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Far Cry 3 Minecraft mod now available for download
The Far Cry 3 mode for Minecraft is now available. The free downloadable mod allows players to explore "the warped tones of Far Cry 3" in the sandbox title. It contains the locations and characters, including Vaas, Jason and Citra, in it as Minecraft mobs. The map also features over 50 Easter Eggs, hidden through the Islands. It was created by Michael Lambert – Sacr3, creator of Newcraft City, and artists Axel Janssen and Yohann Delcourt. Download it here.
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Check our our July Monthly Circular, featuring a FREE WEBER GRILL with qualifying deck purchase!
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Combining lovely elements like an empire waist, half sheer sleeves, cotton lining, upper nylon tulle and beautiful embroidery, this stunning tea length Nataya 40240 Art Deco Gatsby Dress in Sage will make you look and feel special and glamorous regardless of the event you're attending.
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About this Business
- HoursSunClosedMon1:30 PM - 9:30 PMTue-Thu11:00 AM - 9:30 PMFri11:00 AM - 8:00 PMSat8:30 AM - 1:30 PM
From Our Editors
No matter the class, Twisters Gymnastics Center's instructors always make sure to pack their lessons with a hearty dose of encouragement. They inspire students of all ages and skill levels in a 6,200-square-foot gymnasium outfitted with an array of tumbling and gymnastics equipment, from balance beams and pommel horses to colorful tumbling obstacles and a foam pit. Their introductory Mommy & Me and Mini-Gym classes help kiddies build strength and develop muscle control, and their more advanced sessions, including competitive gymnastics, nurture students' desires to test their skills against others. Each child's safety is equally important as their physical development; every instructor is certified in basic first aid and CPR, is USGF-safety certified, and is proficient in scaring away grizzly bears with nothing but a glare.


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During the 76th Sturgis Motorcycle Rally, four members of the Watertown Fire Rescue will spend time in the Black Hills as part of a special task force made up of officials from all over the state.
At first glance, it looks like nothing more than an ordinary household blanket, or perhaps a rug, made of ordinary material.
At issue: Open government means giving citizens access to legal notices. It's been shown that newspapers and their websites are the best way to accomplish that.
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Caltrain Slams Into Unoccupied Car In BurlingameAn unoccupied car was hit by a commuter train in Burlingame on Thursday morning, disrupting Caltrain service in the area, Caltrain officials said.
Pokémon Go Player Caught On Camera Crashing Into Police CarA Maryland man said he was playing Pokémon Go behind the wheel when he crashed his vehicle into a police car, the latest incident involving distracted driving and the gaming phenomenon.
Hit-And-Run Driver Slams Into Walnut Creek Police Car, Injuring OfficerPolice said the officer was in a patrol vehicle at Parkside Drive and Hillside Avenue when another vehicle hit the patrol car.
8 Out Of 10 Drivers Admit To Road Rage In AAA StudyThe AAA Foundation for Traffic Safety says nearly 8 out of 10 U.S. drivers it surveyed admit expressing anger, aggression or road rage at least once in the previous year.
CHP Motorcycle Officer Hurt In Wine Country CrashAll lanes of Interstate Highway 80 have reopened after a California Highway Patrol officer was injured in a crash while responding to a pursuit this afternoon in unincorporated Solano County near American Canyon, CHP officials said.
Child In Stroller Dies After Getting Hit By Truck Near SonomaA collision between a vehicle and a child killed the child Sunday evening in unincorporated Sonoma County near Sonoma, California Highway Patrol officials said.
Transient Killed In San Jose Bicycle AccidentA transient bicyclist died in a collision with a vehicle early Saturday morning in San Jose, police and the Santa Clara County medical examiner said.
Rafting Accident On American River Claims Life Of San Francisco WomanA San Francisco woman died Saturday during a rafting accident in the American River in unincorporated El Dorado County, sheriff's officials said.
Driver Crashes Into Pole In Fremont, Accused Of DUIA man was arrested for allegedly driving under the influence of alcohol early Wednesday morning in Fremont after his car crashed into a pole, according to police.
8 Hurt In Bus Crash On I-80 In PinoleAt least eight people have been transported to a hospital after a bus crash on Interstate Highway 80 in Pinole this afternoon, according to the CHP.
1 Killed, 2 Hurt In Separate Motorcycle Crashes Near GilroyOne person died and two people were injured in separate motorcycle collisions this afternoon in unincorporated Santa Clara County west of Gilroy, according to the California Highway Patrol.
Man Arrested For DUI After Crashing Car Into Tree, Injuring Passenger In PetalumaA man was arrested early Saturday morning in Petaluma after allegedly driving under the influence of alcohol, hitting a tree and injuring a passenger, according to police.


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Westerville, OHUSA
Hometown: Columbus and Paris, France
January 10, 2008
I dividemy timebetween Columbus, and my home near Lizy sur Ourcq, France (in the Champagne region). I'm always looking to improve my French (it needs a lot of improvement!) and stay in touch with French culture.
Recevoir et le groupe d'entrain! Je me suis amusé!
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I am sorry that you experienced what you did. I am sorry, too, for feeling rushed by your husband to heal. It seems that this is a normal reaction by those who are close to us. They fluctuate between believing and wanting to fix things for us...and then disbelief and all its accompanying emotions...it stinks and can be quite frustrating and confusing.
With you as you process this through...you are not alone...
i had few drinks prior to going over, I wasnt drunk when I went over. I dont know if I was drugged, Im not even sure if he got in me (GROSSES THOUGHT EVER) my husband came in see guy walk out in underwear with hard on and I passed right out naked. I was stupid for not going when I realized what may have happened to get screened. I pray my husband got there in time. I understand why he told ppl plus git ppl cause he was about to kill him.Im glad hes here and trying but it gets hard when he flips to disbelieve and thinks I just took my clothes off myself. I blame myself for going over, I blame myself for my love of beer, i blame myself for not going home earlier when my husband text me. i also blame that guy for not taking the offer of other neighbour to take me home. I dont know what to believe my husband was told i was clothed passed out prior to finding me naked. that y
i dont understand y my husband thinks I could of undressed myself.
Not to excuse anyone's behavior but maybe he cannot yet wrap his mind around it all and escaping to not believing you gives him a time out. I don't know you our your husband but I do know that often times when a wife or girlfriend is assualted, the husband or boyfriend feels a sense of responsibility and therefore failure to protect you. Even if there was nothing realistic they could have done. Chances are he could benefit from talking to someone also. Either with you or on his own. He was not asualted but he must still feel tons of emotions he is probably not sitting well with. You need to do what you need to do in your time. Please dont second guess yourself. You handle what you can when you can. Hopefully he will come to understand that.
Please take gentle care of yourself.
Recent Entries
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Recent Comments
need info pleaseSusanna - Jan 09 2014 01:12 PM
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Starring: Nacho Vidal, Britney, Debbie White, Nesty, Boroka Balls, Christy, Baby Silver, Jennyfer, Lili Cross, Keiran Lee, Anastasia Dreams, Katala, Madison P.
Studio: Rocco Siffredi
Director: Rocco Siffredi
Rocco's Lost Movie is a kinky collection of five unreleased scenes ranging from intimate settings to the crazy group scenes that the Italian Stallion is famous for instigating. Rocco himself shoots three semi-POV scenes, each featuring the theme of an 50-something female "teacher" getting in on the three-way action as she turns out a young "student" for the Master. There's also an Eastern European schoolgirl vs. eurostuds orgy, and a bizarre visit to an underground sex clinic that has a straitjacketed patient bursting his bonds to have his way with the nurse and two female in-patients. It's another wild ride from the world's most famous porn stud!


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A pretty pastel shade punctuated by an exposed zipper in back refreshes a dress tailored from fine stretch cotton. An inverted front pleat accentuates the ladylike fit-and-flare silhouette.
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Altar Of Oblivion
Last Known Status: Active
Latest Altar Of Oblivion News
Below is our complete Altar Of Oblivion news coverage, including columns and articles pertaining to the band. Some articles listed may be indirectly related, such as side projects of the band members, etc.
Altar Of Oblivion Streams "State Of Decay"
Altar Of Oblivion is streaming a new single titled "State Of Decay" and taken off the upcoming "Barren Grounds" EP. Shadow Kingdom Records comments:
"It's been eternity until the masters of Denmark doom metal have come out of their cave to unleash some new doom upon our souls! It was certainly well worth the wait! This is remarkable! A much more in-depth description will be released soon, but for now, let the music speak for itself! This is going to be another MASTERPIECE!"
