primers were manually designed with: 19-23 bp length, 30-70% GC content and a short amplicon size (90-110 bp). Their sequences are available upon request. Real time PCR was performed in triplicate using a 7500 Fast Real-Time PCR System (Applied Biosystems). Reaction mixture consisted of a 20 ul volume solution containing 10 ul of Power SYBR Green PCR Master Mix (Applied Biosystems), 500 nM of each primer and 100 ng cDNA. The PCR conditions were 95°C for 10 min followed by 40 cycles of 95° for 15 s and 60° for 1 min. Melting curve analysis was performed for each gene to check the specificity and identity of the RT-PCR products.
For each primer set, the efficiency of the PCR reaction (linear equation: y = slope + intercept) was measured in triplicate on serial dilutions of the same cDNA sample. The PCR efficiency (E) was calculated by the formula
Three control genes (
Proteomic analysis
Hep-2 cells and stromal fibroblasts were seeded at a density of 1 × 106 cells/mL per 75 cm2 culture flasks in complete medium and in conditioned medium, as described for RASH experiments. Hep-2 cells and fibroblasts were cultured for 5 and 3 days, respectively, and harvested by centrifugation at 3200 rpm for 5 min at 4°C. Cells were disrupted by sonication, proteins were isolated and two-dimensional electrophoresis (2-DE) was performed, as described by de Marqui et al. (2006)
Samples were analyzed using MALDI Q-TOF (Matrix Assisted Laser Desorption Ionization - Quadrupole Ion Filter - Time of Flight) Premier (Waters Corporation, Milford, MA, USA) mass spectrometer (MS/MS). Duplicate or triplicate runs of each sample were made to ensure an accurate analysis.
For protein identification, the resulting MS/MS data were interpreted by MASCOT software (MS/MS Ions Search)
Data Handling and Statistical Analysis
Quantification of apoptotic cells was performed with a high magnification objective (×40) counting cells in 100 μm2 areas and reported as mean ± SEM per group. Densitometric analysis for the immunofluorescence staining used an arbitrary scale ranging from 0 to 255 units. Statistical differences between groups were determined by analysis of variance followed, if significant, by the Bonferroni test.
Stromal fibroblasts: selection and immunofluorescence analysis
Fetal calf serum concentration and culture time provided a simple method of selecting fibroblasts from a primary carcinoma of retromolar area. Fibroblast cultures at passage 78 still showed spindle-shaped cells, which displayed the typical fibroblast markers, weak cytokeratin and intense vimentin immunoreactivity in cytoplasm, after immunofluorescence analysis (Figure
Figure 1
Immunofluorescence analysis of cytokeratin and vimentin in stromal fibroblasts and Hep-2 cell line
Immunofluorescence analysis of cytokeratin and vimentin in stromal fibroblasts and Hep-2 cell line. (A and D) Absence of immunoreactivity in sections incubated with control nonimmune mouse serum. Stromal fibroblasts (B and E) and Hep-2 cell line (C and F) were positive for vimentin and cytokeratin, respectively. (G): Densitometric analysis of immunofluorescence reaction to vimentin and cytokeratin in stromal fibroblasts and Hep-2 cell line. Scale bar, 20 μm.
Ultrastructural analysis showed that the stromal fibroblasts present large euchromatic nuclei, more granular endoplasmatic reticulum, mitochondria and nucleoli than normal fibroblasts (data not shown). Therefore, the spontaneously immortalized cell line of fibroblasts retained the characteristics of stromal cells and may correspond to cancer-associated fibroblasts (CAF).
Conditioned medium inhibits proliferation and induces apoptosis
Growth curves of Hep-2 cells treated with FCM showed decreased proliferation (Figure
Figure 2
Growth curve of Hep-2 cell line
Growth curve of Hep-2 cell line. Hep-2 cells were cultured in complete medium, treated with self-conditioned medium (HCM) or with conditioned medium from fibroblast cultures (FCM) and collected 1, 3, 5 and 7 days after medium replacement. Data are means ± s.d. of two independent experiments in duplicates. *
The immunohistochemistry reaction with AnxA5 antibody showed the presence of gold particles on the cytoplasm of the Hep-2 apoptotic cells (Figure
Figure 3
Immunohistochemistry reaction with AnxA5 antibody showed the presence of gold particles on the cytoplasm of apoptotic cells
Immunohistochemistry reaction with AnxA5 antibody showed the presence of gold particles on the cytoplasm of apoptotic cells. Hep-2 cells (A) without treatment and (B) treated with conditioned medium from fibroblast culture (FCM) show AnxA5 immunoreactivity. Apoptotic cells immunolabeling for AnxA5 can be seen in Hep-2 cells treated with FCM (arrows). Staining with haematoxylin. Scale bar, 20 μm.
