text:
After Lynn left, Craig and I decided to take a couple days visiting Cambridge and London, returning to Ann and Geoff's each night. We could pretty much see most of Cambridge in one day, but with London we'd have to be choosy.
As we're not great planners or decision-makers when it comes to travel, we decided to put off being choosy for a day-- so Cambridge came first.
We saw King's College, and spent most of our time in the Chapel. It's a beautiful thing, of course, with a fabulous fan-vaulted ceiling.
Outside Trinity College, we saw a tree that is supposedly descended from Newton's famous apple tree-- it was planted below the room Newton studied in.
And we dropped by St. John's College, where one of my heroes, Mr. Paul Dirac studied. You can find my interview with Dirac's biographer, Graham Farmelo, here.
This is the Bridge of Sighs, at St. John's College. Wikipedia says it was built by Henry Hutchinson, but word on the Cambridge streets was that Sir Christopher Wren is the real architect. Who knows? Anyway, it's beautiful and named after a Venetian bridge that is also covered.
Since students actually live in these colleges (they are essentially student halls, as well as being architectural historical wonders), there were signs all over the place asking non-residents not to go past this point or that point. There were cordons and various other devices set up. But, they hadn't managed to block off every path to the Bridge of Sighs, so Craig and I actually managed to sneak on and walk through. When we got to the other side, though, an stuffy-looking old guy in a really nice suit looked at us sternly as we left the building. You could tell he was British, though, because even though he didn't like us, he made way for us before he stepped into the building that he probably actually has a right to be in...
This is apparently a super-famous new clock. Have you heard about it? It's called the Corpus Clock. There are no numbers on it; blue LEDs indicate the time. Oh, and a giant ghastly mechanical locust crawls atop it, munching up time. It reminded me of the They Might Be Giants song.
"You're older than you've ever been, and now you're even older, and now you're even older, and now you're older still," John Linnell sings.
"Munch, munch," says the locust.
And my heart grows cold.
An apparently super-popular thing to do in Cambridge is to go "punting." I'd never heard of it before, and I'm afraid I rather let the sails out of the tourist information official when I told him that. He was like, "Well, it is a very famous thing to do in Cambridge, but apparently it's not that famous in... where are you from?"
Canada. Definitely Canada.
Anyway, "punting" is actually just taking a boat down the river that runs through Cambridge-- but there's a catch. You can't row the boat. You have to stand on the end of it and use a stick to guide yourself along. Until you're capable of that, you have to fall in the river and capsize your boat, taking all your friends with you. Former friends, possibly.
Given Craig's history of (eerily, almost willfully) falling into just about any nearby body of water, I wasn't about to get in a boat with just him and a stick. But I took a picture of the very famous activity for you, at least.
Craig was intrigued that some of the people doing the actual punting were total hipsters. They were clearly hired to punt for less-courageous tourists (like me), and they served as guides, as well, sort of telling folks what buildings they were passing by, etc. But they were wearing hip sunglasses and skinny jeans and flip-flops, sporting extra cute haircuts. For some reason, it interested him.
But Craig and I didn't have the time (or the money) to hire one of these obliging hipsters, and honestly, we've had enough boat-time in this year, considering Thailand and Cambodia, so we let it slide.
The last thing on our list was to see the Cavendish Laboratory, where Rutherford and Maxwell and other geniuses did their pioneering fancy-pants genius work. It is, oddly, on an unnamed alley with rubbish bins out the front.
Pretty exciting stuff-- or so we thought until we learned about Manchester's scientific history (see this post for more on that).
Well, I can hear my own locust munching my last night of freedom from lectures away, so I better leave London, and my Manchester adventures of this weekend, for later. Until then, dear readers...


text:
I'm at Renaissance on a Sunday night when Pastor Jeff makes this statement, "'…let him deny himself, take up his cross, and follow Me.' …To deny yourself is to turn your back on self-determination." God continued the conversation in my own heart: "Self-help hinders intimacy with Me; it's equivalent to spiritual masturbation."
Shocker? Not to me. It actually pointed toward a much greater understanding that started with my mom several years back. My mom loved Jesus and loved His church. She lived much of her devotional life praying through the tension that existed between the church she saw in the bible and the church she saw in America. Within the last year of her life God gave her this dream:
While walking up an old country road she came upon a beautiful young woman reclined on the dusty ground, masturbating. So disturbed by the site, she quickly looked away to Jesus. He was standing over the woman with pleasures-forevermore resting in His right hand. She asked Him, "What is this I am seeing?" He replied, "This is my bride, the church, pleasuring herself."
True Love
If the love of God is true for us we can be assured it has been scheduled within our destinies' encounters of suffering in the deep rural areas of our journey here on earth. In the 15th chapter of John, Jesus gives His disciples insight into their futures. Written throughout their stories and ours will be repeated visitations of pruning from a Vinedresser who is our Father. There will be places on the side of the road called Destiny where everything that has made us successful in Jesus will be cut clean from us. Everything that has been good and effectual and prosperous will be severed, and He will call it an act of His true love.
When the Vinedresser comes, He will cut away the good stuff, the stuff that has been making Him famous through us. I am not talking about the pruning of sins and weaknesses. He forgives our sins and empowers our weaknesses, but He cuts off our fruit. He comes after the stuff that has proven we follow Him (John 15:8). Perfect in wisdom, He cuts away the very fruit the two of us had been enjoying together.
Pruned, we stand naked before the world with nothing to show for our faith. Our dedicated lives will look like foolishness and will feel like punishment. For a time, our scriptural platitudes will stop proving effective in our lives. Familiar resources will run dry. Precious relationships will untie from us, and it will be for all the best reasons. This is a mysterious place of prospering. He prunes us fruitless so we will be ready to bear even greater fruit—the answers to "whatever you ask in My name.
To escalate our experience from challenging to despairing, the One whom we love removes what we love most, His presence. It is at this point we have to be cautioned. The desperation of the pruning season will tempt us to help ourselves right out of it. If we do we will forfeit the greater work. The evil tempter will be waiting to make his move. His plan is to come to this weak and vulnerable place with one goal in mind: to make us unfaithful to the Lover of our soul.
Self-Help Hinders Intimacy
So back to the introduction. Self-help? Equivalent to a spiritual masturbation? It's simple really. Our world's self-help philosophies seduce us to manipulate our motives and our environments promising us that we can lay hold of our own gratification. That, my friends, is the masturbation of self-help. It's within the pruning season we will be relentlessly tempted to turn away from the innermost intimate life we were enjoying in the Vine. We will be tenaciously enticed to stimulate something, anything, to encounter our own worth and secure for ourselves some level of happiness once again.
How can we know a self-help philosophy when we hear it? It is when the wisdom and encouragement we hear in our ears fails to point to Jesus as our source and ultimate gratification. Rather, the reasonings point us back to ourselves as the alpha and omega of our own lives and happiness. In other words, coaching self-interest attracts the selfish ambition already warring within us. We discover justifications of entitlement then draw upon the power of our own determination to perform a work that while it may benefit others, ultimately gratifies ourselves. Self-help can easily look like success at all the wrong things.
The bible relays that in the last days people will be lovers of self, sensual pleasures, and amusement rather than lovers of God (2 Tim 3:2). Our airways buzz with the tireless pursuit of happily-ever-after. This buzz competes for the affection of our souls. Widely celebrated experts on life and success, those who appear good or even godly, work at genius levels of insight to awaken our selfish ambitions. They promise that if we give all our heart, all our soul, and all our strength to the American dream, we can have it. Did you hear it? Did you hear the enticement to become unfaithful? They use the very promises we vowed to God to turn us away from our intricacy woven life in the Vine and divorce ourselves from Him.
Helplessness Is Our Great Reward
Jesus told the story of the Vinedresser to infuse us, the beloved branches, with courage (John 16:1). He doesn't want us to consider it strange when we lose our fruit. Pruning is a daring act of love our Father performs to safeguard our hearts from independence. God calls pruning our reward for being fruitful, and He puts Himself at risk of being misunderstood by us.
Let's be honest, pruning hurts. There is no way around it. It is what God calls suffering for righteousness (for all the right reasons). However, if we told another truth we would have to confess our strong aversion to suffering for what is right. This aversion is currently robbing His Bride of the resurrection power she desperately needs to be effective where she lives.
True Love's answer to our masturbation of self-help is to move in, closer than our skin, and tenderize us with helplessness. Our own natural abilities to avert the suffering will be far removed from us. Our abilities to arouse successes and fruitfulness will fail to gratify us. Every familiar source we've drawn from in the past to attain happiness will be emptied from us. Eventually we will find ourselves laid out on the side of our road called Destiny. We will be desperate to "help" ourselves into a happily-ever-after existence that was only intended to build while on earth not climax.
The American church, as an example, seems to be afraid to suffer for what it right. She suffers endlessly for the wrong reasons of sin and sickness, but she will not move into suffering for what is right. She will not follow her Lover in suffering the loss of reputation and comfort when following Him is what is right. She will not suffer the fog of the unreasonable that accompanies the impossible when obeying Him is what is right. She will not suffer personal downsizing and go without when loving Him with her whole life is what is right.
We "self-help" in suffering. We need to remember that Jesus' Father is our Father. Jesus, as a son, learned obedience through the things He suffered. To love Him is to obey Him and that includes fellowshipping with His sufferings. What do we do with a God who writes suffering into the stories of His own children's lives for the sake of righteousness?
In the pruning seasons it would be best for us to stop averting and start embracing the helplessness, the loneliness, the emptiness, the disorientation, the loss of reputation, the loss of presence, the loss of hope in a future, the loss of worth, the loss of zeal, the humiliations, and the deep sadness. Pruning is not a time to self-medicate. If we do, we will numb ourselves from the truly intimate encounter of rising again.
Love Stands by Watching
As we attempt the masturbating of self-help, Love is standing by watching. True Love is faithful, and He promised to watch over us always. When the lack of gratification finally exhausts us, His right hand with pleasure-evermore gently extends casting its cooling shadow, and He whispers His most fervent invitation, "abide in Me."
Andrew Murray said, "The intercourse (in Christ) was not only meant to be unbroken, but most intimate and complete." Our saving grace? We will feel the compromise. Our past fruitfulness has ruined us for anything less than a full and intimate relationship with Him and Him alone. We have encountered the divine life that intercourses between our life in Him and His life in us. Our helplessness will return us to Him and to the one thing that makes our lives worth living: His pleasure. The masturbation of self-help will be impotent to reproduce the intimate life that we have relished from life in the Vine before.
Why? The love is so much better when two become one.
Going back to the comfort of a risk-free life is a temptation not an option. At our weakest points the enemy of our love will pressure us to drop all we've been believing for. He will offer every reasonable reason to quit. He will promise immediate relief from the pruning if we would just drop our ridiculous hopes. Don't retreat! Refocus, remember what God has said in the seasons of romance, and hold on.
Yes, the pruning will be too hard. Yes, it will take too long. Yes, it will be more than you can handle. It has to be in order for you to penetrate further into His great grace surging through the Vine. Don't despise your new level of helplessness, it will make it possible for the rooting and grounding of your faith to deepen. Your intimacy with Him will be unhindered and His power will overflow through you more than ever. From this point on you will not be uprooted by the lengthening and widening of the fruit about to blossom in your life.
When you have been tenderized by helplessness and have endured your losses without offense, He will call you My bless-ed (Luke 7:23). And you will call Him my Life.

