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Golfing World: Dredge finding form in 2014
Resurgent Welshman Bradley Dredge sits down for a chat with Golfing World after a couple of very impressive European Tour performances
Golfing World: Catching up with Shane Lowry
We have a chat with recent runner-up at the BMW PGA Championship and Ryder Cup 2014 outsider Shane Lowry.
Golfing World: Henrik Stenson player profile
Golfing World catches up with Mr. 2013, Henrik Stenson, and takes a close look at his return to prominence.
Golfing World: Catching up with Henrik Stenson
We catch up again with Mr. 2013, Henrik Stenson, as he plans to re-find his very best form again in 2014
Stenson captures double-double, but was it a historic event?
Henrik Stenson was the first to win the FedEx Cup and Race to Dubai, but where does it rank among golf's accomplishments?
Dubuisson making big push for Ryder Cup in RTD
Just a couple weeks ago, Victor Dubuisson wasn't a name known to most American golf fans. Now, the Frenchman has a chance to claim back-to-back victories in the Race to Dubai's Final Series.
Bet on it: Poultz top Stenson in Race to Dubai?
GolfChannel.com writer Ryan Lavner joins Win McMurry to preview the DP World Tour Championship and pick a player outside the top three in the standings that could walk away as the Race to Dubai champion.
#AskLav: Stenson closes in on $20 million in 2013
This week's #AskLav dives into Henrik Stenson's dream season, in which he can bank close to $20 million.
No. 8 Newsmaker of 2013: Henrik Stenson
GolfChannel.com writers Rex Hoggard, Randall Mell and Ryan Lavner sit down to discuss Henrik Stenson and why he's the eighth biggest newsmaker of 2013.and Ryan Lavner. We're doing our year-end newsmakers and we've arrived at Henrik Stenson , a phenomenal year and when you consider the PGA Tour didn't give out a comeback player of the year in 2013 but if they did, I think Henrik Stenson would have been do maybe for the comeback player of the decade considering everything he did. He began the season 56th infirst Euro title and then two months later, he walks out the golf course 'cause he cannot keep the ball on the golf course . You know, slumps, loses his game, rises back to number four and then slumps and loses his game, drops to number 230 and then wins the FedExCup and the Race to Dubai, it's remarkable. -I think if you're looking in the last decade, you could see a player like Steve Stricker who couldn't even get the ball off the tee. Obviously, you've seen the longevity that he has had, looking now at Jarrod Lyle, twice a leukemia survivor, trying to work his way back on the tour but Henrik Stenson is an unbelievable story to deepen of the 230s just a year and a half ago, all the way up now to
Stenson, Poulter make Final Series more interesting
Henrik Stenson and Ian Poulter decided to make the Race to Dubai's Final Series a little more interesting.
A look back: O'Grady recaps the season
George O'Grady discusses the transition that the European Tour went through in 2013.European Tour went through a period of transition in 2013 and it ended on a really, really positive note. First and foremost, we announcedbeen a powerful year in a television sense. Many different markets have extended agreements but none more so than our groundbreaking agreement in The United States of America. Many source have a scholarship and covered on NBC for the first time for domestic event. The 10-year extension to our deal with The Golf Channel and NBC which I think is groundbreaking, very positive news for all our tournaments and all our sponsors and certainly for the membership. We signed additional agreements in 2013 in developing markets OSN in the Middle East, ViaSat in Russia and other agreements in India, in China and in Thailand. I think that's the key point about those that gives everybody who sponsors tournament on the European Tour or promotes events great confidence that they get this visibility throughout the world. We launched the European Tour TV at the BMW PGA Championship at Wentworth in May. I think this is a very important announcement because it means on your PC, you can watch European Tour golf in all those areas of the world where it isn't coming through a recognized broadcaster.
Dubai scoring page
Henrik Stenson shot 64 Friday to grab the lead at the DP World Tour Champ., and move a step close to winning the Race to Dubai. Scores
Golfing World: Happy Birthday, Lee Westwood
Golfing World wishes Lee Westwood a very happy 41st birthday!On the 24th of April Lee Westwood , one of the game's most popular players will celebrate his 41st birthday. To mark the occasion, let's take a look at some of the highlights of his career so far. The man from Warsaw started out life as a professional back in 1994, and has since amassed an impressive 23 victories on the European tour , starting with the 1996 Volvo Scandinavian masters, age 23. In 1998 he was crowned European tour player of the year, following four victories. He achieved the same feat again in 2000, a magnificent start of the new millennium,
Stenson two back in bid to win Race to Dubai
Despite a three-putt bogey at the last, Henrik Stenson is in position to win the European Tour season finale and the Race to Dubai.


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Record: 2 – 4 – 0
Tampa Bay -
Winnipeg -
Oct. 24 at 7 p.m.
Toronto -
Ottawa -
Oct. 22 at 6 p.m.Boxscore
Philadelphia 5
Pittsburgh 3
Washington 2
Edmonton 3
Buffalo 1
Anaheim 4
NY Islanders -
Boston -
Oct. 23 at 6 p.m.
Pittsburgh -
Detroit -
Oct. 23 at 6:30 p.m.
Vancouver -
St. Louis -
Oct. 23 at 7 p.m.
Chicago -
Nashville -
Oct. 23 at 7 p.m.
Arizona -
Minnesota -
Oct. 23 at 7 p.m.
Carolina -
Calgary -
Oct. 23 at 8 p.m.
Buffalo -
Los Angeles -
Oct. 23 at 9:30 p.m.