Genes identified using the RaSH approach
A total of 81 clones from the Hep-2 cell line and fibroblast libraries were sequenced. In the Hep-2 cell line, forty-one genes exhibited changes in expression levels in response to FCM treatment (33 down- and 8 up-regulated) and, in fibroblasts, 17 genes showed down-regulation in response to HCM treatment. These genes are involved in response to stimulus, apoptosis, cell proliferation and differentiation, signal transduction, transcription, translation and transport (Table
Table 1
Information on biological processes based on Gene ontology.
Cell communication
signal transduction
Cell migration
Cell cycle
Cell proliferation
negative regulation
positive regulation
Developmental process
epidermis development
Response to stimulus
defense response
inflammatory response
response to stress
response to oxidative stress
response to external stimulus
Metabolic process
protein metabolic process
protein modification process
lipid metabolic process
DNA repair
RNA processing
Cellular homeostasis
No classification
Cell proliferation
negative regulation
Developmental process
organ development
Response to stimulus
response to stress
Metabolic process
protein metabolic process
protein modification process
nucleic acid metabolic process
RNA processing
Top down- and up-regulated genes selected by RaSH in Hep-2 samples treated with FCM.
Table 2
Information on biological processes based on Gene Ontology.
Cell communication
signal transduction
Cell adhesion
Cell proliferation
positive regulation
Developmental process
organ development
epidermis development
Response to stimulus
defense response
response to stress
Metabolic process
protein metabolic process
RNA processing
No classification
Top down-regulated genes selected by RaSH in CAF samples treated with HCM.
Real-time PCR validation of differentially expressed genes
Nine genes displaying down- (
Figure 4
Real-time PCR gene expression in a conditioned medium-treated neoplastic cell line and in primary tumors
Real-time PCR gene expression in a conditioned medium-treated neoplastic cell line and in primary tumors. (A) Expression of
No differences were observed in respect to clinicopathological features between samples presenting up- and down-regulation of
Additional file 1
Clinicopathological features of 24 patients with larynx SCC and of 23 patients with tongue SCC.
Click here for file
Proteomics approach
Comparison between 2-DE patterns from treated cells and controls revealed approximately 80 spots with significant differences in intensity. Seven proteins (Figure
Figure 5
Enlarged 2-DE gels of proteins from conditioned medium-treated Hep-2 cells and stromal fibroblasts
Enlarged 2-DE gels of proteins from conditioned medium-treated Hep-2 cells and stromal fibroblasts. Five proteins (arrows), tubulin beta (A-B), alpha enolase (C-D), aldolase A (E-F), glyceraldehyde-3-phosphate dehydrogenase (G-H) and heterogeneous nuclear ribonucleoprotein C (I-J) were down-regulated in Hep-2 cell line treated with fibroblast conditioned medium (A, C, E, G and I) and two proteins (K-L), vimentin (arrow on left) and actin (arrow on right), were underexpressed in fibroblasts treated with Hep-2 cell line conditioned medium (K).
Additional file 2
Underexpressed proteins in Hep-2 cells and fibroblasts treated with conditioned medium from fibroblasts (FCM) and Hep-2 (HCM), respectively.