subset: commoncrawl


text:
Paris attacks fugitive Salah Abdeslam 'escaped hiding place in cabinet'
PARIS attacks suspect Salah Abdeslam may have avoided police capture by hiding in a cupboard.
It is thought that Belgian-born Abdeslam may have been smuggled from his hiding place in Belgium in a piece of furniture going into a removal van, or a car.
It is believed that he played a central role in the ISIS terrorist attacks on November 13 which killed 130 people.
Belgian broadcaster RTBF has claimed he may have escaped his Belgian bolthole by taking advantage of the traffic to sneak out in a car or even hide in furniture.
RTBF claimed: "This leads to the conclusion that the suspect managed to flee before security forces intervened...the most likely theory is that he was smuggled out by his accomplices."
It added that this was the "top theory embraced by investigators".
"the most likely theory is that he was smuggled out by his accomplices"RTBF
A raid on his hideout could not be carried out on the night of the attacks because of a Belgian law banning night time raids.
He had disappeared by the time their searched the sites.
On the Sunday and Monday following the attacks Belgian security forces raided locations in Molenbleek where Abdeslam lived.
RTBF stated that signs were detected that he has been at one of the locations before they were searched.
The Belgian government announced that they would be changing the ban on night time raids after the attack.
The change is so they can investigate terrorism cases but it won't come into effect until next year.
Some countries implemented the ban as a measure to ensure that police powers of arrest are not abused.


text:
Roundtable: As Crimea Threatens Secession, Does East-West Split Hasten Ukraine's Political Divide?; Debate: Do Venezuelan Protests Reflect Popular Discontent, or the Old Qualms of a Divided Elite?
March 7, 2014
Putin Rejects U.S.-EU on Ukraine; Crimea Sets Referendum for Secession
Senate Rejects Independent Oversight of Sexual Assault
Top Army Prosecutor Suspended for Alleged Sexual Assault; General Pleads Guilty to Lesser Charges
FBI Probing Senate Panel for Seizing Classified CIA Docs on Torture
"Deporter-in-Chief" Obama Faces Pressure to Issue New Reprieve for Undocumented Immigrants
Duke Energy Ordered to Stop Pollution at N.C. Coal Plants
Mall Shooting Wounds 1 in Tennessee; Idaho Approves Concealed Guns on College Campuses
Colorado Prisoner Avoids Death Penalty with Backing of Victim's Father
Rep. Ryan: Lunch Programs Yield "Full Stomach, Empty Soul"


text:
Springfield Welcomes New Salesman
July 28, 2008
Murphy Tractor is pleased to welcome new salesman, Kelly Russell to the Springfield branch. His territory will include a 12 county area north and east of Springfield.
Kelly was born and raised in southwest Missouri. He spent time in Houston, TX for school, then New Jersey and Panama with the US Army. After the first desert storm, he returned to Missouri and began working for Conco Quarries where he worked for 3 good years doing everything from driving a stockpile truck to Quality Control. Kelly began his sales career with the local Springfield Aggregate equipment and Volvo dealer where he spent the last 11 years in sales and management (the last 5 yrs Kansas City).
Welcome Kelly!
Tags: Kelly Russell, Announcement, New Salesman.


text:
We drove out to French Nowheresville today for the yearly family corn roast thing. It was a nice day, warm but not unpleasantly so, and the clouds were fantastic, big and puffy and dramatic. We turned a corner on the lonely country highway and were faced with a gigantic one that looked exactly like a penis. Now usually I seem to miss sexual innuendos, but that one was just too obvious, I couldn't help feeling affected. I kept my eyes on the road and hoped my mom wasn't looking up. When we passed the little Papanack Zoo, I yelped, "Hey look! Llamas!"
Anyway, we turned some more remote corners and got to my uncle's place, a cottage out in the tamed, farm-covered lands slightly east of the city, and voila! The corn roast faces me down, armed with its various ranks of half-known uncles and aunts. All of them are French, and not just a little French, very French. And the language of Quebec is different, full of slang and weird pronunciations – that on top of the fact that French in general is tough to speak with competence when it's your second language. So I end up sitting there, understanding most of what's being said but also terrified to try to speak it myself. My usual strategy is just to say "Bon, merci" when the relatives ask how I've been and hope that satisfies them. Rarely does anyone try to engage me in conversation, outside of the small circle of people I like, and who like me back, I imagine – but when they do I get along with short answers and lots of smiling and nodding. I thought that was only a thing in bad TV shows but you know, it isn't; you can smile and nod your way through lots of real-life things.
The corn roast was long and fraught with mosquitoes, but otherwise it was nice. Everyone was friendly. I'm not sure to what extent the family knows of my LUBR (Large Uncomfortable Boy Revelation) and I'm also not sure how the future will go, as I duke it out with backwoods Quebec. Honestly it unnerves me, to imagine how they'll face the fact that I'm going to grow up and become a man, not a woman. I'm not exactly front and center in the family, I imagine my place is off to the side somewhere, a floating speck of no particular importance, nice and quiet and inoffensive – so I guess they could all just ignore it and let it be. Everyone has always seemed basically nice, and have never been rude to me; the opposite, they've always been friendly and I hope I'm a positive figure in their minds. But they're also conservative and traditional, excepting a few of the younger aunts, uncles, and cousins, and excepting my grandma. And it's hard for anyone to accept the thing I have, even people like my English grandparents, who have always been kind and intelligent and progressive. I'm worried about when the hormones I'm taking really kick in and nobody can imagine away the fact that I have a thing and it's actually going to affect them, in whatever small way. For instance I literally can't even begin to imagine a certain great-aunt I have ever doing the pronoun switch, not even when I have sideburns and a deep voice. And that is going to be extremely difficult, when it happens, because I'm so sure she won't switch. I just don't want to be alienated. I like these people, even if I only see them once in a blue moon; I really do like some of them, and I'm happy that I can go to the corn roasts and be accepted and everything. I never want that to end, I appreciate it and I appreciate them.
But that, as well as many other things, I'm learning, is out of my control. I can send everybody LGBT leaflets – every obscure aunt and uncle from here to the northern end of Ontario – but in the end how they react is up to them. I guess all I can do in the meantime is worry about myself and my immediate friends and family, and hope it works out without me.
You know I think I've talked about this before, but I want to go over it again – the thing about sexuality, how it's not related to gender, but how everyone thinks it is. First everyone thought I was a lesbian because of how I dressed. Then, coming out as transgender, people slowly began to assume that I really did like girls after all, being a boy. Now I have to explain to them that I don't, in fact, explicitly like girls, but in fact just sort of like who I like, with no real preference. I bring it up because of a thing that has happened recently: it's that one of my friends (take note, he was the one who told me he had a crush on me in grade five) sent me a cat emoticon with a heart over its head in a Facebook message after we had a conversation about me being transgender. (He asked me slyly about it after I sent him a story I'd written.) Now, before you shake your head (I see you beginning to think about shaking it, or perhaps you already have), let me tell you that no, I don't put much store in cat emoticons. I think they're pretty cute, but that's not relevant, is it. It's just that of ALL the cat emoticons, why the one with the heart over its head? Sure, you could say it's an expression of support, of caring (by the way I had a small heart attack of relief after he said he was utterly fine with my big revelation), but could you not also come to the conclusion that it is an expression of liking? Well? You know, I hope it is. I've had an on-off liking of him for years. And I'm tired of Zuko being my pretend boyfriend, a real one would be great. A real girlfriend would be great, too. I kind of maybe have briefly entertained fantasies about the ridiculously cool girl at the video game store downtown who looks like she jumped right out of Scott Pilgrim v.s the World. If she doesn't seem like she's maybe twenty or so, and if I was less Asperger's, I would ask her out in a jiffy. Then we would play Zelda Twilight Princess and watch artsy movies together all day. That would be great.
And also, wouldn't you know, school approaches. Oh yes, indeed, she does, upon her chariot of death, eyes aflame, wielding the scythe of misery in one cruel hand! Cower before this demon – all ye children hide yourselves, 'fore she sweeps upon you and steals you away, to suck the lifeblood from your lovely veins and deposit your creaking bones 'pon the bed of heartless society. That was a bit over the top. I think school is more like a wolf, and we are sheep, running blindly from its snapping heels and losing ourselves in the wilderness of vapid education. Although last year's English class wasn't so bad – I happened upon a pretty great teacher, not one of those badly-constructed androids that seem so common. Anyway yes indeed, I'm headed back to school in nine days, although my brain won't process that reality quite yet, and I'll be trying for my last English credit and my first arts credit. Should be anxiety-ridden and horrible as always, but at least I'll see my friends.
Anything else? I'm pretty tired now, it's almost midnight and the corn roast sucked most of the life out of me. I wrote all this on my last 10% or so. I apologize for not doing any posts for the last couple of months – sometimes life is very hard to fit into a 1000-some blog post that a handful of people may or may not read, and besides that I get lazy and overwhelmed. I'll try to write more often again, but no promises – in fact when I make promises like that, more often than not they just make doing it harder. I do write stories, as ever, although most of them I end up abandoning for one reason or another – and I've also been doing some stuff on my music blog, darksideoftheroom, if you happen to be interested in that, and if you've been reading this far. Oh and also, I made an album on my Bandcamp, you can buy it if you want, or you can ask me if I can send you the link to it for free so you don't have to pay, which I'll do. Is that it? That's it. Do come again, and thanks as always for taking the time to read this large mess of words.
(Psst: I don't always wear sunglasses, but when I do, they're rainbowy sunglasses.)
– Brynn