The "Barren Grounds" EP is coming August 30th, 2016 and will feature the following track listing:
1. State of Decay
2. Serenity
3. Barren Grounds
4. Lost More...
Altar Of Oblivion Streaming Song
Shadow Kingdom Records has announced that Altar Of Oblivion's "Grand Gesture of Defiance" will be released on September 11th. Altar Of Oblivion was formed in 2005 and plays epic doom metal. The band thrives on tragedies of war and captures the spirit and atmosphere of this genre in a compelling and credible manner. The tracklisting of the six-song release follows, along with a stream of the track "The Graveyard of Broken Dreams."
1. Where Darkness Is Light
2. The Graveyard Of Broken Dreams
3. In The Shadow Of The Gallows
4. The Smoke-Filled Room
5. Sentenced In Absentia
6. Final Perfection


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The Man of My Dreams: A Novel Paperback – Apr 10 2007
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From Publishers Weekly
Sittenfeld's poignant if generic follow-up to her bestselling debut, Prep, similarly tracks a young woman's coming-of-age, but rather than navigating an elite school's nasty and brutish social system, this time the narrator contends with a dysfunctional family and her own yearnings for love. Fourteen-year-old Hannah Gavener is abruptly shipped off from Philadelphia to live with her aunt in Pittsburgh when her mercurial, vindictive father breaks up his marriage and family, which includes Hannah's older sister, Allison, and their browbeaten mother. Sweet but insecure and passive, Hannah had "been raised... not to be accommodated but to accommodate," an upbringing that hobbles all her subsequent relationships. The novel follows Hannah through her teens and late 20s (from 1991 to 2005), as she searches for romantic fulfillment, navigates friendships (e.g., with her larger-than-life cousin Fig) and alternately tries to reconcile with her father and distance herself from him. But the most influential connection Hannah makes is with her psychiatrist, Dr. Lewin, whom she begins seeing her freshman year at Tufts. Although the novel aspires to be taken seriously and Hannah is a sympathetic protagonist, she remains a textbook case of a young woman who wants "a man who will deny her. A man of her own who isn't hers."
Copyright © Reed Business Information, a division of Reed Elsevier Inc. All rights reserved. --This text refers to the Hardcover edition.
From Booklist
Sittenfeld's second novel is as sharply written as her first, Prep(2005), which chronicled one girl's experiences at an elite private high school. The heroine of her follow-up novel is Hannah Gavener, who, at age 14, is grappling with her mother's decision to stand up to her controlling father. The storyline follows Hannah through college and afterwards as she tries to find the kind of romance she believes she's looking for. Her glamorous, beautiful cousin, Fig, never seems to have trouble finding guys, and it is one of Fig's on-again, off-again boyfriends, Henry, who captivates Hannah and becomes her ideal. She goes through two boyfriends--one who is smitten with her, and one who isn't able to stay faithful--before deciding to pursue Henry and find out if he really does hold the key to her happiness. As is often the case in life, things don't work out quite according to plan, and the result is a novel that rings completely true. The magic of this coming-of-age tale lies in how it captures a generation of young women's anxiety and confusion about finding love and direction in their lives. Kristine Huntley
Copyright © American Library Association. All rights reserved --This text refers to the Hardcover edition.
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Top Customer Reviews
Sittenfeld's novel is certainly all about unfounded hopes, and for her protagonist, Hannah Gavener, the man of her dreams is Henry, a former boyfriend of Hannah's beautiful cousin Fig. Sittenfeld astutely captures Hannah's long adoration of Henry as well as Henry's alternating layers of uncanny emotional awareness and casual dishonesty. But Henry could also be what Hannah has always most truly desired: "a man who will deny her; a man of her own who isn't hers."
The man of Fig's dreams is a man we never meet, we only see her planning to fly out to California to spend time with him, and in a much later chapter we see her reveal two things that startle Hannah: (1) that she no longer remembers this man; not his name, not his profession, not anything about him, and (2) after years of being the object of stunned worship from multitudes of men, Fig has fallen in love with a woman.
But this novel suffers a kind of death when it's reduced to a story line; the real news about Sittenfeld is that she is such an honest and usefully detached writer that it can be an extreme pleasure reading her dissections of sex, first sex, humiliation, resentment, wistful envy, rueful ire, and the formal surprises that come with forgiveness.Read more ›
Most Helpful Customer Reviews on Amazon.com (beta)
Ms. Sittenfeld is an excellent writer and I hope that in her next outing she tackles a different theme ("Dreams" was essentially a carbon copy of "Prep", thematically) and that she follows a more traditional, linear format just to see if she can pull it off.
Curtis Sittenfeld, unlike a lot of young writers with one successful novel on the shelves, didn't fail her audience with The Man of My Dreams. The story follows Hannah from childhood through adulthood, in and out of relationships as she struggles to find a way to be happy and comfortable and true.
The writing is wonderful, capturing the moods of a bad camping trip, of being trapped in a car with a bullying father, of the ambivalence a woman feels with a too-doting lover. Hannah rings very true to life; there is nothing exaggerated or false.
The writing and character developments may be great, but the book feels very much like a short-story collection, like a variation on Melissa Bank and her latest book The Wonder Spot. This isn't a failure, but it's a surprise. A reader could rightfully expect something more original from Sittenfeld, something more profound.
Being pleasantly entertained is wonderful, but one hopes that Sittenfeld reaches a little out of her comfort zone for her next book.
It would be great if she could channel the writing into something that is more than the sum of its parts.
Ironically, this second novel is perhaps a more authentic snapshot of the murkier, less rarified adolescence many experience; I recall being acutely conscious that the voices of Prep's youthful characters were imbued with a suspiciously advanced wealth of insight and mastery of language. Nonetheless, it is the crystallization of such insight that makes most memoir-style/coming of age stories worth reading, not merely the excrutiating--and often colorless--'real-time' detail of the journey.
I have the sense that Ms. Sittenfeld, no doubt justifiably energized and enthused by her debut success, has rushed into an 'afterthought' sophomore effort, retreading what feels like unused surplus material from earlier ideas. I hope that she will take a good long trashy summer vacation...an autumn sabbatical...whatever further hiatus may be necessary for her to refresh and further refine her obvious gifts into richer, more fully-realized work.
Hannah Gavener has suffered from low self-esteem plagued by insecurity and mistrust for men since the age of 14, when her parents marriage falls apart. Over the next decade or so you watch as she grows up, explores the different types of relationships out there - casual, unrequited, platonic, open, and meaningful - and how she functions within them. Along the way you learn about Hannah's distaste for society's false social cues, and her secret idealism for others. The book is supported by several interwoven storylines from sub-characters including her gorgeous yet wild cousin Fig, her kind-hearted yet irritating sister Allison, and other various family members including her parents as they rebuild their lives without one another.
Ultimately I can't review this book without touching on Prep - since that is where most people will have heard of Sittenfeld and its how most people will come across this book.
The two are quite similar in that they are divided into 9-10 'chapters' which are really interweaving short stories set at different points in the main character's life. They both feature a narrator (although Lee is in first person, Hannah is almost entirely written in third person) who suffers from insecurity and self-doubt, particularly when it comes to the realms of socialibility and relationships. They both feature daughters straining to find autonomy from their slightly estranged parents.
They're different though, based solely on the age gap. Lee is in high school, and as such all of the events in Prep take place at one school, over four years, with many of the same characters reoccuring and building on the story. Hannah's story takes place in four different locations over a much larger span of time - representing the varied experiences of adulthood quite fully...but also disparingly. Much like in TV shows that fail to ignite fans during the high school to college transition, the Man of my Dreams suffers slightly since the storyline is not so tightly knit with an established cast of characters.
The other main difference is apparent between the attitudes of Lee and Hannah. While both are somewhat 'outsiders', Lee is relatable again and again to virtually every teenager's feelings of insecurity - past or present. Hannah on the other hand, is often so far gone off the unsociable deep end that you want to give her a firm shake and tell her to just suck it up and be friendly!
That being said - Man of My Dreams is still a great book as it straddles quite eloquently the realms of fiction, 'biography', literature, and to a small degree, chick lit. Curtis Sittenfeld is not your average writer and perhaps my favourite thing about her books is not the characterization or the plots but the acute attention to detail. The observations her characters are so mundane and ordinary, or at other times extremely insightful, you can't help but appreciate their cleverness. I look forward to reading more from this author!


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"We know that Andrew is a competitor and deserves to be playing somewhere, and frankly it just didn't appear he would get into many games for us down the stretch. Minnesota was looking to add a player like Andrew to their roster so the deal made sense for both the clubs and the individual."
Related quotes
"I am an exhibit. Joe is exhibit A. There's a game somewhere. Penn State and Florida State are playing. We're just an exhibit."
"A lot of times, the guy that's out there pitching, you probably played with him somewhere already, ... Nowadays, it seems like everybody knows everybody."
"I'd love to come back, but I think they're pretty well set. I'll be playing somewhere. The league is going to have to drag me off the floor."