Click here for file
The molecular crosstalk between neoplastic and the surrounding tissue induces several stromal changes, including neoangiogenesis and immune/inflammatory reaction, as well as new extracellular matrix formation and the activation of fibroblast-like cells, a process known as desmoplasia
In the present study, we investigated the influence of soluble paracrine factors produced
Down-regulated genes in neoplastic cells treated with FCM are involved in signal transduction (
In fibroblasts treated with HCM, the biological processes of down-regulated genes include signal transduction (
Two genes exhibited similar patterns in both cells (
After literature analysis, nine genes (
The product of
Calreticulin (coded by
Guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1 or Rack1 (coded by
Sequestosome 1 or ubiquitin-binding protein p62 (coded by
Another gene down-regulated by fibroblast-conditioned medium is
With respect to proteomic analysis, few differences (mostly quantitative) between treated and non-treated cells were detected. Among the proteins differentially expressed, alpha-enolase, heterogeneous nuclear ribonucleoprotein C C1/C2, aldolase A, tubulin beta and glyceraldehyde-3-phosphate dehydrogenase were down-regulated in neoplastic cells treated with FCM and vimentin and actin were down-regulated in fibroblasts treated with HCM. These proteins, produced by neoplastic cells or fibroblasts, may affect tumorigenesis. For example, the glycolytic enzyme alpha-enolase and its enzymatically inactive isoform MBP-1 (c-
Fibroblasts, as other cells in tumor microenvironments, need to maintain close communication with cancer cells, promoting proliferation, recruitment of inflammatory cells and acquisition of invasive characteristics. Similarly, cancer cells may influence stromal cells to generate a favorable and supportive environment, which would supply them with nutrients and factors necessary for developing the tumor and spreading of metastasis. In the present study, we observed both positive and negative effects exerted by fibroblasts on Hep-2 cells, favoring or not the former. A significant and common denominator in the results was the potential induction of signaling changes associated with immune or inflammatory response in the absence of a specific protein. In fact,
The complexity of the tumor microenvironment is immense and much information is still necessary for better understanding how the relationship between stroma and carcinoma cells can be used for diagnostic and prognostic evaluation and a target for therapy.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
FCR-L participated in the design of the study and analysis of the data, carried out cell culture, RaSH experiments and drafted the manuscript. PPJr helped with RaSH experiments. AV and GMP carried out proteomics analysis. JVM was responsible for sample collection and processing. JC-R carried out cloning and sequencing of the samples. BRC carried out cell culture experiments. TH helped with manuscript preparation. CFS performed the real time PCR experiments. RAPT and SMO carried out immunofluorescence and immunohistochemical analysis. EEF and PMJr carried out clinical data analysis for sample selection. MBdC carried out clinical data analysis for sample selection and drafted the manuscript. GENCAPO team members were responsible for sample collection and initial on-site sample processing, provided the pathological analysis of the cases, obtained the informed consent and discussed the findings. EHT participated in the study design and coordination, carried out the analysis and interpretation of the data and drafted the manuscript. All authors read and approved the final manuscript.
We acknowledge the financial support from Fundação de Amparo à Pesquisa do Estado de São Paulo/FAPESP (Grants 04/12054-9 and 06/60162-0), Rede Proteoma do Estado de São Paulo (Grant FAPESP 04/14846-0/FINEP 01.07.0290.00), The Ludwig Institute for Cancer Research, and the researcher fellowships from FAPESP (FCR-L) and Conselho Nacional de Pesquisas/CNPq (EHT, SMO).
The GENCAPO (Head and Neck Genome) Project authors are the following: Cury PM7, de Carvalho MB8, Dias-Neto E3,14, Figueiredo DLA9, Fukuyama EE5, Góis-Filho JF5, Leopoldino AM15, Mamede RCM9, Michaluart-Junior P6, Moyses RA6, Nóbrega FG4, Nóbrega MP4, Nunes FD13, Ojopi EPB3, Serafini LN10, Severino P1, Silva AMA8, Silva Jr WA11, Silveira NJF16, Souza SCOM13, Tajara EH2, Wünsch-Filho V12, Amar A8, Bandeira CM4, Braconi MA4, Brandão LG6, Brandão RM11, Canto AL4, Cerione M5, Cicco R5, Chagas MJ4, Chedid H8, Costa A12, Cunha BR2, Curioni OA8, Fortes CS12, Franzi SA8, Frizzera APZ7, Gazito D8, Guimarães PEM6, Kaneto CM11, López RVM12, Macarenco R4, Magalhães MR8, Meneses C4, Mercante AMC8, Pinheiro DG11, Polachini GM2, Rapoport A8, Rodini CO13, Rodrigues-Lisoni FC2, Rodrigues RV2, Rossi L8, Santos ARD11, Santos M8, Settani F5, Silva FAM15, Silva IT11, Souza TB8, Stabenow E6, Takamori JT8, Valentim PJ5, Vidotto A2, Xavier FCA13, Yamagushi F5, Cominato ML5, Correa PMS4, Mendes GS5, Paiva R5, Ramos O6, Silva C6, Silva MJ5, Tarlá MVC11 (also presented in
Affiliations: 1Instituto de Ensino e Pesquisa Albert Einstein, São Paulo; 2Departamento de Biologia Molecular, Faculdade de Medicina de São José do Rio Preto; 3Departamento e Instituto de Psiquiatria, Faculdade de Medicina, Universidade de São Paulo (USP), São Paulo; 4Departamento de Biociências e Diagnóstico Bucal, Faculdade de Odontologia, Universidade Estadual Paulista, São José dos Campos, São Paulo, 5Serviço de Cirurgia de Cabeça e Pescoço, Instituto do Câncer Arnaldo Vieira de Carvalho, São Paulo; 6Departamento de Cirurgia de Cabeça e Pescoço, Faculdade de Medicina, USP, São Paulo; 7Departamento de Patologia, Faculdade de Medicina de São José do Rio Preto; 8Hospital Heliópolis, São Paulo; 9Serviço de Cirurgia de Cabeça e Pescoço, Faculdade de Medicina de Ribeirão Preto, USP; 10Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, USP; 11Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, USP; 12Departamento de Epidemiologia, Faculdade de Saúde Pública, USP, São Paulo; 13Departamento de Estomatologia, Faculdade de Odontologia da USP, São Paulo; 14Centro de Pesquisas do Hospital AC Camargo. São Paulo, 15Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP; 16Departamento de Ciências Exatas, Universidade Federal de Alfenas, UNIFAL, MG; Brazil.
Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing
Know thy neighbor: stromal cells can contribute oncogenic signals
Tumor microenvironment: the role of the tumor stroma in cancer
Distinctive gene expression of prostatic stromal cells cultured from diseased versus normal tissues
Stromal gene expression predicts clinical outcome in breast cancer
Cross-talk between paracrine-acting cytokine and chemokine pathways promotes malignancy in benign human prostatic epithelium
Tenascin-W is a novel marker for activated tumor stroma in low-grade human breast cancer and influences cell behavior
Tumour-stroma interaction: cancer-associated fibroblasts as novel targets in anti-cancer therapy?
Molecular aspects of stromal-parenchymal interactions in malignant neoplasms
14-3-3 zeta protein secreted by tumor associated monocytes/macrophages from ascites of epithelial ovarian cancer patients
Functions of paracrine PDGF signaling in the proangiogenic tumor stroma revealed by pharmacological targeting
Friends or foes - bipolar effects of the tumour stroma in cancer
Extracellular matrix metalloproteinase inducer up-regulates the urokinase-type plasminogen activator system promoting tumor cell invasion
The tumour microenvironment as a target for chemoprevention
Inflammation and cancer: role of nuclear factor-kappaB activation
Infections as a major preventable cause of human cancer
In vitro and in vivo studies on CCR10 regulation by Annexin A1
Annexin 1: differential expression in tumor and mast cells in human larynx cancer
Annexin A1 subcellular expression in laryngeal squamous cell carcinoma
Pathways connecting inflammation and cancer
Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium
Stromal fibroblasts in cancer: a novel tumor-promoting cell type
Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation
Breast carcinoma-associated fibroblasts and their counterparts display neoplastic-specific changes
Diversity, topographic differentiation, and positional memory in human fibroblasts
Fibroblasts in cancer
The myofibroblast: phenotypic characterization as a prerequisite to understanding its functions in translational medicine
Tumor induction of VEGF promoter activity in stromal cells
The stromal proteinase MMP3/stromelysin-1 promotes mammary carcinogenesis
Stromal fibroblasts in colorectal liver metastases originate from resident fibroblasts and generate an inflammatory microenvironment
Integrin- and cadherin-mediated induction of the matrix metalloprotease matrilysin in cocultures of malignant oral squamous cell carcinoma cells and dermal fibroblasts
NF-kappaB involvement in tumor-stroma interaction of squamous cell carcinoma
Collagen-based co-culture for invasive study on cancer cells-fibroblasts interaction
Fibroblast-derived MT1-MMP promotes tumor progression in vitro and in vivo
Targeting the tumor microenvironment
Biomechanics and biophysics of cancer cells
Collagen alpha1(XI) in normal and malignant breast tissue
OX40 agonist therapy enhances CD8 infiltration and decreases immune suppression in the tumor
Drug insight: VEGF as a therapeutic target for breast cancer
Targeted therapy in advanced non-small-cell lung cancer
Cytoskeletal rearrangements in synovial fibroblasts as a novel pathophysiological determinant of modeled rheumatoid arthritis
Stable chromosome changes in human malignant melanoma
Epidermal growth factor receptors on cultured neoplastic human thyroid cells and effects of epidermal growth factor and thyroid-stimulating hormone on their growth
A novel calcium-dependent proapoptotic effect of annexin 1 on human neutrophils
RaSH, a rapid subtraction hybridization approach for identifying and cloning differentially expressed genes
A new mathematical model for relative quantification in real-time RT-PCR
Solubilization of proteins from human lymph node tissue and two-dimensional gel storage
Pathophysiologic significance of host reactions in human cancer tissue: desmoplasia and tumor immunity
Role of tissue stroma in cancer cell invasion
Desmoplasia measured by computer assisted image analysis: an independent prognostic marker in colorectal carcinoma
Primary head and neck cancer. Histopathologic predictors of recurrence after neck dissection in patients with lymph node involvement
Alterations of BRMS1-ARID4A interaction modify gene expression but still suppress metastasis in human breast cancer cells
Activated microglia modulate astroglial enzymes involved in oxidative and inflammatory stress and increase the resistance of astrocytes to oxidative stress in Vitro