text:
The Theater Division launched our new professional theater in partnership with the City of Dublin, Ohio so that we can offer our students continuous theater experience, extending the tradition started by Elizabeth Evans Baker in 1956. Tantrum Theater (so called because a group of Bobcats is sometimes referred to as a tantrum) produced three productions this summer. The season started with Little Shop of Horrors, directed by Daniel Dennis, Tantrum's Artistic Director, with a creative team of professional OHIO faculty designers. The cast featured current performance students and professionals. Next up was Tammy Faye's Final Audition, a new play by alumnus and new Assistant Director of the School of Dance, Film, and Theater, Merri Biechler, featuring head of performance Shelley Delaney in the title role, directed by our Directing Program Head, Dennis Delaney, and also featuring a creative team of professional OHIO faculty designers. The final production was Dancing at Lughnasa directed by Sheila Daniels with a creative team entirely from the Seattle area, featuring Assistant Professor Brian Evans, current students and recent alumni in the cast. All three productions were staffed by students and faculty, rehearsed and built in Kantner Hall on campus, and moved to the Abbey Theater in Dublin for performances. Over 100 students, faculty, staff and professionals were employed by Tantrum this summer. Check out Tantrum's website: www.tantrumtheater.org.
Back on our home turf, we begin our 2016-2017 season with The Library, directed by Shelley Delaney, a play that debuted at New York Public Theater in 2014, which explores the issues of violence and guns in America's schools. Next up is Charles Morey's adaptation of a Georges Feydeau gut-busting farce, The Ladies Man, directed by Dennis Delaney. Rounding out fall semester are our two "style projects", Machinal and Mrs. Packard, directed by second year grad directors Ben Stockman and Allison Epperson respectively.
Spring semester begins with Stupid F***ing Bird, a wonderful modern take on Chekhov's The Seagull, by Aaron Posner, directed by David Haugen. We follow Bird with The Rover, written in 1677 by Aphra Behn, the first female playwright to earn a living as a playwright. The Restoration comedy has surprisingly modern themes and will be adapted by one of our graduate playwrights. It will be directed by Brian Evans. As has been the case for the last 23 years, we end the 2016-17 season with the Seabury Quinn Jr. Playwrights' Festival.
Last but certainly not least, for the last eight months, we have been in discussions with the Acting Company of New York City to be the anchor school in a new consortium of partner schools in the region. The Acting Company is the only permanent, professional touring repertory company dedicated to the development of classical actors. As soon as the official documents are signed this fall, we will schedule visiting artist events throughout the 2016-17 and 2017-18 academic years, culminating in performances of a tour of two Acting Company productions on our stages. Stay tuned for updates.
Please join us for one or all of our productions in 2016-2017!
Michael Lincoln
Artistic Direct/Head


text:
Citing both Apple Computer and Sony's intentions to become the hub of the digital lifestyle, Popular Science has pitted an US$1,800 800MHz flat-panel iMac up against a $3,400 Sony Vaio PCV-MXS10 to see which really comes out on top. The new comparison, penned by Steve Morgenstern, is called Put up your digital dukes.
Over six pages, Morgenstern outlines the advantages and disadvantages of each system. There's too much content to go into depth here, but the bottom line is that the Sony PC -- almost twice as expensive as the iMac -- just narrowly edged out the iMac, with an overall score of 42 to 40.
Morgenstern concluded that both systems get the job done, each delivering on the digital hub promise in their own unique ways. He favors the Sony, however, since "it's a better-rounded system for the digital content that matters" to him.
Overall, however, the iMac edged out the Vaio when it came to ripping CDs to MP3, loading music onto a portable MP3 player, making a DVD, accessing the Internet, and downloading pictures. The Vaio, meanwhile, gets the edge on listening to music, watching movies, editing home movies, dealing with PDAs and touching up and organizing photos.
For the full details, visit Popular Science's Web site.