Columbus -
San Jose -
Oct. 23 at 9:30 p.m.
Winnipeg Free Press - ONLINE EDITION
Ovechkin gets Olympic send-off from Kerry, scores 40th to lead Capitals past Jets 4-2
WASHINGTON - Secretary of State John Kerry, the most powerful politician in the house, performed a ceremonial puck drop to send the Olympians on their way.
Alex Ovechkin, the biggest hockey star in the arena, scored his league-leading 40th goal to give his team the lead in the third period.
Set aside those two larger-than-life names for a moment, however, and look at the trio who occupy the score sheet for the crucial tying goal early in the third period of the Washington Capitals' 4-2 win over the Winnipeg Jets on Thursday night.
Patrick Wey (second NHL game). Julien Brouillette (first NHL game). Tom Wilson (second NHL goal).
"It was a simple play, but it was nice that we ended up scoring," Wey said. "And I'm really excited."
Simple but effective. Wey passed cross-ice to Brouillette, who then deliberately shot wide of the net to set up a carom off the boards that eventually made its way to Wilson.
"The play Brou made, banking it off the boards like that, I thought that was a great play," Washington coach Adam Oates said. "And obviously it worked, the bounce was right. ... It gave us a lot of life."
Wey and Brouillette had arrived in town the night before, having been recalled from Hershey of the AHL to serve as the third defensive pairing because the Capitals had run out of healthy bodies.
"From what I expected," Oates said, "they were fantastic."
The Wey-Brouillette-Wilson goal made the score 2-2, setting the stage for Ovechkin. The Russian forward lifted the puck over a pile of bodies in the crease with 12:14 remaining, following up after Nicklas Backstrom had taken two whacks at a rebound before sliding into goaltender Ondrej Pavelec.
Troy Brouwer then added his second goal of the game in the closing minutes, and Braden Holtby made 34 saves for the Capitals, who are trying to generate at least a modicum of momentum headed into the Olympic break. Washington had dropped 10 of its previous 13, including a 1-0 shutout loss to the New York Islanders on Tuesday.
Bryan Little and Dustin Byfuglien scored, and Pavelec stopped 26 shots for the Jets, who had a three-game winning streak snapped. Winnipeg dominated play in the second period before allowing three goals in the third, dropping the team to 9-3 since coach Claude Noel was fired on Jan. 12 and replaced by Paul Maurice.
"I think they just did a little bit to us what we've been doing to (other) teams," Maurice said. "They weathered that second-period storm."
With the NHL's Olympic break beginning after Saturday's games, Kerry was on hand to recognize the four players from each team headed to Sochi. He then dropped the puck at centre ice between two U.S. Olympians, Winnipeg's Blake Wheeler and Washington's John Carlson.
Kerry also met with Capitals coaches and players in the locker room before the game.
"I had a good talk with him," Oates said. "But he's obviously a very busy man. He comes with his entourage; it's pretty tough to have a chat."
Notes: Little's goal was actually put into the net by Washington's Martin Erat, something the Czech forward has yet to do for his own team this season. Erat has 21 assists but no goals for the Capitals. ... Oates said Holtby was in goal because scheduled starter Michal Neuvirth wasn't feeling well. ... Brouillette and Wey were needed because D John Erskine (off-season knee surgery) was given a rest day and D Mike Green was still sidelined with a concussion. ... Carlson played in his 272nd consecutive game, breaking Calle Johansson's Capitals record for a defenceman.
Live Winnipeg Jets Coverage
@WFPHockey: Jets-Capitals summary bit.ly/NcXDbw #NHLJets 7:46 am on Feb 7
@WFPHockey: Jets let one get away bit.ly/1iwQvB6 #NHLJets 7:46 am on Feb 7
@WFPHockey: Coach's orders: simply move on bit.ly/1lFdIWm #NHLJets 7:46 am on Feb 7
@WFPEdTait: #NHLJets fall to Caps 4-2 at Verizon Center. Three-game win streak is snapped and now 9-3 under Paul Maurice. 8:38 pm on Feb 6
@WFPEdTait: Troy Brouwer with his second, and 100th of his career, puts the Caps up 4-2 on #NHLJets. 8:31 pm on Feb 6
@WFPEdTait: #NHLJets have been out-shot 10-4 in this period... not generating much offensively, let alone second chances. 8:27 pm on Feb 6
@WFPEdTait: Alex Ovechkin puts the Caps ahead 3-2 over the #NHLJets. That's his 40th, the sixth time he's hit that mark in his career. 8:13 pm on Feb 6
@WFPEdTait: Caps tie #NHLJets 2-2 on goal by Wilson. Winnipeg running around a bit in its own zone. 8:06 pm on Feb 6
@WFPEdTait: #NHLJets lead Caps 2-1 after 2 and have out-shot Wash 30-18. 7:42 pm on Feb 6
@WFPEdTait: #NHLJets playing with fire sending Caps to the PP again. Byfuglien off for tripping. 7:25 pm on Feb 6
@WFPEdTait: Dustin Byfuglien, taking a solid pass from Setoguchi, beats Holtby upstairs and it's 2-1 #NHLJets. 7:20 pm on Feb 6
@WFPEdTait: #NHLJets Eric Tangradi pulled up lame on that last shift and has headed to dressing room. 7:15 pm on Feb 6
@WFPEdTait: #NHLJets and Caps tied 1-1 after 1. Jets out-shoot Wash 15-11. 6:52 pm on Feb 6
@WFPEdTait: #NHLJets Dustin Byfuglien takes stick to the face near his eye. Still on bench being attended to by staff. 6:45 pm on Feb 6
@WFPEdTait: Great pass from Ladd to Little, his chance stopped but puck goes in off Martin Erat. 1-1 tie. Caps #NHLJets 6:38 pm on Feb 6
@WFPEdTait: Troy Brouwer opens scoring for Caps as two #NHLJets, Olli Jokinen and Toby Enstrom, go fishing. 1-0 Washington. 6:20 pm on Feb 6
@WFPEdTait: #NHLJets starters: Ladd, Little, Frolik, Bogosian, Enstrom and Pavelec. Holtby now starting for Caps as Neuvirth has flu. 5:58 pm on Feb 6
@WFPEdTait: Trade deadline drawing close, so make up your own rumors aft seeing scouts' list for tonight's #NHLJets game pic.twitter.com/MQh6UQbtzs
4:59 pm on Feb 6
@WFPEdTait: Caps recall D-men Julien Brouillette and Patrick Wey from Hershey. #NHLJets on the ice at Verizon in a couple hours. 8:38 am on Feb 6
@WFPHockey: NHL singing new loonie tune bit.ly/1lCzGt1 #NHLJets 7:58 am on Feb 6
@WFPHockey: Olys chance for Jets, coach to talk contract bit.ly/1iu2q2z #NHLJets 7:58 am on Feb 6
@WFPHockey: The Kladno kids: Frolik and Pavelec bit.ly/1kZkgv6 #NHLJets 7:58 am on Feb 6
Updated on Thursday, February 6, 2014 at 7:20 PM CST: Updates score, changes headline.