Identification of chromatin remodeling genes Arid4a and Arid4b as leukemia suppressor genes
Foxp3 in control of the regulatory T cell lineage
Gene expression changes associated with altered growth and differentiation in benzo[a]pyrene or arsenic exposed normal human epidermal keratinocytes
Arsenic and benzo[a]pyrene differentially alter the capacity for differentiation and growth properties of primary human epidermal keratinocytes
Arsenite suppresses Notch1 signaling in human keratinocytes
Arsenic trioxide, arsenic pentoxide, and arsenic iodide inhibit human keratinocyte proliferation through the induction of apoptosis
The ins and outs of calreticulin: from the ER lumen to the extracellular space
Calreticulin exposure dictates the immunogenicity of cancer cell death
Recognition ligands on apoptotic cells: a perspective
Calreticulin enhances porcine wound repair by diverse biological effects
Differential calreticulin expression affects focal contacts via the calmodulin/CaMK II pathway
Altered expression of calreticulin during the development of fibrosis
RACK1 targets the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway to link integrin engagement with focal adhesion disassembly and cell motility
The RACK1 scaffold protein: a dynamic cog in cell response mechanisms
RACK1 regulates Src activity and modulates paxillin dynamics during cell migration
RACK1 inhibits colonic cell growth by regulating Src activity at cell cycle checkpoints
Molecular cloning of a phosphotyrosine-independent ligand of the p56lck SH2 domain
The signaling adaptor p62 is an important NF-kappaB mediator in tumorigenesis
The interaction of p62 with RIP links the atypical PKCs to NF-kappaB activation
The atypical PKC-interacting protein p62 channels NF-kappaB activation by the IL-1-TRAF6 pathway
The atypical protein kinase C-interacting protein p62 is a scaffold for NF-kappaB activation by nerve growth factor
Identification of interleukin 1 receptor-associated kinase as a conserved component in the p75-neurotrophin receptor activation of nuclear factor-kappa B
p62 forms a ternary complex with PKCzeta and PAR-4 and antagonizes PAR-4-induced PKCzeta inhibition
Analysis of intracytoplasmic hyaline bodies in a hepatocellular carcinoma. Demonstration of p62 as major constituent
p62 overexpression in breast tumors and regulation by prostate-derived Ets factor in breast cancer cells
Cytosolic overexpression of p62 sequestosome 1 in neoplastic prostate tissue
The ubiquitin-binding protein p62 is expressed in breast cancers showing features of aggressive disease
Activity profiling of deubiquitinating enzymes in cervical carcinoma biopsies and cell lines
Targeting the ubiquitin-proteasome pathway in breast cancer therapy
Activity-based ubiquitin-specific protease (USP) profiling of virus-infected and malignant human cells
Over-expression of genes and proteins of ubiquitin specific peptidases (USPs) and proteasome subunits (PSs) in breast cancer tissue observed by the methods of RFDD-PCR and proteomics
RING domains: master builders of molecular scaffolds?
RING for destruction?
Control of c-myc regulation in normal and neoplastic cells
Structural analysis of alpha-enolase. Mapping the functional domains involved in down-regulation of the c-myc protooncogene
c-Myc: linking transformation and genomic instability
Foxp3 represses retroviral transcription by targeting both NF-kappaB and CREB pathways
RACK-1 expression and cytokine production in leukocytes obtained from AD patients
Signal integration and diversification through the p62 scaffold protein
Gpnmb is induced in macrophages by IFN-gamma and lipopolysaccharide and acts as a feedback regulator of proinflammatory responses
Pre-publication history
The pre-publication history for this paper can be accessed here:


text:
Wow Surprise Room Escape game
Wow Surprise Room Escape is another new point and click room escape game from wowescape. You are trapped inside a Surprise room. The door of the room is locked. Try to escape from the room by finding useful object, and hints. Find the right way to escape from the Surprise room. Good Luck and Have Fun!
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text:
- Description
This Vixen DG-DX Ring has 37mm threads and will screw onto digital cameras that present the same thread size. The DG-DX Ring connects to the Digital Camera Adapter, which is sold separately.
Vixen 37mm Deluxe DG Ring
This ring fits a variety of digital cameras and video cameras by Sony, Ricoh, Canon, Sharp, and Victor...check our complete description for a list of camera models.
Vixen 37mm Deluxe DG Ring Fits.