text:
Lung cancer is the leading cause of cancer deaths worldwide, with a five-year overall survival rate of only 15%. Cancerous inhibitor of PP2A (CIP2A) is a human oncoprotein inhibiting PP2A in many human malignancies. However, whether CIP2A can be a new drug target for lung cancer is largely unclear.
Methodology/Principal Findings
Normal and malignant lung tissues were derived from 60 lung cancer patients from southern China. RT-PCR, Western blotting and immunohistochemistry were used to evaluate the expression of CIP2A. We found that among the 60 patients, CIP2A was undetectable or very low in paratumor normal tissues, but was dramatically elevated in tumor samples in 38 (63.3%) patients. CIP2A overexpression was associated with cigarette smoking. Silencing CIP2A by siRNA inhibited the proliferation and clonogenic activity of lung cancer cells. Intriguingly, we found a natural compound, rabdocoetsin B which is extracted from a Traditional Chinese Medicinal herb Rabdosia coetsa, could induce down-regulation of CIP2A and inactivation of Akt pathway, and inhibit proliferation and induce apoptosis in a variety of lung cancer cells.
Citation: Ma L, Wen Z-S, Liu Z, Hu Z, Ma J, Chen X-Q, et al. (2011) Overexpression and Small Molecule-Triggered Downregulation of CIP2A in Lung Cancer. PLoS ONE 6(5): e20159. https://doi.org/10.1371/journal.pone.0020159
Editor: John D. Minna, Univesity of Texas Southwestern Medical Center at Dallas, United States of America
Received: January 31, 2011; Accepted: April 12, 2011; Published: May 31, 2011
Copyright: © 2011 Ma et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by the National Key Program for Basic Research (973, 2010CB529201), the Key Project of Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX1-YW-R-26 and KSCX2-YW-R-235), the National Natural Science Foundation of China (81071930, 30871110), the National Major Scientific and Technological Program for Drug Discovery (2009ZX09103-101), and the Science and Technology Planning Project of Guangzhou (2009Y-C011-2). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Nearly 1.5 million people were diagnosed with lung cancer and 1.4 million people were estimated to die from it in 2007 [1]. The two major forms of lung cancer are non–small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). About 85% of lung cancers are NSCLC, which can be divided into three major histological subtypes: squamous cell carcinoma, adenocarcinoma, and large-cell carcinoma. SCLC represents about 15% of lung cancers [2]. Smoking causes all types of lung cancer but is most strongly linked with SCLC and squamous-cell carcinoma; adenocarcinoma is the most common type in patients who have never smoked [2]. Current treatment is determined by the histologic type of lung cancer and stage at diagnosis, including surgery, platinum doublet therapy, radiation therapy and targeted therapy. Unfortunately, the prognosis of lung cancer is poor with a only 15% of five-year overall survival rate for all stages combined [1]. Therefore, there is an urgent need to identify more effective molecular targets and new targeted therapies for lung cancer.
Cancerous Inhibitor of PP2A (CIP2A) is a human oncoprotein that stabilizes c-Myc by inhibiting protein phosphatase 2A (PP2A)-mediated dephosphorylation of MYC at serine 62 [3]. In addition to inhibiting the degradation of c-Myc, CIP2A appears to be regulated in a positive feedback loop with c-Myc by promoting each other's expression [4]. CIP2A is over-expressed in human neck and head carcinomas, colon, breast, and gastric cancer, and is inversely correlated with disease outcome in gastric cancer [3]–[7]. However, whether CIP2A could be a new drug target for cancers is not fully investigated, and the anti-tumor activity of CIP2A-targeting agents remains largely unknown. We studied the expression of CIP2A in lung cancer and screened for lead compounds that could target CIP2A [8]. Here we show that CIP2A is markedly upregulated in lung cancer tumors compared to patient-matched adjacent normal lung tissues, and report that a natural compound which triggers downregulation of CIP2A exhibits significant antitumor activity in NSCLC cell lines.
CIP2A is over-expressed in lung cancer and is associated with cigarette smoking
We tested the expression of CIP2A at protein level in nonmalignant and malignant cells, and found that CIP2A was highly expressed in lung cancer cell lines (A549, H1975, 95D and L78) compared to normal human embryonic lung fibroblasts (HLF and MRC5) and normal human bronchial epithelial cells (HBEpiC) (Figure 1A). We then analyzed CIP2A in 60 lung cancer samples from patients came from southern China whose baseline characteristics were listed in Table 1. We showed that CIP2A was over-expressed in 38 (63.3%) tumor specimens assayed by western blotting (Figure 1B). However, in the 60 patient-matched adjacent normal lung tissues, CIP2A was undetectable in 57 (95%) samples, and was weakly expressed in 3 (5%) specimens where its expression was much lower than that in tumor samples of the same patients. In samples from 2 patients with inflammatory pseudotumor, CIP2A was not detected in both the pseudotumor and adjacent lung tissues (Figure 1C). Immunohistochemistry assay confirmed that CIP2A was dramatically elevated with a higher immunoreactivity score in tumor samples in 26 out of 39 patients (66.7%) tested (Figure 1D). At mRNA level, CIP2A was also over-expressed in lung tumor tissues compared to normal lung tissues in 39 of 58 (67.2%) patients tested (Figure 1E). Taken together, CIP2A is dramatically elevated in lung cancer tumor samples compared to paired normal lung tissues.
(A): Western blot analysis of CIP2A in human lung cancer cells (A549, H1975, 95D and L78), embryonic lung fibroblast cells (HLF and MRC-5), and normal human bronchial epithelial cells (HBEpiC). (B): Western blot analyses of CIP2A protein in primary lung tumors (T) and adjacent normal lung tissues (N). β-Actin is used as a loading control. Representative results are shown and numbers are referred to individual patients. (C): Western blot analyses of CIP2A protein in inflammatory pseudotumor (P) and adjacent normal lung tissues (N). β-Actin is used as a loading control. (D): Representative images (left panel) and score (right panel) of immunohistochemistry staining for CIP2A expression in primary lung tumors (T) and adjacent normal lung tissues (N). (E): RT-PCR analyses of CIP2A mRNA in primary lung tumors (T) and adjacent normal lung tissues (N). GAPDH is employed as a loading control. Representative results are shown and numbers are referred to individual patients.
CIP2A overexpression is associated with cigarette smoking
We analyzed the correlation between CIP2A overexpression and some clinicopathologic variables. The data demonstrated that CIP2A overexpression in lung cancer was significantly correlated with histologic type of squamous cell carcinoma (p = 0.003) and male gender (p = 0.008) (Table 1) that were shown to strongly link with cigarette smoking [2], [9]. Moreover, our data clearly showed that the high expression of CIP2A was associated with smoker patients (p = 0.004). No significant difference in the CIP2A status was observed according to the pathological stage (p = 0.639) and age (0.082) (Table 1). In an attempt to clarify the most significant factors related to CIP2A overexpression, the multivariate analyses were performed, and the multivariate logistic regression analysis (Table 2) demonstrated that cigarette smoking was the only significant variable associated with CIP2A overexpression in lung cancer (p = 0.008).
The nitrosamine 4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone, or the nicotine-derived nitrosamine ketone (NNK), is a key ingredient of tobacco smoke carcinogen which systemically induces tumors of the lung in rats, mice, and hamsters and also plays a major role in lung carcinogenesis [10], [11]. We then investigated whether NNK could directly induce upregulation of CIP2A or not. To do this, HBEpiC (Figure 2A) and BEAS-2B (Figure 2B) bronchial epithelial cells were exposed to NNK at indicated concentration for indicated time points, lysed, and western blot was performed to analyze the expression of CIP2A. The results showed that treatment with NNK at 0.1 to 10 µM for up to 18 days could not perturb CIP2A expression (Figure 2, A and B). In this study, we did not test the long-term effect of NNK on CIP2A expression in vitro or in vivo.
(A): HBEpiC cells were treated with NNK at various concentrations for indicated time points, and western blot was performed to analyze CIP2A expression. (B): BEAS-2B cells were treated with NNK at indicated concentrations for indicated time points, and western blot was conducted to analyze CIP2A expression. 0.05% and 0.1% DMSO were used as a solvent control corresponding to 5 µΜ and 10 µΜ NNK, respectively.
CIP2A is required for lung cancer cells growth and transformation
CIP2A specific siRNA was employed to evaluate its roles in lung cancer pathogenesis, and the results showed that as compared to negative control (NC), CIP2A silencing (Figure 3A) led to inhibition of clonogenic activity of A549 cells, detected by foci formation (Figure 3, B and C) and soft agar colony formation (Figure 3, D and E) assays [3]. These phenomena were confirmed by results of CIP2A silencing in L78 cells (Figure S1, A through E). Next, A549 cells were transfected with NC or CIP2A-specific siRNA (Figure 3F), and injected subcutaneously into the right and left flanks of 8 nude mice respectively, and tumor volumes were estimated every two days [12]. Interestingly, CIP2A-specific siRNA significantly inhibited tumor growth as compared to NC-siRNA (Figure 3, G through I). Together, these data indicate that CIP2A is essential to lung cancer proliferation and tumorigenesis, and could be an effective therapeutic target.
(A): Western blot analysis of CIP2A protein expression in A549 cells 72 h after transfection with negative control (NC) or CIP2A-specific siRNA. (B and C): Flat plate clone formation assay for the clonogenic activity of A549 cells 72 h after transfection with NC or CIP2A-specific siRNA. (B): representative light miscroscopy images. (C): Quantitation of foci counting. Shown is mean+SD of four independent experiments. (D and E): Soft-agar colony formation assay for A549 cells transfected with NC or CIP2A-specific siRNA. (D): representative light miscroscopy images. (E): Quantitation of foci counting. (F): Western blot analysis of CIP2A protein in A549 cells transfected with NC or CIP2A-specific siRNA for 72 h. (G): Nude mice injected subcutaneously with A549 cells transfected with NC or CIP2A-specific siRNA. (H): The tumor growth curve for the experiment shown in (G). Shown is mean+SD of the mean tumor volumes. (I) Image of xenograft tumors obtained from mice shown in (G). * p<0.01, Student's t test.
Rabdocoetsin B is a CIP2A-targeting natural compound
Our central goal was to identify new drug targets and provide lead compounds for drug development. We screened for CIP2A-targeting small molecules by analyzing their effects on CIP2A expression, and identified a natural compound extracted from a Chinese medicinal herbal Rabdosia coetsa, rabdocoetsin B [13], [14] (Figure 4A), could downregulate CIP2A at protein level at 5 to 20 µM in A549 cells (Figure 4B). In H1975 cells, Rabdocoetsin B also triggered downregulation of CIP2A at 5 to 10 µM (Figure 4C). We analyzed the mechanism of CIP2A down-regulation caused by Rabdocoetsin B, and found that rabdocoetsin B significantly inhibited the transcription of CIP2A in a dose-dependent manner (Figure 4D) assessed by real-time RT-PCR.
(A): Structure of rabdocoetsin B. (B): A549 cells were treated with rabdocoetsin B (RdB) at various concentrations for 48 h. Western blots were used to detect the expression of CIP2A protein (upper panel) and CIP2A protein expression were quantified and normalized against β-actin expression (lower panel) (C): H1975 cells were treated with rabdocoetsin B at various concentrations for 24 h. Western blots were used to detect the expression of CIP2A protein (upper panel) and CIP2A protein expression were quantified and normalized against β-actin expression (lower panel) (D): A549 cells were treated with rabdocoetsin B at various concentrations for 48 h, and the mRNA expression of CIP2A was analyzed using real-time RT-PCR.
Rabdocoetsin B inhibits CIP2A-modulated phosphorylated Akt
In hepatocellular carcinoma cells, CIP2A up-regulates phospho-Akt (pAkt) and decreases Akt-related PP2A activity, whereas silencing CIP2A re-activates PP2A [15]. Since Akt is constitutively active in lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation [16], [17], we examined the effect of CIP2A on pAkt in lung cancer. We showed that CIP2A silencing by specific siRNA resulted in down-regulation of pAkt but not pERK, PCNA, β-catenin, EGFR or Src (Figure 5A). We then investigated whether rabdocoetsin B could modulate the expression of pAkt, and found that treatment with rabdocoetsin B at 5 to 10 µM also down-regulated pAkt in A549 (Figure 5B) and H1975 (Figure 5C) cells. We further showed that in H1975 cells upon rabdocoetsin B at 10 µM, CIP2A was markedly downregulated in 6 to 12 h and became undetectable in 48 h (Figure 5D), while pAKT was decreased in 18 h (Figure 5D). These results were confirmed in A549 cells treated with rabdocoetsin B (Figure 5E), indicating that this compound can inhibit the CIP2A-Akt pathway in lung cancer.
(A): A549 cells were transfected with NC or CIP2A-specific siRNA for 72 h, and the expression of indicated proteins were detected using Western blots. (B and C): A549 (B) and H1975 cells (C) were treated with rabdocoetsin B (RdB) at indicated concentrations for 48 and 24 h, respectively, and Western blots were performed to analyze the expression of proteins indicated. (D and E): H1975 (D) and A549 (E) cells were treated with rabdocoetsin B (RdB) for indicated time points, and Western blot analysis was carried out with antibodies specific for the indicated proteins. β-actin is used as a loading control.
Effects of Rabdocoetsin B on lung cancer cells
We evaluated the effects of rabdocoetsin B on lung cancer cells expressing wide-type (WT) or mutant EGFR. We showed that rabdocoetsin B exhibited significant cytotoxic effects on A549, NCI-H1975, HCC827, SPC-A-1, GLC-82, L78 and 95D lung cancer cell lines (Figure 6, A and B). Rabdocoetsin B induced apoptosis of A549 cells (Figure 6C) with activation of casp-8 and casp-9 and cleavage of PARP (Figure 6D). Rabdocoetsin B also caused activation of casp-8 and casp-9 and cleavage of PARP in NCI-H1975 cells (Figure 6E). These results indicate that rabdocoetsin B inhibits proliferation and induces apoptosis of lung cancer cells via activating the endogenous and exogenous apoptosis pathways.
(A): Lung cancer cells were treated with increasing concentrations of Rabdocoetsin B (RdB) (from 1 µM to 10 µM) and cell viability was measured 48 h by the MTT assay. (B): the IC50 of cells treated with rabdocoetsin B. (C): Rabdocoetsin B induces apoptosis of A549 cells, assayed by flow cytometry. (D and E): Western blots were performed to detect the expression of apoptosis regulators in A549 (D) and H1975 cells (E).
CIP2A is an auto-antigen [7] that is over-expressed in human neoplasms [3]–[7], [18]–[21]. Peng [22] showed that in American-based patients, CIP2A is increased in 61 of the 72 (84.7%) lung cancer tissue specimens, which is significantly higher than in normal lung tissues (14.3%, 9/63). Recently, Dong et al [23] reported that in 29 patients from Northern China, CIP2A is over-expressed at mRNA level in 24 cases (82.7%) in tumor samples compared with their corresponding normal tissues; CIP2A protein (detected by immunohistochemistry) is found to be over-expressed in 72.2% of 90 lung cancer samples and correlated with poor survival. We test CIP2A expression in 60 patients from Southern China [8], and report that CIP2A is drastically increased in 63.3% (detected by western blotting) or 67.2% (at mRNA level) of tumor specimens compared to adjacent normal tissues (Figure 1).