7:53 PM: Updates second-period score, changes headline.
8:47 PM: Adds final score, changes headline.
11:26 PM: Replaces pre-game story with post-game coverage.
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News & Public Affairs
New film warns about climate refugees listen01/31/11 Sarah Curran
WMNF Drive-Time News Monday Listen to this entire show:
As the planet changes, the way humans co-exist with it will have to start changing as well. The film Climate Refugees puts a human face on the issue of climate change. Itâs been shown at film festival around the world. Suzanne Haerther is the chapter coordinator for the US Green Building Council Gulf Coast chapter.
1 comments
âWe have had some major climate issues. And whether or not you believe that they are man made or just a natural cycle they are still displacing people and we are going ...
Tampa Theatre organist Rosa Rio dies at 107 listen05/14/10 Seán Kinane
WMNF Drive-Time News Friday Listen to this entire show:
The legendary Tampa Theatre organist Rosa Rio has died at the age of 107. She began playing organ at movie theaters nearly a century ago.
Tara Schroeder, Tampa Theatreâs Director Programming and Marketing, was a co-worker and a friend. "She was loved by everyone," said Schroeder.
Rosa Rio was interviewed for StoryCorps early in 2009. She said, "I just love the organ."
There will be a pub...2 comments
A look inside Islam listen01/19/10 Joshua Lee Holton
WMNF Drive-Time News Tuesday Listen to this entire show:
A new civil rights group for Muslims sponsored a showing of the new film âInside Islam: What a billion Muslims really think.â The film was at USF in Tampa last Saturday night, and was designed to challenge the notion that Islam and the West are on a collision course.
Muslims in 40 countries participated in a Gallup public opinion poll that showed what Muslims think about the most controversial issues surrounding their religion. Last Saturday hundreds of people attended the film at USF...7 comments
Sarasota Film Festival Opens03/21/09 Mary Glenney & Arlene Engelhardt
From A Woman's Point of View
Holly Herrick, Programmer, Sarasota Film Festival discussed the upcoming festival - particularly films of interest to women and discussing issues affecting women.
Deborah Kampmeier, director, "Hounddog" discussed the controversy surrounding the rape scene in her film and the lead played by the young Dakota Fanning. "Hounddog premiered at Sundance in 2007. âHounddogâ, Friday, April 3, 8 p.m. http://www.hounddogmovie.com/
Ingrid Veninger, director, âOnlyâ and writer, produc...Be the first to comment


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Toilet Paper Advertising
I am gonna work in advertising one day and I am prepared to stretch logic, reason and the truth every now and then and around as many corners as necessary.
However, one of my promises to the world will be to never ever use animals for toilet paper commercials. (We will discuss the other promises another time.)
Animals do not use toilet paper. Toilet paper is not made out of animal fur or skin (!?). I see the connection that toilet paper is as soft as a little puppy, cub or a kitten. (Weirdly enough, is seems to be more of a dog or a bear product?)
But that's where that connections already ends. I never sat on the toilet, noticed that I ran out of toilet paper and thought, "I bet my cat used it all…" (And for the record, I also never thought, "Oh I wish my cat would walk in right now so I could solve my little problem…")
I think it's time to get real and if you toilet paper advertisers won't do it, I will.
I will revolutionize an industry (or two).
I think everyone knows that using the bathroom is not cute, fluffy and cuddly. It makes funny noises, it smells and there's a reason why we shake, wipe, flush and light a match.
It's time to stop the bullshit and just tell the truth.
I was thinking of something along those (quilted) lines:
(A campaign is in the works. And it's probably gonna look less like Abercrombie & Fitch Toilet Paper.)