- Sony DSC-P30, P50, S30, S50, MVC-FD92
- Ricoh Caplio-400G, Caplio GX, GX8, GR
- Canon Power Shot S60, S70
- Sony DCR-PC100, 110, 120, DCR-TRV30, 50, 240K, 300K, 820K, TR-290PH, CCD-TRV96K
- Sharp VL-MR1
- Victor GR-DVA30K, 33K
- Additional Information
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SKU VX-3933 Manufacturer Vixen
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text:
With the glut of first basemen at the upper levels of the system it was inevitable that one would be moved.
The following players were released by the club:
Scott Fogleson –RHP
Clark Girardeau –RHP
David O'Hagan –RHP
David Parker –2B
Howie Pence –RHP
Jordan Pickens –OF
Kelvin Vazquez –INF
Fogleson, signed out of the Independent League last year, went 1-0 with a 3.08 ERA in five games, three starts, for Peoria of the Arizona Rookie League.
Girardeau, a seventh round pick in 2003, went 3-9 with a 7.13 ERA in 88.1 innings for High-A Lake Elsinore after going 1-1 with a 4.15 ERA in six starts with Low-A Fort Wayne. He was 15-18 over his career with a 5.24 ERA.
O'Hagan pitched in just eight games with Eugene in 2004 after being selected in the ninth round that year, going 1-0 with a 2.57 ERA. He had surgery in March of 2005 and missed the entire year.
Parker was signed as an non-drafted free agent in 2005, hitting .219 over 44 games with the Arizona Rookie League squad.
Pence, signed as a non-drafted free agent in 2003, went 4-5 with Lake Elsinore, sporting a 4.56 ERA over 49.1 innings of relief. His year ended early when he had to undergo an emergency appendectomy.
Pickens hit .245 with ten homers and 46 RBI's in 77 games for the Lake Elsinore Storm. He has battled the injury bug since being selected in the 12th round of the 2003 draft, specifically to his shoulder.
Vazquez split time between Fort Wayne and Eugene, hitting .320 in 14 games for the Wizards and .254 in 23 games for the Emeralds. He was a 26th round pick in 2004.
David Jay contributed to this report


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Tell the truth: Are you happy being a stay-at-home mom?
Before giving birth to my first child, I was completely convinced that I wanted to stay home with her. It made me really sad to think that I would have to leave my baby at a daycare or in the care of someone else. Luckily, I was able to be a SAHM for the first two and a half years of her life. Even so, I was never exclusively a stay-at-home-mom. I mean, besides taking care of my daughter full time, I always had some kind of project going on the side and I'm pretty convinced it's the reason why I didn't go crazy or became depressed.
Well, according to a new Gallup survey, moms who stay at home with young kids are more likely to feel sad or angry than moms who work outside the home.
In fact, stay-at-home moms are also more likely to report that they've been diagnosed with depression more than moms who work outside the home. I'm not surprised because being a SAHM is the hardest job I've ever done. Seriously.
As I already said, the only reason I survived it is because I was always doing something else in addition to being a mom. Thus, I'm truly in awe of those moms capable of taking care of their children exclusively because I really don't know how they do it.
And yet, I have to admit that it makes me super sad to see that, at least according to the survey, so many stay-at-home moms are so depressed. I'm no expert, but what I do know is that it's always been super important for me to carry out my role as a mom, but it's also imperative that to honor my role as Roxana A. Soto. And I'm convinced that this is what saved me the years I chose to stay home with my daughter.
What do you think of the survey? Are you truly happy as a SAHM?
Image via Joe Scholabotnik/flickr
From Our Amigos
I have been severely depressed on and off since I was 12!!! So staying at home with my son it doesn't surprise me that I have phases of depression. I LOVE being a SAHM!!! It makes me happy that I know how my son is being raised. I wouldn't have it any other way! :) Staying at home with your child is DEFINITELY not for everyone, or most at that.
Being a SAHM is certainly challenging, but I agree that one of the keys to maintaining a balanced sense of mental and emotional stability is to have other things to do as well, either with or without the children. Some alone mommy time or some play dates to get together with other moms while the kids play are always a good way to keep a social connection.


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Interview with Tinker Tailor Soldier Spy director Tomas Alfredson
As a general rule of thumb, acclaimed directors from non-English speaking countries have found the transition to English speaking films to be difficult and laborious. Tomas Alfredson is an exception to that rule.
After receiving international acclaim for his moody vampire movie Let the Right One In, Swedish born Alfredson has assembled an all-star cast for his adaptation of John le Carre's Tinker Tailor Soldier Spy an espionage thriller with emphasis on mood and character rather than action and sex.