We show for the first time that CIP2A overexpression is associated with cigarette smoking. In the 60 lung cancer patients examined in this study, 28 of 36 (77.8%) smoker patients show increased expression of CIP2A in the tumor samples compared to their adjacent normal specimens, whereas 10 of 24 (41.7%) nonsmoker cases exhibit up-regulated CIP2A at protein level (p = 0.004) (Table 1). Currently smoking continues to be more prevalent among men (63%) than women (3.8%) [24]. We report here that CIP2A is over-expressed in 30/40 (75%) and 8/20 (40%) in men and women patients (p = 0.008) (Table 1), respectively. We also find that CIP2A is elevated in 17/19 (89.5%) patients with squamous cell carcinoma, whereas 17/35 (48.6%) patients with adenocarcinoma have over-expressed CIP2A (p = 0.003) (Table 1). The multivariate logistic regression analysis clearly demonstrates that smoking is the only significant variable associated with CIP2A overexpression in lung cancer in our setting (p = 0.008) (Table 2). While the overall smoking rate in the North and Northeast sectors is higher than that in the South and East of China, the prevalence of environmental tobacco smoke exposure among nonsmokers in Northern China is significantly higher than that in nonsmokers in Southern China [24], [25]. These may contribute to the difference in CIP2A expression between patients in our setting and Dong's report [23]. Tobacco smoke which causes approximately 5–6 million deaths per year and 31% and 6% of all lung cancer deaths in middle-aged men and women respectively [26], can induce genetic and epigenetic changes and reduce DNA repair capacity [2]. Zhao et al [27] recently showed that in human gastric cell lines, Helicobacter pylori infection can induce CIP2A expression via bacterial oncoprotein CagA-caused activation of the Src and MEK/ERK signal pathways. Though NNK (0.1 to 50 µM) fails to upregulate CIP2A in HBEpiC and BEAS-2B cells in up to 18 days in our study (Figure 2), we can not exclude the possibility that NNK may perturb the expression of the oncoprotein in a longer exposure time course, and this hypothesis warrants further investigation.
CIP2A is critical for lung cancer cell growth, since CIP2A knockdown by specific siRNA results in significant inhibition of cell proliferation and transformation in vitro and in vivo (Figure 3 and Figure S1). These data indicate that CIP2A could be an attractive target for novel anti-lung cancer drugs. Interestingly, we identify a natural compound rabdocoetsin B, can down-regulate CIP2A protein in lung cancer cells (Figure 4, A through C) by inhibiting its expression at mRNA level (Figure 4D). Studies show that CIP2A can up-regulate pAkt [15], and our results confirm that CIP2A silencing can decrease pAkt (Figure 5A). We further show that rabdocoetsin B also inhibits pAkt (Figure 5, B through E). Rabdocoetsin B inhibits the growth and induces apoptosis of a variety of lung cancer cells (Figure 6, A through E). Our data thus provide a lead compound for CIP2A-targeting anti-tumor therapeutics.
Materials and Methods
Patient samples
Use of the samples was approved by the Institutional Review Board of Institute of Zoology, Chinese Academy of Sciences and The Cancer Hospital, Sun Yat-Sen University. All tumor and adjacent normal tissue samples were obtained with written informed consent from patients at the Cancer Hospital, Sun Yat-Sen University. For RT-PCR analysis, tissue specimens were ground in liquid nitrogen-cooled mortar, RNA was extracted using the Trizol (Invitrogen), and RT-PCR experiments were performed using PrimeScript® One Step RT-PCR Kit Ver.2 (TaKaRa) according to the manufacturer's protocol. The sequences of primers used for CIP2A are as follows: forward 5′-CCATATGCTCACTCAGATGATGT-3′,_reverse 5′-GTGTATCATCTCCACAGAGAGTT-3′ [5].
For Western blot assay, tissue specimens were ground in liquid nitrogen-cooled mortar, tissue powder was suspended in lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM Na3VO4, 1 mM NaF, 1 mM EDTA, 1 mM PMSF, complete protease inhibitor cocktail) and cleared by centrifugation. Equal amounts of samples were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-CIP2A or anti-β-actin antibody.
Immunohistochemical assay and scoring of immunoreactivity were performed as described [28]. Formalin-fixed, paraffin-embedded lung cancer tissue specimens (5 µm) were deparaffinized and subjected to a heat-induced epitope retrieval step for 2 min. The H2O2 (3%) was used to block endogenous peroxidase activity for 10 min. The sections were then washed with PBS. The rabbit polyclonal anti-CIP2A antibody was applied to the slides at a dilution of 1∶500 at 4°C overnight. Detection was achieved with the Immunohistochemistry kit (pv-6001) (Zhongshan Golden Bridge Biotechnology Co., Ltd, Beijing, China) according to the manufacturer's protocol. Sections were colored with 3, 3′-diaminobenzidine (DAB) and counterstained with hematoxylin, dehydrated, treated with xylene, and mounted. CIP2A protein levels were scored as described [29].
Rabdocoetsin B was extracted from Rabdosia coetsa by Professor Han-Dong Sun. The 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di- phenytetrazoliumromide (MTT) was purchased from Amresco, Inc. (Solon, OH). PE Annexin V-7AAD Apoptosis Detection kit was obtained from BD Biosciences (San Jose, CA, USA).
The antibodies used in this study were as follows: anti-β-Actin (Sigma); anti-casp-9 (C9), anti-casp-8 (1C12); anti-PARP, anti-phospho-p44/42 MAPK, anti-EGFR, anti-Src and anti-β-catenin (Cell signaling), anti-CIP2A (2G10-3B5), anti-pAKT and ATK (Santa Cruz Biotechnology); anti-ERK2 (Abcam); anti-PCNA (Abmart); anti-rabbit or anti-mouse HRP-conjugated secondary antibody (Pierce); Rabbit Polyclonal anti-CIP2A (Novus Biologicals, Inc). Detection was performed by using a Chemiluminescent Western detection kit (Cell Signaling).
Cell culture
The lung cancer lines NCI-H1975 and A549 and human embryonic kidney HEK-293 cells were obtained from the American Tissue Culture Collection (ATCC) and human embryonic lung fibroblast MRC-5 cells were purchased from the Cell Resource Center, Chinese Academy of Medical Sciences (Beijing). Normal human bronchial epithelial cells (HBEpiC, Catalog Number: 3210) were purchased from ScienCell (ScienCell Research Laboratories, San Diego, California). Human lung squamous carcinoma cell lines L78 and highly metastatic large-cell lung cancer cell lines 95D were obtained from the Cell bank of Chinese Academy of Sciences (Shanghai), and human embryonic lung fibroblast HLF cells were purchased from Kenqiang Instrument Co., Ltd (Shanghai, China). BEAS-2B bronchial epithelial cells were provided by Professor Hongbin Ji at Shanghai Institute for Biological Sciences, Chinese Academy of Sciences. A549, HLF and BEAS-2B cells were cultured in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS; Gibco/BRL, Grand Island, NY), 100 U/ml penicillin, 100 µg/ml streptomycin. HBEpiC cells were cultured in a serum-free Bronchial Epithelial Cell Medium (BECM, Cat. No. 3211, ScienCell Research Laboratories) containing essential and non-essential amino acids, vitamins, hormones, growth factors and trace minerals. L78, 95D and NCI-H1975 cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS; Gibco/BRL, Grand Island, NY), 100 U/ml penicillin, 100 µg/ml streptomycin. MRC-5 cells were cultured in MEM/EBSS medium supplemented with non-essential amino acids, 10% fetal bovine serum (FBS; Gibco/BRL, Grand Island, NY), 100 U/ml penicillin and 100 µg/ml streptomycin.
Assessment of cell proliferation and apoptosis
Cells were treated with Rabdocoetsin B for indicated concentration and time points. Cell proliferation was determined using MTT assay. Cell viability was estimated by trypan blue dye exclusion. Externalization of phosphatidylserine was tested using a PE Annexin V-7 AAD Apoptosis Detection kit (BD Biosciences, San Jose, CA) according to manufacturer's instruction.
Quantitative real-time PCR
Quantitative real-time PCR was performed in CFXTM96 Real Time System (Bio-Rad) using SYBR® Premix Ex Taq™(Perfect Real Time) (TaKaRa Code: DRR041) according to the manufacturer's instruction. Primers for CIP2A and Actin were as follows: CIP2A: forward sequence, 5′-TGCGGCACTTGGAGGTAATTTC-3′, reverse sequence, 5′-AGCTCTACAAGGCAACTCAAGC-3′; Actin: forward sequence, 5′-ATCGTCCACCGCAAATGCTTCTA-3′, reverse sequence, 5′-AGCCATGCCAATCTCATCTTGTT-3′. Levels of CIP2A mRNA were expressed as the ratio versus actin based on the CT values.
siRNA assays
Using HiPerFect Transfection Reagent (Qiagen, Crawley, UK) according to the manufacturer's protocol, cells were transfected with 100 nM double-stranded siRNA oligonucleotides [3]. The siRNA sequences were 5′-CUGUGGUUGUGUUUGCACUTT-3′ (CIP2A siRNA1), 5′-ACCAUUGAUAUCCUUAGAATT-3′ (CIP2A siRNA2) [3], and 5′-UUCUCCGAACGUGUCACGUTT-3′ (negative control (NC) siRNA).
Clonogenic assay
For foci formation, A549 or L78 cells transfected with negative control or CIP2A-specific siRNA were seeded in triplicate onto 35 mm plates (300 cells per plate). After 14 days of culturing, cells were stained with Giemsa and clones containing more than 50 cells were counted. For soft-agar colony formation assay, cells were suspended in 1 ml DMEM (for A549 cells) or RPMI 1640 (for L78 cells) containing 0.3% low-melting-point agarose (Amresco, Solon, OH) and 10% FBS and plated on a bottom layer containing 0.6% agarose in 35 mm plat (1,000 cells/plate) in triplicate. After 2-3 weeks culturing, plates were stained with Giemsa and colonies were counted using a light microscope.
Murine models
All animal studies were conducted according to protocols approved by the Animal Ethics Committee of the Institute of Zoology, Chinese Academy of Sciences, with the approval ID of AEC2010070202. All mice used in this study were bred and maintained in a specific pathogen-free environment. Nude mice (n = 8) were injected subcutaneously with A549 cells (4×106) transfected with NC or CIP2A-specific siRNA into right and left flanks respectively, and tumor was calculated as described [12].
Statistical analysis
Differences between data groups were evaluated for significance using Student t-test of unpaired data, χ2 test or one-way analysis of variance and Bonferroni post-test. The tumor volume was analyzed with one-way ANOVA and independent sample t test using the software SPSS 12.0 for Windows (Chicago, IL). The association between CIP2A high expression and clinicopathological feature was assessed using either χ2 test or the Fisher exact test, and a backward stepwise multivariate logistic regression analysis was carried out to investigate the most significant variables related to CIP2A over-expression after adjustment. P values <.05 were considered statistically significant. All experiments were repeated at least three times and the data were presented as the mean±SD unless noted otherwise.
Supporting Information
Figure S1.
The effects of CIP2A depletion on the L78 cells' growth and trasnsformation. (A): Western blot analysis of CIP2A protein expression in L78 cells 72 h after transfection with NC or CIP2A-specific siRNA. (B and C): Flat plate clone formation assay for clonogenic activity of L78 cells 72 h after transfection with NC or CIP2A-specific siRNA. (B): Representative light microscopy images. (C): Quantitation of foci counting. Shown is mean+SD of three independent experiments. (D and E): Soft-agar colony formation assay of L78 cells transfected with NC or CIP2A-specific siRNA. (D): Representative light microscopy images. (E): Quantitation of foci counting.
We thank Professors Zhu Chen, Sai-Juan Chen and Zhen-Yi Wang at Shanghai Institute of Hematology for their long-term support. The authors thank Professor Jukka Westermarck at University of Turku, Finland for providing the pcDNA3.1-CIP2A construct, and Professor Hongbin Ji at Shanghai Institute for Biological Sciences, Chinese Academy of Sciences for providing the BEAS-2B cells.
Author Contributions
Conceived and designed the experiments: G-BZ. Performed the experiments: LM ZL ZH Y-QL J-XP W-LX. Analyzed the data: LM G-BZ. Contributed reagents/materials/analysis tools: H-DS. Wrote the paper: LM G-BZ. Clinical study: Z-SW JM X-QC.
- 1.
Garcia M, Jemal A, Ward EM, Center MM, Hao Y, et al. (2007) Global Cancer Facts & Figures 2007. Atlanta, GA: American Cancer Society, 2007. Available: http://www.cancer.org/Research/CancerFactsFigures/GlobalCancerFactsFigures/global-cancer-facts-figures-2007.
- 2. Herbst RS, Heymach JV, Lippman SM (2008) Lung Cancer. N Engl J Med 359: 1367–1380.
- 3. Junttila MR, Puustinen P, Niemela M, Ahola R, Arnold H, et al. (2007) CIP2A inhibits PP2A in human malignancies. Cell 130: 51–62.
- 4. Khanna A, Bockelman C, Hemmes A, Junttila MR, Wiksten JP, et al. (2009) MYC-dependent regulation and prognostic role of CIP2A in gastric cancer. J Natl Cancer Inst 101: 793–805.
- 5. Li W, Ge Z, Liu C, Liu Z, Bjorkholm M, et al. (2008) CIP2A is overexpressed in gastric cancer and its depletion leads to impaired clonogenicity, senescence, or differentiation of tumor cells. Clin Cancer Res 14: 3722–3728.
- 6. Come C, Laine A, Chanrion M, Edgren H, Mattila E, et al. (2009) CIP2A is associated with human breast cancer aggressivity. Clin Cancer Res 15: 5092–5100.
- 7. Soo HL, Zhang JY, Chan EK (2002) Cloning and characterization of a novel 90 kDa 'companion' auto-antigen of p62 overexpressed in cancer. Oncogene 21: 5006–5015.
- 8.
Zhou GB, Ma L, Wen ZS (2010) Cancerous inhibitor of phosphatase PP2A in lung cancer. AACR 101st Annual Meeting 2010 in Washington, DC Permanent Abstract Number. LB-45 p.
- 9. Sharma SV, Bell DW, Settleman J, Haber DA (2007) Epidermal growth factor receptor mutations in lung cancer. Nat Rev Cancer 7: 169–181.
- 10. Hecht SS (2002) Cigarette smoking and lung cancer: chemical mechanisms and approaches to prevention. Lancet Oncol 3: 461–469.
- 11. Lin RK, Hsieh YS, Lin P, Hsu HS, Chen CY, et al. (2010) The tobacco-specific carcinogen NNK induces DNA methyltransferase 1 accumulation and tumor suppressor gene hypermethylation in mice and lung cancer patients. J Clin Invest 120: 521–532.
- 12. Hu Z, Pan XF, Wu FQ, Ma LY, Liu DP, et al. (2009) Synergy between Proteasome Inhibitors and Imatinib Mesylate in Chronic Myeloid Leukemia. PLoS ONE 4: e6257.
- 13. Feng T, Pu J, Hu Z, Liu D, Sun H, et al. (2009) Rabdocoetsin B, a diterpenoid isolated from Isodon coetsa, is a potential proteasome inhibitor and induced apoptosis of t(8;21) leukemia cells. Chin J Biotech 25: 1218–1224.
- 14. Chen YP, Sun HD, Lin ZW (2011) Study on the diterpenoids of Rabdosia coetsa. Acta Bot Sin 32: 292–296.
- 15. Chen KF, Liu CY, Lin YC, Yu HC, Liu TH, et al. (2010) CIP2A mediates effects of bortezomib on phospho-Akt and apoptosis in hepatocellular carcinoma cells. Oncogene 29: 6257–6266.
- 16. Brognard J, Clark AS, Ni Y, Dennis PA (2001) Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res 61: 3986–3997.
- 17. David O, Jett J, LeBeau H, Dy G, Hughes J, et al. (2004) Phospho-Akt overexpression in non-small cell lung cancer confers significant stage-independent survival disadvantage. Clin Cancer Res 10: 6865–6871.
- 18. Basile JR, Czerninski R (2010) The role of CIP2A in oral squamous cell carcinoma. Cancer Biol Ther 10: 700–702.
- 19. Vaarala M, Vaisanen MR, Ristimaki A (2010) CIP2A expression is increased in prostate cancer. Journal of Experimental & Clinical Cancer Research 29: 136.
- 20. Qu W, Li W, Wei L, Xing L, Wang X, et al. (2010) CIP2A is overexpressed in esophageal squamous cell carcinoma. Medical Oncology 1–6.
- 21.
Wang J, Li W, Li L, Yu X, Jia J, et al. (2011) CIP2A is over-expressed in acute myeloid leukaemia and associated with HL60 cells proliferation and differentiation. Int J Lab Hematol. doi 10.1111/j.1751-553X.2010.01288.x. [Epub ahead of print].
- 22. Peng B, Liu SS, Qian W, Chan EKL, Tan EM, et al. (2010) Immune response to a fetal oncoprotein p90/CIP2A in lung cancer. AACR 101 Annual Meeting Abstract Book Abstract # 4792:
- 23. Dong QZ, Wang Y, Dong XJ, Li ZX, Tang ZP, et al. (2010) CIP2A is Overexpressed in Non-Small Cell Lung Cancer and Correlates with Poor Prognosis. Annals of Surgical Oncology 1–9.
- 24. Yang G, Fan L, Tan J, Qi G, Zhang Y, et al. (1999) Smoking in China. JAMA: The Journal of the American Medical Association 282: 1247–1253.
- 25. Gu D, Kelly TN, Wu X, Chen J, Samet JM, et al. (2009) Mortality Attributable to Smoking in China. New England Journal of Medicine 360: 150–159.
- 26. Jha P (2009) Avoidable global cancer deaths and total deaths from smoking. Nature Reviews Cancer 9: 655–664.
- 27. Zhao D, Liu Z, Ding J, Li W, Sun Y, et al. (2010) Helicobacter pylori CagA upregulation of CIP2A is dependent on the Src and MEK/ERK pathways. J Med Microbiol 59: 259–265.
- 28. Wang YY, Zhou GB, Yin T, Chen B, Shi JY, et al. (2005) AML1-ETO and C-KIT mutation/overexpression in t(8;21) leukemia: Implication in stepwise leukemogenesis and response to Gleevec. Proc Natl Acad Sci U S A 102: 1104–1109.
- 29. Remmele W, Stegner HE (1987) [Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue]. Pathologe 8: 138–140.