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Gasp. My Snark clip on tuner battery died! Not that I really need it, but it's so damn useful on those days when my ears insist on hearing things a little sharper. A trip to Home Depot was in the cards anyway for weather stripping (¡¡¡frost warning this weekend in New Albany!!!), but then I pulled the wrong batteries off the shelf – CR2025s instead of CR2032s. So disappointed, and then looked in my writing desk drawer – and I had a spare CR2032 all along. Definitely a day Snark won. But now I have my tuner back.
typewriter maintenance day
Bugging me for months. Sticky keys on the Olivetti finally solved thanks to finding a repair note on google letting us know the bottom pops off. Spraying isopropyl on the linkages removed the gunk that some poor soul added thinking it makes the keys work better to oil them – it doesn't. New ribbon, no sticky keys (but the stubborn "5") and I was inspired to write a letter to a friend just home from jail after a terrible misunderstanding. Now, fresh pages, dark black ribbon, cleaned rollers, and no sticky keys – and I can't wait to type some of the novel as soon as I can get to it.
I am reading a lot of William Carlos Williams. I am going to share my research with the W490 Writing Capstone course I'm teaching.
lucky 13
The fall submission blitz has begun for me – as of today i have 13 subs out, including 4 poetry book prize submissions and 1 chapbook prize submissions. More to go – keep you informed here. Last fall I ended up as second place in the Raynes Poetry Prize judged by Joan Larkin. Hope this fall is as good or better. But you never know.


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|Controlled electroporation and mass transfer across cell membranes
|May, 2010
|Rubinsky et al.
|Animal Model of Prostate Cancer and Use Thereof
|February, 2008
|Lin et al.
|Method for hair removal by electroporation
|September, 2007
|Chornenky et al.
|Apparatus for controlling the generation of electric fields
|May, 2007
|Persson et al.
|Apparatus and method for hair removal by electroporation
|May, 2007
|Chornenky et al.
|Tissue ablation with irreversible electroporation
|February, 2007
|Davalos et al.
|Electroporation controlled with real time imaging
|November, 2006
|Rubinsky et al.
|Apparatus and method for the treatment of benign prostatic hyperplasia
|October, 2006
|Chornenky et al.
|Apparatus and method for treatment of benign prostatic hyperplasia
|September, 2006
|Chornenky et al.
|Methods and apparatus for treating congestive heart failure
|September, 2006
|Demarais et al.
|Controlled electroporation and mass transfer across cell membranes
|June, 2006
|Rubinsky et al.
|Vacuum coagulation probes
|June, 2006
|Whayne et al.
|Controlled electroporation and mass transfer across cell membranes in tissue
|May, 2006
|Rubinsky et al.
|Transurethral needle ablation system
|April, 2006
|Elmouelhi et al.
|Blood detector for controlling anesu and method therefor
|February, 2006
|Apparatus and method for treatment of benign prostatic hyperplasia
|February, 2006
|Chornenky et al.
|Apparatus for controlling the generation of electric fields
|January, 2006
|Persson et al.
|Methods and apparatus for renal neuromodulation
|December, 2005
|Deem et al.
|Open system heat exchange catheters and methods of use
|December, 2005
|Eum et al.
|Systems and methods for selective denervation of heart dysrhythmias
|November, 2005
|Deem et al.
|Method for detecting and treating tumors using localized impedance measurement
|November, 2005
|Johnson et al.
|Apparatus and method for reducing subcutaneous fat deposits, virtual face lift and body sculpturing by electroporation
|August, 2005
|Chornenky et al.
|Electroporation to interrupt blood flow
|August, 2005
|Rubinsky et al.
|Irreversible electroporation to control bleeding
|August, 2005
|Rubinsky et al.
|Cell viability detection using electrical measurements
|August, 2005
|Rubinsky et al.
|Microporation of tissue for delivery of bioactive agents
|July, 2005
|Method and apparatus for delivery of therapeutic agents
|June, 2005
|Bernard et al.
|Apparatus and method for reducing subcutaneous fat deposits, virtual face lift and body sculpturing by electroporation
|May, 2005
|Jaafar et al.
|Device for medicine delivery by intraocular iontophoresis or electroporation
|March, 2005
|Behar et al.
|Electrically induced vessel vasodilation
|March, 2005
|Dev et al.
|Device II
|February, 2005
|McHale et al.
|Device and method for controlled delivery of active substance into the skin
|December, 2004
|Mavor et al.
|Methods and devices for treating atrial fibrilation
|December, 2004
|Delivery of an agent
|November, 2004
|McHale et al.
|Device and method for monitoring and controlling electrical resistance at a tissue site undergoing iontophoresis
|October, 2004
|Miller et al.
|Apparatus and method for reducing subcutaneous fat deposits by electroporation
|September, 2004
|Chornenky et al.
|Electrosurgical apparatus and methods for ablating tissue
|August, 2004
|Diagnosis and treatment of cancer:I
|July, 2004
|Diss et al.
|Site-specific cell perforation technique
|June, 2004
|Karube et al.
|Apparatus and method for the treatment of benign prostatic hyperplasia
|March, 2004
|Chornenky et al.
|Device and method for treating skin
|March, 2004
|Apparatus and method for reducing subcutaneous fat deposits by electroporation with improved comfort of patients
|February, 2004
|Chornenky et al.
|Skin and muscle-targeted gene therapy by pulsed electrical field
|February, 2004
|Dev et al.
|Method for performing intraurethral radio-frequency urethral enlargement
|February, 2004
|McGovern et al.
|Apparatus and method for reducing subcutaneous fat deposits, virtual face lift and body sculpturing by electroporation
|January, 2004
|Jaafar et al.
|Transdermal drug delivery patch system, method of making same and method of using same
|December, 2003
|Eppstein et al.
|Epicardial myocardial revascularization and denervation methods and apparatus
|December, 2003
|Method and apparatus for plasma-mediated thermo-electrical ablation
|November, 2003
|Palanker et al.