Set during the Cold War, the film focuses on George Smiley (Gary Oldman) a semi-retired spy who is asked to investigate whether a Soviet agent has infiltrated MI6. Smiley finds himself navigating a murky labyrinth of treachery and danger in his path to the truth.
Co-starring Colin Firth, Tom Hardy, John Hurt and a host of other premiere British talent, Tinker Tailor Soldier Spy has proven itself to be one of the best films of 2011 and showcases a director able to weave tightly woven suspense and character depth into an engrossing movie experience.
Thomas Alfredson spoke to Matt's Movie Reviews about making Tinker Tailor Soldier Spy, working with Gary Oldman and his career thus far.
After the success of Let the Right One In, you must have received a tonne of directing offers. What made you choose Tinker Tailor Soldier Spy as your follow up?
It's hard to answer, but it's right as you describe…Let the Right One In was my first international success and I was a little paralysed by that and I didn't know what to do for my next film. So I read scripts for almost two years before I decided to go with this.
I tried to read books and scripts with my body rather than with my brain. It's a very physical thing if you see images, if you cry and laugh or get upset or something when you read it. It's something you react physically on and when I first heard this project was on the market (so to say), I was very excited when I heard about it.
I saw the TV series a long time ago in the mid '70s on Swedish television and I remember it very vividly even though I was too young to understand it. Then in my early 20's I read the Le Carre trilogy and I was a big fan of it. So the books and the world had always attracted me.
John le Carre gave you a piece of advice, which was not to shoot the book or remake the miniseries but to make this material your own. With that in mind what kind of rules or boundaries did you give yourself to make sure that this version of Tinker Tailor… would be different to the others but faithful to the source material?
Well you know, when you think of it there are so many ways to look upon the same thing depending on where you stand in the room. If you and I were interrogated tomorrow about our phone call we're having now, and if I were to describe the feeling I would describe a sunny day in London in a cosy hotel and it's noon, and you would describe it totally different. But we have shared this moment in two very different positions.
It's the same when you read something. You get very different images and I think shooting a book is not for the author, it's for a possible audience and John le Carre said it's meaningless if you do the book, because the book already exists and if you make a crappy film the book will still be good. So he really encouraged us to be personal with it and have a different view.
I think it's impossible to do 360 pages like this book is. You have to sort of cut out a filet or pick a side, and I think we have tried to do a very emotional take on this and on what the personal costs were for the soldiers of the cold war rather than trying to be philosophical about the east/west political situation, or the details of the cold war, or the factors behind it. So we decided to do something about the human costs.
"I was looking for an actor that you wouldn't...associate with the kind of character he plays in this film and if you see what Gary has done through the year's it's very, very different portrait of people, and I thought he is the perfect guy for the part ." - Tomas Alfredson
It's funny you say that because my reaction to Tinker Tailor… was that I felt really sorry for these men and what they had to go through. Were you also very sympathetic to these characters?
Yes. You have to love your characters even if you do things you don't sympathise with and I suppose there lives must have been awful in many ways. Someone told me a story, I think it was John le Carre, that some of these spy's were actually decorated. They were invited to a small ceremony in Buckingham Palace and the Queen would give them a medal and they could wear it for 10 minutes and someone would take the medal and put it away in a cupboard because the whole thing was a secret.
The silence and the secrecy that these people had to live in must have been horrifying in many ways, and a lot of them must be still alive walking the streets around us, but we don't know what they've done or what they've experienced and they're not allowed to discuss it with anyone. So it must have been a very strange thing to be a spy.
You've ensemble a terrific cast. Were you amazed about the number of quality actors who wanted to star in this movie?
I was! But to be honest it was quite easy. Everyone we asked wanted to participate. I think 90% of the cast were out first choices and the other 10% couldn't be because of practical reasons. But everyone wanted to be in this project and it was fantastic to have the opportunity to work with the finest actors there is in this country.
I must say it was easy as well to direct it, because it's easy to direct great talent. It's just to let them do what they're good at, so it that way it was very joyful.
The first person cast in the movie was Gary Oldman. He is known more for his ability to play extroverted characters. What made you think he would be perfect for someone as introverted as George Smiley?
Well, I was looking for an actor that you wouldn't, how do you say…. associate with the kind of character he plays in this film. So he should be not connected to other Smiley-esque types and if you see what Gary has done through the year's it's very, very different portrait of people, and I thought he is the perfect guy for the part because he's also a very different choice than Alec Guinness and that was good because Alec Guinness portrait of Smiley is so famous and well done. So we really needed to come up with something different for this interpretation.