text:
Fall always puts me in a reflective mood, and this week I've been thinking about what I've learned since I joined publishing four years ago. This is an incredible time in the industry and what an adventure it has been! Here are a few of my top tips for writers.
1) Publishing success is a marathon, not a sprint. We've all heard the stories of authors who struck gold—winning an agent, a major publishing deal, and solid commercial success with their very first novel. It does happen. Heck, I've worked with a few of those authors. But a far more common scenario goes like this: author writes first novel, hates it, shelves it; writes three more manuscripts, uses them for firewood; submits fifth novel to dozens of agents, finally gets a bite; newly found agent submits manuscripts to publishers; pubs turn it down but agree to look at future work; author gets a modest deal for next three books; agent coaches author through several years of ramen noodles; author writes bestseller and finally "makes it;" several years go by and author hits creative wall, uses new novel for firewood. . . Each variation of this scenario calls for the same advice: dream of overnight success but don't stake your career on it! If you're serious about making a life and career out of your writing, be ready for the same ups and downs you'd see in any profession, and throw your hat in the ring for the long haul.
2) Make peace with the unknown. When I began working in publishing in 2010, there was no such thing as a Kindle Fire or a Nook Color (I thought I was darn cool with my spankin' new Kindle Keyboard). People in the know were saying things like, "we're about to see e-books really take off." (Snort!) Self-publishing and vanity publishing were still considered synonymous, and few "serious" authors would consider going indie except as a last resort. Looking back, the pace of change is mind-blowing. To be successful, authors, agents, and everyone else in the industry will have to stay flexible and embrace the changes; what we can't afford to do is hunker down and hope the tide will change and carry us back to a simpler time. I admit to serious nostalgia for the days of Borders, and I worry about our remaining bookstores. But it's also one of the most exciting times in the history of publishing—a time to explore new opportunities and look forward to the unforeseeable future.
3) Treat writing like a business, but not at the sake of craft. It's vital, in this age of limitless distractions, to stay focused on the most important part of your career—the writing. It seems obvious, but too often writers get so caught up in the pressure to market and be on every existing social media outlet, that their craft suffers. It's in every writer's best interest to stay current and build good business skills, but all the marketing savvy in the world is useless without a stand-out manuscript—or a finished one, for that matter. Similarly, a stand-out author platform will earn you points in a query letter, but only after you've won over the agent with your premise and impeccable writing. Don't let peripheral work crowd out your priority: getting your best work on the page.
4) A big advance doesn't (necessarily) mean take your day job and shove it. Over the past few years, I've had the privilege of working with or meeting a number of authors who made serious waves with their book deals (hats off to their amazing agents!). When that happens, it calls for a major celebration. But a good agent will also throw in a dash of realism—a big advance isn't an automatic ticket to super-stardom, and it means you'll have to work even harder, as the bar has been raised. If your book fizzles, that large sum of unearned royalties can be a major turn-off to your publisher when it comes time for your next book deal. For most authors, success will ebb and flow (see #1). There are too many stories of authors who left themselves with no cushion when their book sales fell short. I'll never tell anyone not to follow their dream—just be sure that when you do quit that pesky day job, you have a decent Plan B to ensure you don't have to go back.
5) Writer's block can strike at any point in your career—roll with it and don't panic. We've all heard about the dreaded "sophomore novel"—the one where you're writing for a real (no longer hypothetical) agent/editor/audience, and you've got that blessed advance to earn out. It's enough to give anyone stage fright! But what I found surprising was meeting highly successful authors with a string of bestsellers and accolades suffering the same problem. Whether it's from mounting pressure or creative fatigue, even the most prolific writers have to face this stumbling block at some point in their career. This is where you find out if your support team (agent, editor, critique group, writing coach, etc.) is worth its salt. Those who are will stick with you through the rough patches, show some tough love, and help get you writing smoothly again. And oh yes, you will!