"Usually if you win a game, two phases have played very well and, somewhere along the line, one of the phases is not able to, ... On a day like today when all three played extremely well - I don't want to say it's rare; I'd like to see it happen more often - but it's difficult to do."


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I hate what just happened but if you step back and try not to be emotional, with the RS and financing behind us, there should be a steady stream of good news ahead including American Diabetes meeting later this month, start and completion of EU trial, finalization of US PMA protocol and start of that trial, possible partner .... Hold your nose and get on board.
I agree, so I picked up some more yesterday.
Sentiment: Strong Buy
Scoop me off the floor, please, someone. An actual voice of reason shows up on this board, and can spell and use grammar properly all at the same time!
hack, you have nothing to add to any post except critique of the other posters. You're like my grandmother, who was an english professor. I doubt you even own shares in this company. Why not just go the hell away?
Sentiment: Hold
I'm tempted to get my feet wet again. But with a huge cash burn of 5.1M per Q, They barely raised 2 Qs of cash, yet diluted 60% to raise it. They may need to raise cash again even before the EU trial results are in, to insure cash doesn't run out like it almost did this Q. I'm not sure where the SP goes from here. Temporarily, once the financing deal closes, we'll probably get a pop, but investors may be back in this situation again 4- 5 months out.
I agree. Some on this board thing a company with no way of generating revenue does not need money for trials. They haven't done the best job, but some very near gains are on the horizon. This secondary is being bought up quickly. A couple more positive updates and this will be back to the R/S level in no time and ready to start pushing up to the double digits.
Which I know is still way low considering they just did a 10/1 split, but it's going to get there with very little.
bigcire, thanks for the comments. Just one point of clarification - the secondary is done and will probably close next week. All of the shares being traded today are separate from the offering and just open market purchases by people like us that think the stock will be moving up after the huge downdraft this week. Based on the high volume there could be some activity from the secondary purchasers who didn't get their full allocations and are filling out their positions at these depressed prices. Unfortunately, I think a lot of the shorts were in there today as well covering their positions way to easily as the emotional sellers handed them their gains.
I agree that their offering is moving quickly; over 2,000,000 in volume. That is a big day even pre R/S. I also agree that they have not done a good job. In fact a shi$$y job of managing money since the $0.95 offering and losing the Platinum financing. My vote is to get someone else in charge of the money. Keep PM but get him the hell away from the money.


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Welcome to the Home of The Basset Hound Club of America
AVMA Animal Health Studies Database
- Last Updated on Thursday, 14 July 2016 22:11
- Published on Thursday, 14 July 2016 22:09
- Written by Administration
The AVMA Animal Health Studies Database is the first, and only, database that includes listings of veterinary clinical studies being conducted in all species of animals and most disciplines of veterinary medicine.
Each dog breed has a likelihood for higher incidences of particular medical conditions, and Basset Hounds are no exception. The database lists many studies that are looking for specific breeds, and it is fully expected that there will be many more studies to come that, in many cases, just want a sample of blood or tissue with which to conduct genetic analyses.
Take a few minutes to check out the site at http://www.avma.org/findvetstudies, and consider participating in a study to improve the health of our breed.
AKC Outstanding Sportsmanship Award
- Last Updated on Thursday, 14 July 2016 21:55
- Published on Thursday, 14 July 2016 21:54
- Written by Administration
Attention Members! Each year BHCA presents a member with the AKC Outstanding Sportsmanship Award. Any individual member or club in good standing can nominate another member for the award.
Judges election for 2017 Nationals
- Last Updated on Tuesday, 19 July 2016 22:14
- Published on Friday, 10 June 2016 09:26
- Written by Administration
The following BHCA member judges have been nominated by a vote of the membership to appear on the ballot to choose the judges for the 2017 BHCA Nationals:
Regular and Non-regular classes:
Peter Martin
Brian Pechtold
Carol O'Bryant
Anne O'Reilly
Joani Rush
Jim Tysseling
Margie Wikerd
The following BHCA members have been nominated by a vote of the membership to appear on the ballot to choose the Sweepstakes judge for the 2017 BHCA Nationals:
Beverly Barringer-Ruggeri
Debbie Moore
Monica Rush
BHCA Elections 2016
- Last Updated on Friday, 22 July 2016 17:48
- Published on Monday, 23 May 2016 01:23
- Written by Administration
The following BHCA members will appear as candidates for election as officers and board members in 2016:
President: Bobbi Brandt
2nd VP: Brian Pechtold
Corresponding Secretary: Anne Testoni
Board members:
Marianne Lovejoy (West Region)
Jacqueline Nolan (West Region)
Sue Smyth (East Region)
2016 Nationals website
- Last Updated on Monday, 23 May 2016 01:31
- Published on Monday, 26 January 2015 14:35
- Written by Administration
It's not too early to prepare for the 2016 Nationals in St. Louis, and that means checking out this year's Nationals website.
The site contains hotel information for both the Holiday Inn Six Flags Eureka, the host hotel; and the Travelodge Six Flags/Gray Summit, the overflow hotel. A preliminary schedule of Nationals judging and other events is also available.
Information continues to be added to the site as we approach Nationals at the beginning of October.
Check out the site at http://www.2016bhcanationals.com/.


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A Day in the Life of the Network Engineer:. Advanced knowledge of network security, policies, and procedures. Cisco Certification in network technologies - CCNA...View job on Indeed.com


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1 of 2 people found the following review helpful
An Overdose Janis,
This review is from: Janis Joplin's Greatest Hits (Audio CD)
�Oh Lord, won`t you buy me a Mercedes Benz, my friends all drive Porsches, I must make amends," Janis Joplin sang so outragedly, if it would be seriousness and not satire (knowing very well, that, occasionally, some wishes are not fulfilled). �Worked hard all my lifetime, no help from my friends," - that does not matter anyway and applies to God (if there is one) not as an argument: Proof of it is clear to find. Janis Joplin has slaved away and lived, intensive as a lunatic -- this has not changed the mind of God to let her get 100 years old. Perhaps it was not God, however, who set forcefully a final stroke to this style of protesting impudently (�Oh Lord, won`t you buy me a colour TV, ...I wait for delivery, each day until three"), it seems, that Janis arrived at the dead end, because she once took pure, separate heroin (her main dealer was just on holiday). �Oh Lord, won't you buy me a night on the town, I'm counting on you Lord, please don't let me down, prove that you love me, and buy the next round..." There was not any next round, after she had taken this song ("Mercedes Benz") in the studio - a capella - the instruments should get mixed the next day. But in the night of October 4th 1970 Janis Joplin died. Eric Burdon commented: "Janis did not die of an overdose heroin. She died of an overdose Janis"
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Hutchinson-Gilford progeria syndrome (HGPS), a devastating premature aging disease, is caused by a point mutation in the lamin A gene (LMNA). This mutation constitutively activates a cryptic splice donor site, resulting in a mutant lamin A protein known as progerin. Recent studies have demonstrated that progerin is also produced at low levels in normal human cells and tissues. However, the cause-and-effect relationship between normal aging and progerin production in normal individuals has not yet been determined. In this study, we have shown in normal human fibroblasts that progressive telomere damage during cellular senescence plays a causative role in activating progerin production. Progressive telomere damage was also found to lead to extensive changes in alternative splicing in multiple other genes. Interestingly, elevated progerin production was not seen during cellular senescence that does not entail telomere shortening. Taken together, our results suggest a synergistic relationship between telomere dysfunction and progerin production during the induction of cell senescence, providing mechanistic insight into how progerin may participate in the normal aging process.
Cellular senescence, the finite division capacity of normal somatic cells in vitro, has long been used as a cellular model for understanding mechanisms underlying normal aging (1, 2). A popular hypothesis of cellular senescence is that progressive attrition of telomeric DNA results in loss of telomere capping proteins, exposing DNA breaks that activate cell cycle arrest and senescence (3). Telomeres are the specialized structures at the chromosome ends, made up of many kilobases of a simple DNA repeat (TTAGGG)n bound by a multiprotein complex known as shelterin (4). Telomeres cannot be copied to their extreme termini by DNA polymerase, and so undergo progressive shortening unless elongated by a ribonucleoprotein named telomerase (5, 6). Telomerase is composed of a reverse transcriptase (TERT) and an RNA component (TERC) that serves as a template for telomere elongation (6, 7). In somatic cells lacking telomerase, gradual telomere loss and ultimate senescence are inevitable (8). Consistent with this model, there is a strong association between cell immortalization and persistent telomerase expression (9). Besides telomere shortening, cellular senescence can be triggered by a variety of environmental and intracellular stimuli, including γ-irradiation, oxidative stress, and overexpression of certain oncogenes, such as H-rasV12 (10–12). Moreover, mutations in certain genes, such as lamin A (LMNA; accession no. NM_170707), have been linked to premature senescence (13, 14).