|Method and device for predicting physiological values
|November, 2003
|Dunn et al.
|Cell separation using electric fields
|October, 2003
|Mangano et al.
|Method for intracellular modifications within living cells using pulsed electric fields
|September, 2003
|Gundersen et al.
|Methods and systems for applying multi-mode energy to biological samples
|September, 2003
|Unger et al.
|Capillary electrokinesis based cellular assays
|September, 2003
|McCormick et al.
|Monopolar and bipolar current application for transdermal drug delivery and analyte extraction
|August, 2003
|Avrahami et al.
|Electrosurgical device
|August, 2003
|Jones et al.
|Impedance controlled tissue ablation apparatus and method
|July, 2003
|Pearson et al.
|Apparatus and methods for monitoring tissue impedance
|May, 2003
|Tu et al.
|Controlled electroporation and mass transfer across cell membranes
|May, 2003
|Rubinsky et al.
|Apparatus and method for treatment of benign prostatic hyperplasia
|March, 2003
|Chornenky et al.
|Apparatus for thermal treatment of tissue
|February, 2003
|Device for tumor diagnosis and methods thereof
|January, 2003
|Tu et al.
|Method and apparatus for the treatment of cosmetic skin conditions
|December, 2002
|Smith, III
|Methods for electrosurgical spine surgery
|December, 2002
|Underwood et al.
|Percutaneous electrical therapy system with electrode position maintenance
|December, 2002
|Leonard et al.
|Cell/tissue analysis via controlled electroporation
|November, 2002
|Rubinsky et al.
|Method and apparatus for treating cardiac arrhythmia
|October, 2002
|Ideker et al.
|Apparatus and method for selectively removing a body fat mass in human body
|September, 2002
|July, 2002
|Dev et al.
|Implantable device and method for the electrical treatment of cancer
|June, 2002
|Schroeppel et al.
|Controlled electroporation and mass transfer across cell membranes
|June, 2002
|Rubinsky et al.
|May, 2002
|Atala et al.
|Electrical impedance tomography to control electroporation
|May, 2002
|Rubinsky et al.
|Method for inducing electroanesthesia using high frequency, high intensity transcutaneous electrical nerve stimulation
|February, 2002
|Neuro-stimulation to control pain during cardioversion defibrillation
|February, 2002
|Electrically induced vessel vasodilation
|February, 2002
|Dev et al.
|Method and apparatus for intracellular electro-manipulation
|January, 2002
|Method and apparatus for intracellular electro-manipulation
|December, 2001
|Schoenbach et al.
|Apparatus and method for treatment of vascular restenosis by electroporation
|November, 2001
|Controlled electroporation and mass transfer across cell membranes
|October, 2001
|Electrodes and electrode arrays for generating electroporation inducing electrical fields
|August, 2001
|Ultra-wide band RF-enhanced chemotherapy for cancer treatmeat
|July, 2001
|Radio frequency ablation device for treatment of the prostate
|June, 2001
|Lundquist et al.
|Iontophoresis, electroporation and combination catheters for local drug delivery to arteries and other body tissues
|April, 2001
|Brown et al.
|Method of programming an array of needle electrodes for electroporation therapy of tissue
|April, 2001
|Method for treating tumors near the surface of an organ
|April, 2001
|Methods for electrosurgical dermatological treatment
|April, 2001
|Olsen et al.
|Electroporation apparatus with connective electrode template
|March, 2001
|System for attenuating pain during bone marrow aspiration and method
|December, 2000
|Strauss et al.
|Electroporetic gene and drug therapy
|October, 2000
|Apparatus and method for analyzing particles
|September, 2000
|Multimodality instrument for tissue characterization
|August, 2000
|Mah et al.
|Apparatus for thermal treatment of tissue
|August, 2000
|Blewett et al.
|Device for suction-assisted lipectomy and method of using same
|August, 2000
|Electrosurgical instrument
|July, 2000
|Goble et al.
|Biopotential measurement including electroporation of tissue surface
|July, 2000
|Weaver et al.
|Method of Selectively applying needle array configurations
|May, 2000
|Hofmann et al.
|Apparatus for addressing needle array electrodes for electroporation therapy
|April, 2000
|Hofmann et al.
|Cell separation using electric fields
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This application is a divisional of our earlier filed application Ser. No. 11/340,176, filed Jan. 25, 2006, which is continuation of our earlier filed application Ser. No. 10/407,580 filed Apr. 3, 2003 (now abandoned), which is a continuation of our earlier filed application Ser. No. 09/863,117 filed May 22, 2001 (issued as U.S. Pat. No. 6,562,604 on May 13, 2003), which application is a continuation of our earlier filed application Ser. No. 09/358,510 filed Jul. 21, 1999 (issued as U.S. Pat. No. 6,300,108 on Oct. 9, 2001), which applications are incorporated herein by reference and to which applications we claim priority under 35 USC §120.
This invention resides in the fields of electroporation and mass transfer across cell membranes.
Electroporation is a technique that is used for introducing chemical species into biological cells, and is performed by exposing the cells to an electric potential that traverses the cell membrane. While its mechanism is not fully understood, electroporation is believed to involve the breakdown of the cell membrane lipid bilayer leading to the formation of transient or permanent pores in the membrane that permit the chemical species to enter the cell by diffusion. The electric potential is typically applied in pulses, and whether the pore formation is reversible or irreversible depends on such parameters as the amplitude, length, shape and repetition rate of the pulses, in addition to the type and development stage of the cell. As a method of introducing chemical species into cells, electroporation offers numerous advantages: it is simple to use; it can be used to treat whole populations of cells simultaneously; it can be used to introduce essentially any macromolecule into a cell; it can be used with a wide variety of primary or established cell lines and is particularly effective with certain cell lines;-and it can be used on both prokaryotic and eukaryotic cells without major modifications or adaptations to cell type and origin. Electroporation is currently used on cells in suspension or in culture, as well as cells in tissues and organs.