text:
.....who LOVES all things unicorn at the moment!! ;D
Wednesday, 3 June 2015
In honour of my little miss....
Posted by Pocketful of Patterns at 8:30 am 0 comments
Labels: Emma Truman Designs, fairytale, fun, giftbag, greetings cards, juvenile, magical, pink, rainbow, unicorn
Tuesday, 2 June 2015
Progressive Greetings Live
Crikey - it's a windy ol' day for June, isn't it - let's hope the forecast is right and Summer finally takes hold after today!!!
If you're in or around the London area, then why not pop down to Progressive Greetings Live where a selection of my work is currently available and on display with Inspire Art Licensing, along with a collection of other fabulous designers and illustrators...it's our debut too, so fingers crossed it's a successful outing for us ;) Here's some birthday designs that I have been working on in the run up....
Posted by Pocketful of Patterns at 3:25 pm 0 comments
Monday, 1 June 2015
Wedding season....
June....a time for weddings.....here's a few little bespoke bits and pieces I was asked to create for a lovely couple earlier this year; I thoroughly enjoyed designing these and they were very well received - hope I get the opportunity to do more soon ;)
Posted by Pocketful of Patterns at 10:28 pm 0 comments
Labels: bespoke, cushion, Emma Truman Designs, handmade, Mr & Mrs, prints, stationery, tote bag, wedding
Subscribe to: Posts (Atom)