LMNA encodes 2 intermediate filament proteins, lamin A and lamin C. Together with lamin B, they form a dynamic meshwork located just inside the nuclear inner membrane, named the nuclear lamina. The lamina provides important mechanical support to the nuclear structure and also influences chromatin organization, gene expression, and DNA replication (15–17). To date, at least 13 human diseases (referred to as the laminopathies) have been associated with the mutations in lamin genes (13). Among them, Hutchinson-Gilford progeria syndrome (HGPS) has received the most attention because of its striking premature aging phenotype, including alopecia, diminished subcutaneous fat, premature atherosclerosis, and skeletal abnormalities. Children with HGPS die at an average age of 12 years, usually from heart attack or stroke (18). The vast majority of HGPS cases are associated with a de novo nucleotide substitution at position 1824 (C→T) in the LMNA gene (14, 19). This mutation does not affect the coded amino acid (and is thus generally referred to as G608G), but partially activates a cryptic splice donor site in exon 11 of LMNA, leading to the production of a prelamin A mRNA that contains an internal deletion of 150 base pairs (14). This transcript is then translated into a protein known as progerin, which lacks 50 amino acids near the C terminus (14). Indeed, progerin has been found to accumulate in multiple tissues in biopsies from HGPS patients, including skin, tongue, breast, heart, liver, kidney, stomach, bladder, diaphragm, pancreas, spleen, thyroid, adipose tissue, joint cartilage, bone, skeletal muscle, heart, and large and small arteries (20).
The cellular phenotypes in HGPS include blebbing (i.e., abnormal shape) of nuclei, thickening of the nuclear lamina, loss of peripheral heterochromatin, clustering of nuclear pores, and premature senescence (21). Interestingly, prior to developing these obvious nuclear morphological changes, fibroblasts from HGPS patients exhibit broad abnormalities in histone modification patterns (22). HGPS fibroblasts also show global changes in gene expression (23, 24) and a delayed response in DNA-damage repair (25).
Gene transfer experiments have left no doubt that progerin acts as a dominant negative. Progerin expression induces multiple defects during mitosis: cytokinesis delay, abnormal chromosome segregation, and binucleation (26, 27). Several Lmna mutant mouse models have been created. A transgenic model carrying the G608G mutated human LMNA in a bacterial artificial chromosome (BAC) shows progressive loss of vascular smooth muscle cells in the medial layer of large arteries, closely resembling the most lethal aspect of the human phenotype (28).
The molecular mechanism of progerin toxicity is at least partially understood. Normal lamin A is farnesylated at its C terminus, and that posttranslational modification is thought to play a role in targeting lamin A to the inner nuclear membrane. But subsequently, the ZMPSTE24 endoprotease cleaves off the last 18 amino acids at the C terminus of lamin A, including the farnesyl tail. This releases lamin A from its membrane anchor and allows it to take its place in the nuclear scaffold (29). The 50–amino acid internal deletion in progerin includes the ZMPSTE24 cleavage site, and thus progerin remains permanently farnesylated (27). Recently, treatment with farnesyl transferase inhibitors (FTIs) has been shown to improve progerin-induced cellular phenotypes in vitro and in several HGPS mouse models (13, 27, 30–34). A clinical trial with an FTI in children with HGPS was initiated in May 2007.
It has always been of great interest to determine the biological relevance of HGPS to normal aging. This interest was heightened by the detection of progerin mRNA and progerin protein in cells obtained from healthy individuals, which indicates that the cryptic splice site activated by the HGPS mutation is also capable of being used in the presence of the normal sequence of exon 11 (35). Recently, progerin transcripts and progerin were identified in vivo in skin biopsies from healthy individuals, and a recent analysis suggested that progerin transcript levels increase in late-passage cells from HGPS patients and parental controls (36, 37).
Using a reporter construct, Scaffidi et al. showed that the normal LMNA sequence at G608G could also be used as a weak splice donor to produce progerin in normal individuals (35). We have previously shown that a significant percentage of progerin-positive cells from normal individuals are morphologically abnormal, exhibiting phenotypes that resemble HGPS cells (26). This suggests that activation of progerin expression in normal cells, triggered by some unknown signal, may contribute to senescence.
However, the cause-and-effect relationship between normal aging and progerin production in normal individuals has not been determined. In the present study, we assayed the effects of cellular passage and donor age on the activation of progerin production and found that the cryptic splice donor site that produces progerin is activated in senescent cells. Screening various primary and transformed cell lines revealed an interesting inverse correlation between cell immortalization and progerin transcription, and ectopic expression of telomerase in normal fibroblast cells resulted in a significant decrease in progerin production. In addition, we found that progerin production was not induced in telomere-independent, oncogene-driven senescence, further supporting a potential causal relationship between telomere-induced senescence and progerin production. Consistent with this model, we found that elevated levels of progerin were induced in fibroblast cells whose telomeres had been uncapped. With splicing-sensitive exon microarrays, we further showed that extensive changes in alternative splicing of multiple genes, including LMNA, occurred as telomeres shortened and cells approached senescence. Taken together, our findings are suggestive of synergism between telomere damage and progerin production in induction of cellular aging and provide what we believe to be the first genome-wide analysis of the changes in alternative splicing during cell senescence.
To elucidate the cause-and-effect relationship between aging and progerin production, we designed a splicing reporter assay that permits observation of the immediate effects of cell passage or donor age on the activation of the progerin cryptic splice donor site. The cryptic splicing reporter originally described by Scaffidi et al. (35) was PCR amplified and subcloned into a bicistronic vector (pIRES-DsRed-Expression 2) (Figure (Figure1A).1A). This 2-color progerin splicing reporter was then transiently transfected into normal fibroblast cells. In this assay, all transfected cells expressed red fluorescence protein, and cells using the cryptic splice site also expressed GFP (Supplemental Figure 1, A and B; supplemental material available online with this article; doi: 10.1172/JCI43578DS1). At 96 hours after transfection, we sorted those fibroblasts with fluorescence-activated cell sorting (FACS) analysis and quantified 2 subpopulations (Figure (Figure1B1B and Supplemental Figure 1A): the fraction positive for both GFP and DsRed, consisting of transfected cells that had activated the cryptic splice site in LMNA (referred to herein as double-positive), and the fraction positive for DsRed only, containing the transfected cells that did not use the cryptic splice (referred to herein as DsRed-only). To examine the effect of cellular passage and donor age, we performed the reporter assay in 6 independent primary fibroblast lines, derived from healthy donors ranging 10–92 years in age, at increasing cellular passages. Percent double-positive cells relative to total DsRed cells was used as an indicator of the progerin splicing activity and plotted against cell passage and donor age. In all tested cell lines, we consistently observed that double-positive cell percentage increased with cell passage (Figure (Figure1C1C and Supplemental Figure 2), which suggests that cellular senescence correlates with activation of progerin splicing. Furthermore, in agreement with previous reports (35, 36, 38, 39), we did not observe an obvious correlation of progerin production with donor age (Supplemental Figure 2).
To gain mechanistic insights into the activation of the progerin cryptic splice site in cellular aging, we next compared telomere length in the double-positive and DsRed-only subgroups after FACS analysis. The telomeres were labeled using FISH with a peptide nucleic acid probe against telomere repeat sequence (PNA-FISH). The telomere length of each cell was quantified as the total fluorescence intensity from PNA-FISH in each nucleus, which was defined by the DAPI staining of DNA (Figure (Figure2A).2A). We examined 2 normal fibroblast cell lines, AG06299 at passage 35 (p35) and HGFDFN168 at p18. Because both cell lines demonstrated similar results, we showed results only from HGFDFN168. Box plot analysis indicated that the telomeres in the double-positive subgroup were significantly shorter than those in the DsRed-only subgroup from the same cell line (P < 0.0001; Figure Figure2B).2B). Taken together, these results indicate that the progerin-producing double-positive cells define a subpopulation of cells with short telomeres, which suggests a potential connection between telomere erosion and progerin splicing activation.
To further test the connection between progressive telomere shortening and progerin production, we measured the amount of progerin mRNA in immortalized cancer cells with stable telomeres as a result of telomerase expression as well as in primary cells lacking telomerase expression. A RT-PCR assay with progerin- or lamin A–specific primers was designed to assess relative use of the endogenous lamin A cryptic splice site (Figure (Figure3A).3A). Paired with a forward primer at the exon 7–8 junction, these primers specifically amplified truncated progerin or lamin A products with the expected sizes. A high level of specificity was demonstrated using in vitro–purified cDNAs (Supplemental Figure 3). DNA sequencing verified the identities of these PCR products (data not shown). We tested a total of 16 cell lines, including 12 primary cell lines derived from 3 cell types (fibroblasts, aortic SMCs, and B lymphocytes) and 4 immortalized cell lines from various telomerase-positive cancers (Figure (Figure3B).3B). Consistent with previous reports (40), quantitative RT-PCR (qRT-PCR) analysis revealed substantial variation in LMNA expression in different cell types, with the lowest expression found in B lymphocytes (15-fold less than fibroblasts; data not shown). To directly compare the level of progerin mRNA among these different types of cells, we normalized the amount of progerin mRNA to the LMNA mRNA in each cell line. Interestingly, we found that progerin mRNA was essentially absent in immortalized cells with stable telomeres (Figure (Figure3B). 3B).