"The silence and the secrecy that these people had to live in must have been horrifying in many ways, and a lot of them must be still alive walking the streets around us, but we don't know what they've done or what they've experienced and they're not allowed to discuss it with anyone." - Tomas Alfredson
Gary Oldman has said that he wants to play Smiley again. Can you foresee you two reunite on another Le Carre novel? Perhaps something like Smiley's Affair?
We have discussed it and it would be great to do that. We just have to find the right timing for it and John le Carre has said that he has an idea on how to combine The Honorary Schoolboy and Smiley's People into one film adaptation. I would be happy to participate and I think that the rest of the creative team behind this one are more than interested in doing it.
You worked on a lot of comedic films in Sweden before Let the Right One In. Can you see yourself return to comedy in the future?
Yes. I try to…genres I think are for marketing people. To label things whether they're tragic or funny or scary or dramatic, it's not for me to decide. I try to do as different things as possible and as hard things as possible for me to do. So if it would be something that makes you laugh or cry, it doesn't matter really.
You come from a movie family, with your father an actor and director. Was directing something you always envisioned you would do?
Yes, and my brother to. He's actually done part two and part three of the Girl with the Dragon Tattoo series in the Swedish version. So we're three directors in the family.
I think maybe it's…my father was doing films since he started in the early '60s so I was really born into this and all of my summer holidays I spent on different movie sets. So I had the opportunity to learn a lot just watching and participating. I never considered doing anything else really, because it's been such an important part of my family life.
Created and Edited by Matthew Pejkovic / Contact: email@example.com
created by Colony Graphic Design / Copyright © Matthew Pejkovic


text:
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The above graph displays service status activity for Blinkbox.com over the last 10 automatic checks. The blue bar displays the response time, which is better when smaller. If no bar is displayed for a specific time it means that the service was down and the site was offline.
Service Status History
|Ping Time
|Ping Time
|264.37 ms.
|170.87 ms.
|171.01 ms.
|264.23 ms.
|264.95 ms.
|263.49 ms.
|264.39 ms.
|263.06 ms.
|263.69 ms.
|171.83 ms.
* Times displayed are PT, Pacific Time (UTC/GMT 0) | Current server time is 10:47
We have tried pinging Blinkbox website using our server and the website returned the above results. If blinkbox.com is down for us too there is nothing you can do except waiting. Probably the server is overloaded, down or unreachable because of a network problem, outage or a website maintenance is in progress...
If the site is UP but you cant access the page, try one of the below solutions:
Browser Related Problems
Force a full refresh for the site. This can be achieved by pressing CTRL + F5 keys at the same time on your favourite browser (Firefox, Chrome, Explorer, etc.)
Clear the temporary cache and cookies on your browser to make sure that you have the most recent version of the web page. For instructions choose your browser :
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A Domain Name System (DNS) allows a site IP address (192.168.x.x) to be identified with words (*.com) in order to be remembered more easily, like a phonebook for websites. This service is usually provided by your ISP.
Clear your local DNS cache to make sure that you grab the most recent cache that your ISP has. For Windows - (Start > Command Prompt > type "ipconfig /flushdns" and hit enter). For details choose your operating system :
If you can access a website at office or from a 3G network yet it's not working on your computer, it is a good idea to use an alternative DNS service other than your ISPs. OpenDNS or Google Public DNS are both excellent and free public DNS services.
Check our help page for step-by-step instructions on how to change your DNS.
is blinkbox playing up today?
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I've not had any problems before, but I currently cannot download or stream my TV shows.
Blinkbox is down... started watching movie last night, but can't watch the rest! States down for maintenance, but doesn't give any indication if this will take minutes, hours, or days! Try this link to bombard them with moans! blinkbox.zendesk.com/anonymous_requests/new
Blinkbox is useless. I have forgotten my password and it is not coming up with the option to send me a link to remind me of it or to create a new one. Actually at first it did send me a link and then when I put it a new password it tells me to set up a new profile then continually tells me that my email or clubcard is attached to another account. It is only attached to me. What is up? Cannot even submit a question as it says I am not logged in but I cannot log in! Useless.
Just rented a movie, and keep getting "Sorry we are unable to play this movie or TV show" Error code 0. They should have a refund button if a movie won't play when you want to watch it.
not happy ive paid £3.49 for a movie ive been unable to watch, I got a code 209 then a code 5 but theres nothing I can do, nobody to contact or email? shocking service wont be using it again!!!
This service is unreliable and causes no end of problems. I am attempting to watch a streamed but purchased movie and keep getting either Error Code 4 (another device is streaming on your account effectively, but nothing else is in use!) or Error Code 5, their servers are unavailable. Having PAID for movies, not much less than the DVD price, you expect to be able to access your movies! Rubbish service!
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