text:
GO Keyboard 0.7.0 Apk
GO Keyboard 0.7.0 Apk | 4.8 MB | Mediafire Download
Requires Android:1.6 and up
GO Keyboard is the best and the most popular keyboard on the android market! GO Keyboard can make your typing faster and smarter. It is a essential choice for android mobile.
v0.7.0 update:
1. (Optimized) Setting menu option.
2. (Optimized) Chinese and English dictionary, add lots of new words.
3. (Fix) The error for install themes.
5. (Fix) The error for search plug-in in google market.
6. (Fix) The error for recover the user dictionary.
●Main features:
★Predict in multiple languages and keyboard layouts
★Has accurate dictionaries
★Import contacts
★Import SMS To user dictionary
★Support fantasy text output
★Support Emoji
★Support a variety of themes
★Smart to set skin and back ground
★Auto-memory and correct as you type
★Multi-point touch keyboard
★Support the slide input
Bagikan :
Description: GO Keyboard 0.7.0 Apk Rating: 3.5 Reviewer: Admin ItemReviewed: GO Keyboard 0.7.0 Apk 11:51 PM
Sampaikan Komentar Anda disini :


text:
Thailand Birdwatching
Choose where you sleep
Despite the development that has taken place in many parts of Thailand over the past decade, there are still many spots of awesome undisturbed natural beauty. Kayaking is one great way to discover life away from the roads where nature is left to flourish.
This kayaking expedition takes you to two very different such places, both of them designated national parks, where the wildlife and natural environment remains pristine.
Khao Sok National Park is an area of rainforest with a vast lake created by a dam. Because it was already a protected area before the area was flooded, there is no human encroachment along the lakes shores, offering us an unparalleled opportunity to get close to the forest while remaining silently on the water. The wildlife appears to be unconcerned by our presence and thus the results are spectacular! Long-tailed macaques, pig-tail macaques, dusky langurs, banded langurs, up to five species of hornbill and even gibbons are seen on almost every trip. There are too many other bird species that you will see to mention here. Orchid lovers will find countless species clinging to the trees in the shallow waters.
Accommodation is in floating raft houses tethered close to the shore, and your bath is a dive in, climb out, soap down and dive in affair - truly back to nature.
Despite its remoteness, the raft's chef produce excellent Thai food and there is always a supply of cold beer!
This component can be taken on its own if you do not have enough time for the whole trip. See Khao Sok Bird & Wildlife Adventure.
Part 2 of the expedition takes us out to sea to the Tarutao National Marine Park. A different kind of nature experience here with numerous uninhabited islands fringed with sparkling white sand beaches set in turquoise waters.
Tarutao is a marine national park is on the border between Thailand and Malaysia. In fact, Langkawi, one of Malaysia's most popular beach destinations, is just five miles from Tarutao.
Designated by UNESCO as an ASEAN Heritage site, Tarutao consists of 51 islands, including seven large ones. The main island, Koh Tarutao, features tidal rivers, old-growth mangrove forests, old-growth hardwood forests, primary nipa palm forests (this palm's fronds are customarily used for roof thatching), limestone karst islands and lots of wildlife. Crab-eating Macaques (monkeys) are common on Tarutao. We often see them walking the mudflats at low tide searching for crabs or whatever else they can eat. Dusky langurs, cute black monkeys with white rings around their eyes, are common too. We frequently see monitor lizards. They get as big as 60 pounds and as long as six or seven feet! We do see a huge one occasionally, but the smaller one are much easier to see.
There is a park headquarters with bungalows where we land. The long beach in front of the park adds much beauty to an already stunning scene. A tidal river next to the park is where we do our first bit of exploring. Actually, you could spend several days exploring just this one area. We paddle in a mangrove forest beside steep limestone cliffs. There are lots of birds in the mangrove. Brown-winged Kingfishers, bright orange birds with brown wings and a blue back, are often seen. Oriental Pied hornbills and Southern Pied hornbills regularly cross over the mangrove channels or soar along the side of the massive limestone mountains. Greater Ratchet-tailed Drongos, medium-sized black birds with two very long tail feathers, flutter between trees at the park headquarters in their constant search for insects.
Some of the islands outside the park have small fishing communities whose lifestyles have scarcely changed in centuries, offering us a cultural encounter that is becoming increasingly hard to find.
Scuba divers might consider extending their stay in Thailand to take a 4 day liveaboard dive trip to the world class dive sites of the Similan Islands National Park, further north - famed for whaleshark and manta ray sightings.


text:
S: lonnie and starr 2-18-2006
BC: i love you more today than yesterday... ...but not as much as tomorrow! Lonnie, you are "My Hero" | february 2011
FC: i love you more today than yesterday... ...but not as much as tomorrow
1: Lonnie, This book is a small representation of our life together and i look forward to filling many more! You are my hero... ...my best friend! Your wife, Starr | Saturday, February 18, 2006... | lonnie and starr
2: you are my best friend
9: F a m i l y
10: lsu | let the games begin
11: go tigers
12: Go Saints!
13: kern's the name football's the game | who dat
15: Nights at Zepher Staduim | zepher stadium
16: family fun
18: Happy
19: merry | Holidays | christmas
20: friends
22: Love and Happiness!
24: I Love Us!
25: Together... F o r e v e r


text:
I never got the memo about Pine Brothers before I did this review and had the chance to sample their products. They have been around for awhile now, since 1870. I'm really not sure how this company escaped me, since I pride myself on being rather up on Americana and all. I'd say that a company that has been around that long needs to be a part of Americana! But apparently moms from all over know that Pine Bros. has them covered when it comes to their family's sore throats and coughs.
When I got the Throat Drops, I knew this was something for my class of boys to try out. I call them boys, even though they would never want me to say it in front of them or others. They are too independent (or at least trying to show that they are) to let a woman treat them like boys, because they are men, don't ya know. The first one that tried them was my associate, Shane, who was feeling under the weather that day. I told him, like I was his mom, to try one of these Softish Throat Drops. It happened to be the Honey Flavor. Unfortunately, he didn't enjoy the taste. But he did say that his throat started feeling better later on. I'm not sure if it was the drops or just the illness running its course. Next, I gave the Wild Cherry flavor drops to some of the boys in the class. They were a positive hit with everyone, and they actually started begging me (like little boys) for them. One boy said that it helped him deal with his smoking urges. (Sorry to say he is already a user and smoker at 16) Well, that was an unexpected benefit of the throat drops, a smoking inhibitor. I also prefer the Wild Cherry over the Honey flavor, which tastes a bit too medicinal for me.
S. Grant was President, the population of America hovered at 39
million, and the telephone was still a pipe dream, when in 1870 J.
Herman Pine hung out a confectionery shingle on 8th Street above Walnut
in Philadelphia and, along with his brother, sold their first batch of
what they called 'Pine Brothers Glycerine Tablets.' In an attempt to
relieve his own sore throat and those of his family members, Mr. Pine, a
former German confectionery maker, determined that by combining the
soothing natural qualities of glycerin with the healing properties of
gum acacia, he could sooth and mitigate the mild discomfort of a sore
throat. Add in some natural flavoring and a sweetener and voila, Pine Brothers Softish Throat Drops were born.
Another little discovery that I made is that I found the drops in the bags to be a little softer than the drops in the cannisters for some reason, although it is very convenient to have a small container to put in your purse without worrying about spilling.
Pine Brothers Softish Throat Drops come in 4 flavors - natural honey, lemon citrus, licorice, and wild cherry!
Disclosure: I was given product in exchange for my honest review and opinion!


text:
After struggling with his 3-point shooting for much of the NBA's Las Vegas Summer League, former Maryland forward Jake Layman hit five of eight 3-pointers in Monday night's championship game in the Portland Trail Blazers' 110-98 loss to the Los Angeles Lakers, who played without Lonzo Ball, the summer's league Most Valuable Player.
Layman finished with 21 points, shooting 8-for-19 overall, to go along with seven rebounds in 29 minutes. Layman, who played sparingly for the Trail Blazers as a rookie last season after being acquired in a draft-night trade with the Orlando Magic (who picked him at No. 47), averaged 15 points in the summer league.


text:
Hankyu Tourist Pass (1-Day or 2-Day) for Osaka, Kyoto, KobeVoyagin VerifiedAdd to Wishlist
ShareID: 9547
Best Seller
Use a Hankyu Tourist Pass for unlimited rail travel on Hankyu lines between Osaka, Kobe, and Kyoto. Enjoy convenient airport pick-up and the choice of 1-day or 2-day validity, for easier sightseeing in western Japan.
- Choice of collection points: Kansai Airport, or OCAT Building in central Osaka
- Choose a 1-day or 2-day pass for Hankyu Railway lines
- Valid on all Hankyu Railway lines except Kobe Kosoku Line
- Unlimited Hankyu line travel between Osaka, Kyoto, and Kobe
- Freedom to get on or off at any station to explore at your leisure