Next, we examined how changes in telomere length would influence progerin production. We studied 2 human TERT–immortalized normal fibroblast cell lines, hTERT-HGFDFN090 and hTERT-AG09838, and their parent cells. qRT-PCR analysis with human TERT primers confirmed the presence of TERT mRNA in the immortalized fibroblast cells, whereas no TERT mRNA was detected in their nonimmortalized counterparts because of its extremely low amount in differentiated primary fibroblasts (data not shown). In addition, quantitative telomere PNA-FISH analysis demonstrated that stable infection of human TERT resulted in elongated telomeres (Figure (Figure3C3C and Supplemental Figure 4). Consistent with our finding in immortalized cancer cells, we found that the ectopic expression of telomerase in normal fibroblasts led to significant downregulation in progerin production: progerin mRNA decreased 3- and 10-fold in hTERT-AG08398 and hTERT-HGFDFN090 fibroblasts, respectively, compared with their nonimmortalized passage-matched counterparts (Figure (Figure3D).3D). Therefore, we concluded that forced elongation of telomeres in primary fibroblast cells leads to suppression of progerin production. Together, our data demonstrated an inverse correlation between telomerase expression (cell immortalization) and progerin production and suggested that telomeres serve as the upstream signals in regulating progerin production.
To examine whether progerin splicing is activated in cellular senescence that does not entail telomere shortening, we analyzed progerin production in premature senescence induced by oncogenic ras (12). We chose 2 well-characterized normal fibroblast lines, AG08470 and HGFDFN168, and introduced an activated ras allele (H-rasV12) to those cells using the pBABE retrovirus, as previously described (12). The vector coexpresses a puromycin-resistant gene that allows selection for transduced cells in 4–5 days. After selection, those cells were grown in media without puromycin for an additional 6 days. Expression of H-rasV12 induced morphological changes in both normal fibroblast cell lines. A substantial portion of H-rasV12–transduced cells became flat and enlarged and exhibited increased SA–β-gal activity (Figure (Figure4A).4A). No changes in morphology were detected in cells transduced with vector control (Figure (Figure4A).4A). Consistent with previous studies (12), quantitative telomere PNA-FISH analysis demonstrated no obvious changes in telomere length in H-rasV12–induced cells compared with that in control vector–induced cells (Figure (Figure4B).4B). Importantly, although the morphological features of H-rasV12–induced cells closely resembled cells that have passed their proliferative capacity and become senescent, qRT-PCR analysis indicated no upregulation of progerin production in either cell line tested (Figure (Figure4C).4C). In addition, we found no significant upregulation of progerin mRNA production in the premature senescent cells that had been induced by treatment with 0.5 mM sodium butyrate, a histone deacetylase inhibitor (41), for 2 weeks (data not shown). Together, these data suggest that upregulation of progerin production is not a common feature for all types of senescence, but appears specific for telomere-driven senescence.
To further test the hypothesis that short telomeres activate progerin production in senescent cells, we used 2 fibroblast lines, JH-1 and JH-2, derived from patients with dyskeratosis congenita (DC) (42). These cells carry a heterozygous loss-of-function mutation (K902N) in exon 11 of TERT that results in haploinsufficiency of telomerase and accelerated telomere shortening (42). DNA sequencing verified the presence of K902N in both DC fibroblast lines (Supplemental Figure 5). Quantitative telomere PNA-FISH analysis confirmed accelerated telomere loss during cellular senescence in these DC cells compared with passage-matched normal fibroblasts (P < 0.0001; Figure Figure5A).5A). Using qRT-PCR on cells at p12–p15, we found a more than 2-fold increase in progerin production in these DC fibroblasts compared with normal passage-matched control cells (P = 0.013; Figure Figure5B).5B). Interestingly, both JH-1 and JH-2 cells exhibited a more than 2-fold increase in cell blebbing (Figure (Figure5,5, C and D; see Supplemental Figure 6 for passage-matched normal fibroblast control), reminiscent of the hallmark cellular phenotype in HGPS. In addition, we noted a significant increase in JH-1 and JH-2 cells containing micronuclei (small unincorporated chromosome fragments; Figure Figure5,5, C and D), potentially caused by prior defects in cell division, which has also been reported in HGPS cells (26, 27). Immunostaining of the JH-1 and JH-2 cells with an anti-progerin antibody revealed positive progerin staining inside micronuclei in those abnormal cells (Figure (Figure5E).5E). The specificity of the anti-progerin antibody was verified using immunostaining in HeLa cells expressing either GFP-progerin or GFP–lamin A (Supplemental Figure 7). Interestingly, we observed positive cytoplasmic signals of progerin in a small percentage of DC cells (Figure (Figure5E),5E), which contrasts with the predominant nuclear lamina localization of WT lamin A and lamin C. Our data in DC cells provide further support for a model in which short telomeres act as an upstream signal to activate progerin production in primary fibroblast cells.
To address further the effect of telomere-directed senescence on progerin production, we used TRF2 inhibition as a tool to induce immediate telomere dysfunction in normal cells. TRF2 is a major component of the telomere-capping complex, shelterin (4). When TRF2 is dislodged from telomeres by a dominant-negative allele (TRF2ΔBΔM), a fraction of chromosome ends lose their protection, leading to a growth arrest that is indistinguishable from replicative senescence (43). We infected normal human fibroblasts with retroviruses expressing TRF2ΔBΔM or WT TRF2. Cells were kept under puromycin-containing medium for 5 days before being used for analysis. qRT-PCR analysis with progerin- and lamin A–specific primer pairs indicated no significant changes of WT LMNA mRNA level, but more than 8-fold upregulation of progerin mRNA was observed in TRF2ΔBΔM-expressing cells compared with control cells (Figure (Figure6,6, A and B). Western blotting analysis with the anti-progerin antibody revealed a distinct band corresponding to progerin in cells infected with TRF2ΔBΔM, but not in control cells infected with WT TRF2 (Figure (Figure6C).6C). Similarly, immunofluorescence analysis revealed positive progerin staining in the TRF2ΔBΔM cells (Figure (Figure6D).6D). Interestingly, we found that progerin staining in TRF2ΔBΔM cells was quite diffuse in the nucleoplasm and was even seen in the cytoplasm in some cells. This differs with the predominant nuclear lamina localization of WT lamin A/C. We reasoned that the apparent lack of signal from the anti–lamin A/C antibody in the progerin-positive cytoplasm might be just a sensitivity issue, since there is such a large amount of lamin A/C in the nucleus. Indeed, simply by increasing the gain of the signal for lamin A/C, we observed colocalization of signals from anti-progerin and anti–lamin A/C antibodies (Supplemental Figure 8). In summary, these data support the conclusion that uncapped dysfunctional telomeres activate the cryptic splice site in normal fibroblast cells to produce progerin.
Next, we asked whether telomere dysfunction induces a broader set of alternative splicing changes, beyond the effects on LMNA. To monitor global effects on alternative splicing, we used the human Exon 10ST array, a splicing-sensitive microarray with probes targeted to individual exons or exon junctions, although it is not designed to detect use of the progerin cryptic splice site. This design allowed us to detect RNA isoforms expressed while simultaneously profiling gene expression. The expression level of each exon on each array was detected independently and analyzed using XRAY microarray analysis software.
The profiles of exon usage were compared between the same fibroblast lines with either long or short telomeres (group A) and between passage-matched fibroblasts where no significant variations in telomere length were present (group B), as controls (Figure (Figure7A).7A). Group A included 2 comparisons between early- and late-passage normal fibroblast cells (groups A1 and A2) and 2 comparisons between passage-matched human TERT–immortalized and nonimmortalized cells (groups A3 and A4). Group B comparisons included 4 comparisons between passage-matched normal and HGPS fibroblasts. Using a false discovery rate (FDR) threshold of 0.01, we identified numerous changes in gene expression in each comparison, even between the passage-matched fibroblasts from 2 normal individuals (data not shown). Surprisingly, changes in alternative splicing were much more prominent in group A, in which significant telomere shortening had occurred (Figure (Figure7A7A and Supplemental Table 1). Between 10% and 50% of the genes showing changes in alternative splicing also exhibited differential regulation of gene expression (Figure (Figure7A),7A), which suggested distinct but also partially overlapping programs of transcriptional and posttranscriptional regulation. An overlap of 82 genes was observed in the 4 comparisons of group A (Figure (Figure7B7B and Supplemental Table 2), which implies that 1 or more common regulatory pathways might be activated in these cells by shortening of telomeres. Statistical analysis (see Methods) indicated that the overlap of genes showing altered expression and altered splicing within all 4 group A sets was not significant (P > 0.75); however, the overlap of 82 genes with altered splicing between the 4 sets was highly significant (P < 0.0001). We were unable to identify any obvious pattern or sequence motifs in the set of genes that showed alternative splicing in the presence of telomere shortening.