Electroporation is currently performed by placing one or more cells, in suspension or in tissue, between two electrodes connected to a generator that emits pulses of a high-voltage electric field. The pore formation, or permealization, of the membrane occurs at the cell poles, which are the sites on the cell membranes that directly face the electrodes and thus the sites at which the transmembrane potential is highest. Unfortunately, the degree of permealization occurring in electroporation varies with the cell type and also varies among cells in a given population. Furthermore, since the procedure is performed in large populations of cells whose properties vary among the individual cells in the population, the electroporation conditions can only be selected to address the average qualities of the cell population; the procedure as currently practiced cannot be adapted to the specific characteristics of individual cells. Of particular concern is that under certain conditions, electroporation can induce irreversible pore formation and cell death. A high electric field, for example, may thus produce an increase in transfection efficiency in one portion of a cell population while causing cell death in another. A further problem with known methods of electroporation is that the efficiency of transfection by electroporation can at time be low. In the case of DNA, for example, a large amount of DNA is needed in the surrounding medium to achieve effective transformation of the cell.
Many of the problems identified above are a consequence of the fact that the process of electroporation in both individual cells and tissues cannot be controlled in real time. There are no means at present to ascertain in real time when a cell enters a state of electroporation. As a result, the outcome of an electroporation protocol can only be determined through the eventual consequences of the mass transfer process and its effect on the cell. These occur long after the mass transfer under electroporation has taken place. These and other deficiencies of current methods of electroporation are addressed by the present invention.
Also relevant to the present invention are current techniques for the study and control of mass transfer across cell membranes. Knowledge of mass transfer across cell membranes in nature, both in cells that are functioning normally and in diseased cells, is valuable in the study of certain diseases. In addition, the ability to modify and control mass transfer across cell membranes is a useful tool in conducting research and therapy in modern biotechnology and medicine. The introduction or removal of chemical species such as DNA or proteins from the cell to control the function, physiology, or behavior of the cell provides valuable information regarding both normal and abnormal physiological processes of the cell.
The most common method of effecting and studying mass transfer across a cell membrane is to place the cell in contact with a solution that contains the compound that is to be transported across the membrane, either with or without electroporation. This bulk transfer method does not permit precise control or measurement of the mass transfer across the membrane. The composition of the solution at specific sites is not known and is variable. In addition, when an electric field is present, the local field intensity will vary from one point to another. Furthermore, the surface of the cell that is exposed to the solution is not well defined. Cell surface areas vary among cells in a given population, and this leads to significant differences among the cells in the amount of mass transfer. For these reasons, the amount of mass transfer achieved by bulk transfer processes is not uniform among cells, and the actual amount transferred for any particular cell cannot be determined.
Attempts made so far to overcome the limitations of bulk transfer techniques include techniques for treating individual cells that include either the mechanical injection (microinjection) of chemical compounds through the cell membrane or electroporation with microelectrodes. In injection techniques, the membrane is penetrated with a needle to deliver a chemical agent, localizing the application of the chemical agent to a small region close to the point of injection. This requires manipulation of the cell with the human hand, a technique that is difficult to perform, labor-intensive, and not readily reproducible. Electroporation with microelectrodes suffers these problems as well as the lack of any means to detect the onset of electroporation in an individual cell. These problems are likewise addressed by the present invention.
The present invention arises in part from the discovery that the onset and extent of electroporation in a biological cell can be correlated:to changes in the electrical impedance (which term is used herein to mean the ratio of current to voltage) of the biological cell or of a conductive medium that includes the biological cell. An increase in the current-to-voltage ratio across a biological cell occurs when the cell membrane becomes permeable due to pore formation. Likewise, a decrease in the current-to-voltage ratio through a flowing conductive fluid occurs when the fluid draws a biological cell into the region between the electrodes in a flow-through electric cell. Thus, by monitoring the impedance of the biological cell or of an electrolyte solution in which the cell is suspended, one can detect the point in time in which pore formation in the cell membrane occurs, as well as the relative degree of cell membrane permeability due to the pore formation. This information can then be used to establish that a given cell has in fact undergone electroporation, or to control the electroporation process by governing the selection of the voltage magnitude. This discovery is also useful in the simultaneous electroporation of multitudes of cells, since it provides a direct indication of the actual occurrence of electroporation and an indication of the degree of electroporation averaged over the multitude. The discovery is likewise useful in the electroporation of biological tissue (masses of biological cells with contiguous membranes) for the same reasons.
The benefits of this process include a high level of control over the onset and degree of electroporation, together with a more detailed knowledge of the occurrence and degree of permeability created in particular individual cells or cell masses. When applied to individual cells or to a succession of individual cells, this process assures that the individual cells are indeed rendered permeable and are indeed transformed by the introduction of chemical species. The process also offers the ability to increase the efficiency of electroporation by avoiding variations in the electrical environment that would destroy some cells while having an insufficient effect on others.