In an attempt to address the functional relevance of these differentially spliced genes induced by changes in telomere length, gene ontology (GO) enrichment analyses (see Methods) were carried out for these 82 common genes, using the complete set of genes covered by the exon microarray as a reference. Interestingly, among the top 10 most overrepresented categories, 5 were related to cytoskeleton organization and function, including the genes regulating intermediate filaments and microtubules, such as actin, fibronectin, vimentin, and tubulin (Figure (Figure7C).7C). These results indicate that cellular senescence induces extensive changes in alternative splicing in multiple genes and suggest a potential role of differential splicing in cytoskeleton reorganization during cellular aging.
Considerable progress has been made in understanding the connection between HGPS and normal aging. In 2006, Scaffidi et al. first showed that the normal LMNA sequence could produce progerin at a low level in normal individuals, potentially implicating progerin in normal physiological aging (35). With a progerin-specific antibody, we previously demonstrated that the progerin-expressing cells from normal individuals mimic some aspects of HGPS cells, further supporting the idea that the premature aging disease HGPS and normal aging may share a common cellular and molecular basis (26). Furthermore, a recent analysis has shown that levels of progerin mRNA transcript increase in late-passage cells in vitro (37), and progerin protein accumulates with age in vivo in normal individuals (36). However, the cause-and-effect relationship between progerin production and aging has not been clear.
In this study, we applied a splice reporter assay that permits observation of the immediate effects of cell passage or donor age on the activation of the progerin cryptic splice site in LMNA. In normal fibroblasts, we showed that this cryptic site was increasingly activated as cells reached later cellular passage numbers. Thus, cellular senescence correlated with enhanced progerin production. No obvious correlation was found, however, between progerin production and donor age. One potential explanation is that the cells capable of growing in culture from a skin biopsy are relatively early in their in vivo passage number, regardless of the subject's chronological age (39).
We further showed that the fibroblast cells that used the LMNA cryptic splice site had shorter telomeres and exhibited high senescence-associated β-gal (SA–β-gal) activity. The finding that most of the cells with activated progerin splicing were senescent suggests a possible role of the cryptic splice reporter for progerin as a biomarker for cellular senescence. Previously, McClintock et al. found that progerin-expressing normal cells in skin biopsies were restricted to dermal fibroblasts and terminally differentiated keratinocytes and suggested that the population of progerin-expressing fibroblasts in skin might characterize a possible terminal differentiation phenotype in vivo (36). While distinct, both cellular senescence and terminal differentiation share some common characteristics including cell-cycle arrest, flattened and enlarged cell morphology, and changes in chromatin organization and in gene expression (36). It is possible that the progerin splicing events in cultured cells in vitro partially recapitulate the process of progerin production in normal tissues in vivo, and it will be interesting to determine the telomere length in those progerin-expressing cells in skin tissues.
In support of the notion that progerin production is selectively induced in senescent cells with short dysfunctional telomeres, we showed that progerin mRNA was essentially absent in immortalized cells. Significantly, human TERT immortalization experiments in primary healthy fibroblast cells revealed that the forced elongation of telomeres suppressed progerin production, which suggests that telomere shortening lies upstream of activation of the cryptic splice site. This conclusion is consistent with the observation that progerin production was not altered in oncogene-induced cellular senescence, in which telomere shortening was not involved. In further support of this model, an elevated amount of progerin was observed in DC fibroblast cells carrying a null TERT mutation. Notably, cells expressing TRF2ΔBΔM, which induces uncapping of telomeres, showed substantial upregulation of progerin production. Taken together, these observations support the conclusion that shortened or uncapped telomeres act as an upstream signal to activate progerin production in primary fibroblast cells.
Is the reverse pathway also present, i.e., does expression of progerin affect telomere maintenance? The answer to this question has been controversial in the literature. A previous study reported that one-third of the independent isolated fibroblast clones from HGPS patients fail to immortalize despite the exogenous expression of telomerase, which suggests that the growth arrest in HGPS confers resistance to telomerization (44). Another report indicated that telomerase failed to protect progerin-induced DNA damage (45). However, Decker et al. reported shortened telomeres in HGPS fibroblasts, although their finding was limited to late-passage HGPS fibroblasts, where telomere attrition may well have been on the basis of cell passage number rather than being specific for HGPS (46). Most recently, a report by Benson et al. showed that exogenous expression of progerin in human diploid fibroblasts rapidly induces telomere aggregation, appearance of DNA-damage signals at telomeres, and chromosomal aberrations. Importantly, these effects were abolished by telomerase expression (47). Using quantitative telomere PNA-FISH in HGPS fibroblasts, we found no clear evidence of shortened telomeres compared with passage-matched normal fibroblasts (data not shown). Similarly, we found that the telomere length in vascular smooth muscle cells derived from a transgenic mouse model of HGPS was no different from that in nontransgenic control mice (data not shown). In support of the conclusion that progerin's effect on telomeres is independent of shortening of the telomeric DNA, Benson et al. (47) indicated that progerin induces rapid telomere dysfunction well before telomere attrition would be detectable. Therefore, we suggest that progerin induces an acute DNA damage response at telomeres — perhaps on the basis of well-established effects of progerin on disrupting heterochromatin structure (21) — that leads to deprotection of the telomeric 3′ overhang.
Cellular senescence reflects a complex process of gradual deterioration of the molecular components, checkpoints, and cellular structures. Inspired by the progerin results, we hypothesized that alternative splicing, a key pre-mRNA processing step, might be more broadly influenced by senescence. Using splicing-sensitive exon microarrays, we showed that not only LMNA splicing was affected by short telomeres, but a significant number of other genes were also affected. Interestingly, GO analysis further demonstrated that the most overrepresented categories in this set of differentially spliced genes were related to cytoskeleton function. This finding may connect with the longstanding observation that senescent cells demonstrate characteristic changes in cytoskeleton organization, including cell shapes, cell adhesion, and cell mobility. Previous studies reported that fibronectin (48), a cell adhesion protein, and vimentin (49, 50), an intermediate filament protein, are alternatively spliced in senescent cells. Both were identified in our analysis, as were other cytoskeleton-related proteins, such as actin and tubulin. Our studies present what we believe to be the first systematic genome-wide analysis of alternative splicing during cellular senescence and suggest a significant potential role of differential splicing in cytoskeleton reorganization during cellular aging.
To summarize, we demonstrated that progressive telomere loss in normal cells acted as an upstream signal to activate the cryptic splice site in LMNA to produce progerin as well as to induce alternative splicing of a number of other genes. Telomere-induced senescence has previously been primarily attributed to the activation of the tumor suppressor p53 (11). Interestingly, in HGPS cells, progerin-induced senescence has also been partially linked to the activation of p53 (51). Taken together, our data suggest a more complex model: in addition to activation of p53, progressive telomere shortening triggers alternative splicing of a suite of genes, including production of progerin mRNA and progerin protein. These changes, in turn, contribute to cellular senescence (Figure (Figure8).8). The recent report from Benson et al. (47) adds another potential feature to this mechanism — a positive feedback loop between telomeres and progerin.
The specific signaling pathway from dysfunctional telomeres to the spliceosome machinery remains undefined. An attractive hypothesis is that certain telomere-binding/capping proteins are released from telomere ends when telomeres are progressively shortened or damaged, and then influence spliceosome function in senescent cells. One potential candidate for carrying out this function is the shelterin complex, consisting of 6 telomere-binding proteins: TRF1, TRF2, POT1, TIN2, Rap1, and TPP1. Acting together with DNA repair factors, these proteins protect telomere ends by forming a specialized complex that masks the telomeres from being seen as sites of DNA damage (4). The released shelterin proteins could potentially act as signal transducers for alternative splicing in senescent cells. Other factors that may directly influence alternative splicing would include the heterogeneous nuclear ribonucleoproteins (hnRNPs), which bind to both RNA and single-stranded telomere DNA. In fact, hnRNPs have been suggested to participate in the posttranscriptional processing of precursor mRNA (52). Moreover, there is evidence suggesting that the single-stranded DNA binding activity of 2 hnRNPs, A1 and A2, is reduced in senescent cells (53, 54). Given the finding on the activation of extensive alternative splicing in senescent cells, it will be of great interest to determine whether shelterin, hnRNPs, and/or other telomere-binding proteins may play a role in pre-mRNA splicing in cellular aging. In summary, the present studies uncover what we believe to be a novel signaling pathway for cellular senescence and further enhance the conclusion that the study of HGPS can provide critical clues to the normal aging process.