In some of its more specific embodiments, the present invention involves the use of an electrical cell in which a biological cell can be placed and that contains a barrier that directs the electric current flow and hence the ion flow through a flow path that passes through the biological cell while permitting substantially no electric current to bypass the biological cell. In some of these embodiments, the invention involves the use of an apparatus containing two liquid-retaining chambers separated by a barrier that is substantially impermeable to an electric current. The barrier contains an opening that is smaller than the biological cell such that the biological cell once lodged in the opening will plug or close the opening. To achieve electroporation, the biological cell is secured over the opening by mechanical or chemical means, preferably in a reversible manner so that the biological cell can later be removed without damage to the biological cell. Once the biological cell is secured over the opening, a voltage is imposed between the two chambers and across the biological cell residing in the opening. The passage of current between the chambers is thus restricted to a path passing through the opening and hence through the biological cell. By monitoring the current-voltage relation in the electric cell, the onset of electroporation is detected and the degree of pore formation is controlled, to both assure that electroporation is occurring and to prevent excessive pore formation and cell death. The user is thus afforded a highly precise knowledge and control of the condition of and the flux across the biological cell membrane.
In another series of embodiments, this invention is useful in the diffusive transport of chemical species into or out of a biological cell. In these embodiments, the cell is again divided into two chambers separated by a barrier, and the biological cell is lodged across an opening in the barrier in such a manner that the passage of liquid around the cell from one chamber to the other is substantially prevented. A liquid solution of the species to be introduced into the biological cell is placed in one or both of the chambers. The concentration of the species in the solution differs from that in the cell (either higher or lower, depending on whether one seeks to introduce or remove the species from the cell), or the concentration in one chamber differs from that in the other chamber.
In preferred methods of applying this invention to diffusive transport, the solutions in the two chambers differ in concentration such that the driving force for the diffusive transport is between the two chambers themselves rather than between the chambers and the interior of the biological cell. Knowledge and controlled monitoring of the concentrations in each of the two chambers on a periodic or continuous basis as the diffusion proceeds, together with the precise knowledge of the dimensions of the opening, enables the user to precisely observe and control the rate and amount of the species that enters the cell. The diffusion time can be controlled by imposing stepwise changes in the concentrations in either or both of the chambers, thereby imposing or removing the concentration differential. An application of particular interest is the combination of this type of diffusive transport of a chemical species with controlled electroporation as described in the preceding paragraph.
Each of the various embodiments of this invention may be used with two or more biological cells simultaneously, or cell masses such as in tissue, rather than just one cell. The apparatus described above can be adapted for use with two or more biological cells by arranging the barrier such that the current or diffusive transport will be restricted to a flow path that passes through all of the cells while preventing bypass around the cells. A further application of the concepts of this invention is the electroporation of biological cells suspended in a flowing liquid. Electrodes are placed in fixed positions in the flow channel, and a voltage is imposed between the electrodes while current passing between the electrodes is monitored. Biological cells entering the region between the electrodes will lower the current, the impedance serving as an indication of the presence of one or more cells in the region, and optionally also as a signal to initiate the application of a higher voltage sufficient to achieve electroporation.
Among the advantages that this invention offers relative to the prior art are the ability to treat cells individually and to adapt the treatment conditions to the needs of individual cells. In embodiments where voltage is applied, the monitoring of the impedance affords the user knowledge of the presence or absence of pores and shows the progress of the pore formation and whether irreversible pore formation that might lead to cell death has occurred. An advantage of the barrier-and-opening apparatus is its highly efficient use of electrical energy by virtue of its restriction of the current to a current flow path passing through the opening. A still further advantage is the ability of the apparatus and method to be integrated into an automated system whereby the condition of each cell is monitored by instrumentation and individual cells are lodged in the opening and then removed at times governed by the monitored conditions.
These and further features, advantages and objects of the invention will be better understood from the description that follows.
FIG. 1 is a cross section of a microdiffusion device useful in the practice of the present invention for infusing a biological cell with a chemical species without the assistance of an electrical current to effect electroporation.
FIG. 2 is a cross section of a microelectroporation device useful in the practice of the present invention for achieving pore formation in a biological cell, and.
optionally for infusing the cell with a chemical species with the assistance of electroporation.
FIG. 3a is a longitudinal cross section of an electroporation device in accordance with this invention, designed for a mobile suspension of biological cells. FIG. 3b is a transverse cross section of the device shown in FIG. 3a.
FIG. 4 is a plot of current vs. voltage in a series of electroporation experiments conducted using a microelectroporation device of the structure similar to that of FIG. 2.
FIGS. 5a, 5b, 5c, and 5d are plots of current vs. voltage in a further series of electroporation experiments conducted using a microelectroporation device similar to that of FIG. 2.
While this invention extends to a variety of structures, methods, and applications, this portion of the specification will illustrate certain specific structures and methods in detail, from which the concepts of the invention as a whole will become apparent.
The first structure that will be discussed is an electroporation cell with an internal support to hold a single biological cell and an internal barrier that restricts the electric current flow in the electric cell to a flow path that passes through the biological cell. When no voltage is applied, the structure can be used for diffusive transport alone, unassisted by voltage-induced pore formation.
The configuration of the barrier, and the two chambers in embodiments that include two chambers, is not critical to the invention, and can vary widely while still serving the purposes and advantages of the invention. Since biological cells are microscopic in size, however, the preferred type of apparatus for the practice of this invention in each of its various forms is one in which the structure as a whole and/or its chambers are the size of electronic chips, fabricated by microfabrication techniques such as those used in electronic chip manufacture. It is further preferred that the chambers are constructed as flow-through chambers to allow the passage of the liquids in continuous flow, intermittent flow, or flow at the direction of the user, and to allow changes in the concentrations, pressure, and other conditions as needed to achieve close control over the passage of species across the biological cell membrane. Accordingly, a preferred structure and method of manufacture of the apparatus are those that involve the formation of the apparatus in layers or platelets with appropriate openings that form flow passages when the layers or platelets are bonded together.