Primary human dermal fibroblasts were cultured under 5% CO2 (balanced by ambient air) in MEM (Gibco; Invitrogen) supplemented with 15% FBS (Invitrogen) plus 2 mM l-glutamine. The primary fibroblast cell lines used in our studies included AG06299 (normal), AG08470 (normal), AG07306 (normal), AG08048 (normal), AG06277 (normal), AG09838 (normal), and AG09602 (normal) from Coriell Cell Repositories and HGADFN167 (HGPS), HGADFN003 (HGPS), HGFDFN168 (normal), and HGFDFN090 (normal) from the Progeria Research Foundation. See Supplemental Table 3 for detailed information for each cell line. The hTERT-HGFDFN090 and hTERT-AG09838 stable fibroblast cell lines were generated and provided by T. Glover (University of Michigan, Ann Arbor, Michigan, USA). Each cell passage was defined as a 3-fold split. Normal fibroblasts typically grew for about 30 passages, and HGPS fibroblasts grew 15–20 passages, under the culturing condition.
The human coronary vascular SMCs were cultured in SmGM-2 medium (Lonza). In addition, 2 normal primary B lymphocyte cell lines (AG11506 and AG09393) were obtained from Coriell Cell Repositories. The immortalized cells HeLa, HepG2, Mcf7, and 293T were cultured in DMEM (Gibco; Invitrogen) supplemented with 10% heat-inactivated FBS (Gibco; Invitrogen).
Primary DC fibroblast cells were provided from skin biopsies using standard methods by M. Armanios (Johns Hopkins University School of Medicine, Baltimore, Maryland, USA). Briefly, the skin biopsy was minced and grown in MEM with Earle's and 20% fetal bovine serum. After first passage, cells were maintained in complete media containing 10% serum. The study was approved by the Johns Hopkins Institutional Review Board.
The original splice reporter construct was provided by T. Misteli (National Cancer Institute, NIH, Bethesda, Maryland, USA). To subclone the splicing reporter into the pIRES-DsRed-Expression 2 vector, the pPRO reporter previously described (35) was PCR amplified using the following primers: forward, CCGCTCGAGGTACGGCTCTCATCAACTCC; reverse, CCGCTCGAGTTACTTGTACAGCTCGTC. The PCR product was subsequently cloned into the XhoI site of pIRES-DsRed-Expression 2 vector, and the correct orientation of the insert was determined by sequencing.
All transient transfections with the progerin splicing reporter in the primary fibroblasts were performed using the Amaxa nucleofection device and nucleofector kits for adult human–dermal fibroblasts (NHDF; Lonza) according to the manufacturer's instructions.
The antibodies used in the study included a rabbit polyclonal antibody against progerin (provided by K. Djabali, Columbia University, New York, New York, USA), mouse anti–lamin A/C (MAB3211; Chemicon), mouse anti-tubulin (DM1α; Sigma-Aldrich), rabbit anti-GFP (Abcam), and mouse anti-GAPDH (Abcam).
Primary fibroblasts transfected with the progerin splicing reporter were excited at 488 nm and sorted with a FACSAria machine (BD) according to the manufacturer's instructions.
Cells were harvested at approximately 75% confluence. RNA for each sample was isolated with the Qiagen RNAesy kit according to the manufacturer's instructions. RNA was reverse transcribed using the superscript III kit (Invitrogen) according to the manufacturer's instructions. qRT-PCR was performed to measure expression of progerin, LMNA, TERT, and ACTB. All reactions were carried out at least in triplicate on an Applied Biosystems 7900HT Fast Real-Time PCR System using SYBR Green mix (Qiagen) according to the manufacturer's instructions. Reaction conditions were as follows: 1 cycle of 2 minutes at 50°C; 1 cycle of 10 minutes at 95°C; and 40 cycles of 15 seconds at 95°C, 15 seconds at 58°C, and 30 seconds at 72°C. Primers for TERT and β-actin were obtained from R&D and Ambion, respectively. The sequence of the forward primer for amplifying progerin/lamin A is gcaacaagtccaatgaggacca. The progerin- and lamin A–specific reverse primers were designed according to amplification-refractory mutation system strategy, by introducing a mutation at the penultimate base to increase specificity. The progerin-specific primer sequence is CATGATGCTGCAGTTCTGGGGGCTCTGGAC, and that for lamin A is CATGATGCTGCAGTTCTGGGGGCTCTGGAT. To normalize for mRNA input in each reaction, the relative expression values for LMNA, progerin, or TERT were normalized to the mean values of ACTB. Expression values for each gene were expressed as mean ± SD of experimental triplicates.
FISH was performed with the Telomere PNA Kit (Dako) according to the manufacturer's protocol. Confocal images were acquired at room temperature using a Zeiss LSM 510 NLO Meta system mounted on a Zeiss Axiovert 200M microscope with an oil immersion Plan-Apochromat ×63/1.4 differential image contrast objective lens. Excitation wavelengths of 488 nm (4%), 561 nm (7%), and 740 nm (3%) were used for detection of the FITC-tagged telomeric probe, the Cy3-tagged telomeric probe, and DAPI, respectively. Fluorescent emissions were collected in a BP 500- to 550-nm IR blocked filter, LP 575-nm filter, and BP 390- to 465-nm IR blocked filter, respectively. All pinholes were set with a range from 1.21 to 1.39 Airy units, which correspond to an optical slice of 1.0 μm (excluding the DAPI channel, for which a multiphoton laser was used). All confocal images were of frame size 512 pixels × 512 pixels, scan zoom 2, and were line averaged 8 times. Confocal images were postprocessed using MediaCybernetics Image-Pro Plus software (version 6.3). Every image was processed using a custom macro designed to calibrate, filter (Gaussian), segment (touching nuclei or nuclei touching the image border were not included), outline the nuclei, count, and record measurements. Measurements included nucleus area (μmβ) and fluorescent intensity (minimum, maximum, mean, SD, and sum) measurements. Nuclei outlines were obtained using DAPI staining as the template, then propagating that outline throughout the FITC and Spectrum Orange channels. At least 20–30 randomly chosen nuclei were processed per cell line.
Immortalized cells or primary fibroblasts grown on chamber slides (Nunc) were fixed either with 4% formaldehyde in PBS for 20 minutes at room temperature followed by a 5-minute treatment with 0.5% Triton-X 100 in PBS, or with methanol/Acetone (1:1) at –20°C as described previously (26). The fixed cells were rinsed with PBS and blocked with 10% horse serum and 4% BSA in PBS for 30 minutes. Cells were then incubated for 1 hour with the primary antibodies diluted in blocking solution. The secondary antibodies were Alexa 488– or Alexa 594–conjugated donkey anti-rabbit or donkey anti-mouse IgG antibodies (Invitrogen). All samples were also counterstained with DAPI (Vector Laboratories). Cells were observed with a LSM510 or LSM710 confocal microscope (Zeiss) or an Axioplan fluorescence microscope (Zeiss).
Subconfluent primary fibroblasts grown on chamber slides (Nunc) were fixed in 3% formaldehyde and stained overnight at 37°C with X-gal buffered with sodium phosphate at pH 6.0 in the presence of potassium ferrocyanide and potassium ferricyanide as described previously (55).
Total RNAs were prepared according to Affymetrix protocols (Affymetrix). RNA quality and quantity was ensured using the Bioanalyzer (Agilent) and NanoDrop (Thermo Scientific), respectively. For RNA labeling, 1 μg total RNA was used in conjunction with the Affymetrix-recommended protocol for GeneChip WT Sense Target Labeling. The total RNA was first subjected to a ribosomal RNA reduction using the Invitrogen RiboMinus Transcriptome Isolation Kit. The remaining RNA was then reverse transcribed in the presence of dT7N6 primers to generate cDNA. An in vitro transcription was then performed, and the resulting cRNA was purified. A first-strand cDNA was generated form the cRNA in the presence of dUTPs. After RNA hydrolysis, the single-stranded cDNA was fragmented and end-labeled with biotin. The hybridization cocktail containing the fragmented and biotin-labeled cDNAs were hybridized to the Affymetrix GeneChip Human Exon 1.0 ST Array. The chips were washed and stained by the Affymetrix Fluidics Station using the standard format and protocols as described by Affymetrix. The probe arrays were stained with streptavidin phycoerythrin solution (Invitrogen) and enhanced using an antibody solution containing 0.5 mg/ml biotinylated anti-streptavidin (Vector Laboratories). An Affymetrix Gene Chip Scanner 3000 was used to scan the probe arrays. Gene expression intensities were calculated using Affymetrix Expression Console software, and .cel files were generated. Each sample was processed in technical triplicates. Data were deposited in NCBI Gene Expression Omnibus (accession no. GSE28863; http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE28863).
All exon array analysis was performed using XRAY microarray analysis software (version 3.9). Full quantile normalization was performed to minimize technical variance within the data. Only core probe sets (corresponding to high-quality genomic features like RefSeq or Ensembl transcripts) were considered for analysis. Probes with GC counts less than 6 and greater than 17 were filtered from the analysis, and the probe scores were transformed by taking natural logarithm of 0 plus the probe score. Background correction for each probe score was carried out by subtracting the median expression score of background probes with similar GC content. The expression score for a probe set was calculated as the median of all probes within the set, and probe sets with fewer than 3 probes were filtered from further analysis. Mixed model nested ANOVA was used to identify genes with statistically significant group-specific gene expression or alternative splicing. FDR method was used to correct for multiple testing, and FDR of 1.00 or less was considered significant for both differential alternative splicing and gene expression tests. A P value threshold of 0.001 was used to reject the null hypothesis that the average of group expression is not above background. P value filters were also applied to group expression levels for each gene to reduce false positives for alternative splicing. Quality control diagnostics produced for each array before and after normalization proved the good quality of arrays. GO enrichment analysis for the gene lists was carried out using MetaCore (GeneGo). Categories with an FDR of 0.05 or less were considered statistically significant.
To assay the statistical significance of the overlap between the genes showing altered expression and altered splicing, we randomly selected transcripts/genes from the final list of probes considered for the analysis. For example, for group A1, we randomly selected 3,900 of approximately 7,900 total genes and 533 of approximately 89,000 total transcripts represented in the final probe list. We then determined the number of overlaps between these randomly selected genes and transcripts. This was done 10,000 times to generate a distribution of the random overlaps between genes and transcripts for the group A1 set. The overlap of genes showing altered expression and altered splicing within all 4 group A sets was not statistically significant (P > 0.75). A similar analysis was carried out to determine the statistical significance of overlapping 82 genes among the 4 group A sets. In this case, we randomly selected a defined number of transcripts for each of the 4 group A sets and looked for overlap among them. For example, we randomly pulled 533 transcripts from group A1, 659 from group A2, 305 from group A3, and 243 from group A4 and determined the number of shared transcripts. We repeated this analysis 10,000 times to generate a distribution of how often the sets of alternatively spliced genes overlapped at random. In this case, the overlap of 82 genes with altered splicing between the 4 sets was found to be highly significant (P < 0.0001).
Genomic DNA from JH-1 and JH-2 lines was prepared using DNeasy Blood and Tissue Kit (Qiagen). The DNA fragment that flanks the K902N mutation in TERT was PCR amplified with the following primers: forward, CGGGCTGCTCCTGCGTTTGGTG; reverse, ACATCCCTGCGTTCTTGGCTTTCA. The amplified PCR products were sequenced by ACGT Inc.
Retroviral packaging BOSC 23 cells (6 × 106; ATCC) were plated in a 25-cm dish, incubated for 24 hours, and then transfected by Fugene 6 (Promega) with 30 μg retroviral plasmids. The retroviral plasmids pBABE-puro H-rasV12, pBABE-puro control, and pLPC- TRF2 (WT and mutant) were obtained from Addgene. After 60 hours, the retrovirus-containing medium was filtered (0.45-μm filter; Millipore), concentrated 50-fold (from 25 ml to 500 μl), and titrated. The target normal fibroblasts were plated at 70%–80% confluence in a 10-cm dish the night before infection. Each infection used 100 μl of the concentrated virus (MOI 0.5) supplemented with 5 μg/ml polybrene (Sigma-Aldrich). The infected normal fibroblast cells were selected in media containing 2 μg/ml puromycin for 5 days after infection and were then grown in regular media for an additional 6 days for analysis.
Unless otherwise specified, results are presented as mean ± SD. Data were analyzed using 2-tailed Student's t test, and a P value less than 0.05 was considered significant.
We thank Stephen Wincovitch and Amalia Dutra for support of the quantitative telomere PNA-FISH experiments, Stacie Anderson for support of the FACS analysis, Abdel Elkahloun for support of the exon array experiment, Tyra Wolfsberg for help with GO analysis, Darryl Leja for help with figures, Thomas Misteli for the original splice reporter construct, Thomas Glover for the human TERT–immortalized fibroblast cell lines, Mary Armanios for the DC fibroblast cell lines and helpful discussions, Samir Kelada for statistical help, and Karima Djabali for the anti-progerin antibody. This work was supported by NIA/NIH grant R00AG029761 (to K. Cao) and by the intramural program of the National Human Genome Research Institute (to F.S. Collins).
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2011;121(7):2833–2844. doi:10.1172/JCI43578.
Elizabeth G. Nabel's present address is: Brigham and Women's Hospital, Boston, Massachusetts, USA.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Pokemon Battle Revolution
This Revolution can only end in tears.
And spin-offs like Pokémon Battle Revolution? They're the cheap, diluted version of cocaine, the kind that some bastard has cut with flour, or Drano, or rat poison, or god knows what else. Yeah, it'll soothe your addiction-frayed nerves if you're jonesing for a Pokémon fix, but it's gonna kill you, man.
Battle Revolution is a game for Pokémon fanatics alone. No one else has reason to play it, and it makes no effort whatsoever to be accessible to a non-Pokéddicted audience. It consists of nothing but the battling component of the Game Boy and DS RPGs; in fact, the only way to fully make use of its features is to connect a copy of Diamond or Pearl via wireless and copy the monsters you've been training up for the past few hundred hours onto your Wii. The DS connectivity feature is seamless and efficient, but adds an extra $170 to the price of admission -- not to mention the time investment it requires.
So what if you don't own a DS? What if you've never played a Pokémon game before? Tough luck. You can "rent" a set of monsters to fight with from the beginning of the game, but you're initially given no control over your roster -- nor does the game explain the nuances of the combat system and the skills your rented creatures have. Where the handheld RPGs feature a gentle learning curve that allows you a few hours to come to terms with the rock-paper-scissors monster-type balance and gradually rolls out skills and techniques, Battle Revolution drops you right into the thick of battle and expects you to hold your own. Sure, the early challenges are simple enough, but it seems unlikely that a novice player is going to pick up on the more advanced strategies that evolve naturally through the progressive experience that the real Pokémon games provide. If you like trial and error, you'll love Battle Revolution!
Truth be told, battling is only one part of Pokémon's appeal. The rest lies in exploring the world, embarking on the little side quests and subplots that pop up along the way to the grand finale, and developing of a team of creatures that suit your own fighting style. In short, all the things that Battle Revolution abandons in favor of fighting. Honestly, combat in the Pokémon series is dull and plodding, only interesting when you square off against a friend -- something that Battle Revolution allows, sure. But so do Diamond and Pearl. All Battle Revolution really has going for it is the visual overhaul it boasts: Every monster has been translated into polygons, complete with animations instead of simple two-frame motions. But this mainly serves to make the series' already poky conflicts even slower, and frankly most of the added animations are simply silly.
So what's the point of Battle Revolution, then, if all it does is duplicate a portion of the real Pokémon games in a less-fulfilling way? Honestly, not much. Those who have drained every last moment of enjoyment from the DS game might appreciate it as a way to eke a little more challenge from the experience. And really obsessive types might appreciate the fact that the announcer guy from the cartoon performs (highly repetitious) running commentary during in-game battles. Everyone else, though, will simply regard this as a waste of money.
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Welcome! This guide has been created to help you better understand some of the issues we think Christians care about for the upcoming federal election. It is not definitive, but we will keep adding to it as issues develop and policies are released. Is there something we've missed? Let us know.
Click on each of the issues below for our election guide:
A message from Greg Clarke:
Bible Society Australia is a non-denominational and non-political organisation, but we encourage Christian reflection on political, social and ethical issues. We are delighted when the Bible is involved in these reflections, offering wisdom that has helped shape the nation we love, and still provides so much for individuals, families and communities today.
Christians are held together by stronger bonds than politics. We are united in believing that Jesus Christ is the revelation of God to us, the "word made flesh" as the Bible describes him, and the salvation of the world. Any political differences that Christians have are surpassed by this common commitment to Christ which means we can call each other brothers and sisters.
Our unity in Christ enables us to express our different viewpoints in love and fellowship, and this guide hosts a range of different perspectives from Australian Christian organisations and individuals. Please visit the various pages to discover the opinions of leading Australian Christian thinkers on issues affecting the life of the nation. I'm sure you will be informed, intrigued, inspired and perhaps even incensed!
We hope and pray that this material assists you to undertake the great privilege of voting for our nation's next leaders.
Every blessing,
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