Flow-through chambers offer the advantage of permitting the successive entry and removal of individual cells so that large numbers of cells can be treated in succession. Flow-through chambers also permit replenishment of solute-depleted solutions so that concentration gradients can be continuously maintained when desired. A further function that can be served by flow-through chambers is the increase and decrease of pressure, a function that is useful for various purposes as described below.
The support for the biological cell in this structure can be any structure that secures the biological cell in a fixed position and that allows the passage of electric current. The most convenient support is an opening in the barrier. Securement of a biological cell over the opening serves to close, seal or plug the opening, thereby directing the passage of electric current, diffusive transport, or both, through the cell and eliminating or minimizing leakage around the cell. A convenient mechanical means of achieving this is to impose a pressure differential across the opening in a direction that will press the cell against the opening. The diameter of the opening will be smaller than that of the cell, and the cell upon entering the apparatus will pass into one of the two chambers. By increasing the pressure in the chamber in which the cell resides, or lowering the pressure in the other chamber, the cell will be forced against the opening, closing it off. Once the procedure is completed, the cell is readily released from the opening by equalizing the pressures in the two chambers or by reversing the differential such that the higher pressure is in the chamber other than the chamber in which the cell was introduced. The flow of liquid in the chamber in which the cell was introduced will then remove the cell from the opening, exposing the opening for another cell.
An alternative method of sealing the opening with the cell is by the use of a coating on the barrier surface, or over the rim of the opening, of a substance that binds to the cell membrane. Since biological cell membranes are negatively charged, the coating may be a substance that bears a positive charge, such as polylysine, polyarginine, or polyhistidine. The biological cell can be directed to the opening by a pressure differential across the opening, and held in place by the coating. One the procedure is completed, the cell can be released from the coating by momentarily increasing the flow rate of the liquid in the chamber on the cell side of the opening, or by imposing a reverse pressure differential across the opening to urge the cell away from the opening.
The size of the opening is not critical to the invention provided that the opening exposes sufficient surface area on the cell membrane to achieve the desired degree of either mass transfer, the passage of an electric current, or both, within a controllable and economically reasonable period of time. The optimal size will thus vary with the particular cells being treated or studied. In general, the opening is preferably circular or approximately circular in shape, and depending on the cell size, preferably ranges in diameter from about 1 micron to about 100 microns, more preferably from about 1 micron to about 50 microns, and most preferably from about 2 microns to about 20 microns. The barrier in which the hole is formed and which separates the two chambers is preferably of a rigid dielectric material that is impermeable to both water and solutes and that will hold a pressure differential sufficient to secure a cell against the opening. For devices that are manufactured by microfabrication techniques, a convenient material for the barrier is silicon nitride. Other materials that will serve equally well will be readily apparent to those skilled in the art.
A further feature of preferred embodiments of this invention is the use of apparatus made of transparent materials. This enables the user to observe cell interiors and the processes of microdiffusion and microelectroporation through a microscope as they occur.
An example of a microdiffusion apparatus in accordance with this invention for a single biological cell, for transporting materials across the cell membrane without the application of an electric field, is shown in FIG. 1. This components of this apparatus, from the bottom up, are an acrylic base 11, an intermediate silicon layer 12 (1 micron in thickness) with a portion 13 carved out to define the lateral boundaries of the lower of the two liquid chambers, a silicon nitride layer 14 serving as the barrier between the two chambers, a silicon washer 15 defining the lateral boundaries of the upper liquid chamber 16, and a glass cover plate 17. A hole 18 in the silicon nitride barrier serves as the opening, and a cell or contiguous cell mass such as tissue 19 is shown covering the hole. Channels extend through the acrylic base to serve as inlet and outlet channels for the liquids that pass through the upper and lower chambers, as shown by the arrows in the Figure.
When the pressure in the upper chamber 16 is higher than that in the lower chamber 13, the cell will be retained in position over the hole, serving as a plug separating the liquids in the two chambers from each other. When the composition of the solutions in the two chambers differs from that of the cell interior, mass transfer occurs across the cell membrane between the chambers and the cell. When the composition of the solution in one chamber differs from that in the other, mass transfer occurs through the cell from one chamber to the other. By precisely controlling the compositions of the solutions in the two chambers, one can precisely control the mass transfer rate and direction within the cell. Since the diameter of the opening 18 is known, one can precisely determine the mass transfer that occurs through the opening.
The numerous applications of this microdiffusion device will be readily apparent. For example, the device can be used to infuse a cell with a cryopreservative such as glycerol by filling the upper chamber 16 with physiological saline and the tower chamber 13 with glycerol. When using a cell 19 for which the mass transfer coefficient of glycerol across the cell membrane is known, one can readily calculate the amount of glycerol that will enter the cell and adjust the concentrations and exposure times to infuse the cell with the amount that is known to be required for cryopreservation.
An example of a microelectroporation apparatus in accordance with this invention for a single biological cell, is shown in FIG. 2. The apparatus is similar in construction to the microdiffusion apparatus of FIG. 1. Its structural components, from the bottom up, are an acrylic base 21, a lower silicon layer 22 with a portion carved out to define the lateral boundaries of the lower liquid chamber 23, a silicon nitride layer 24 (1 micron in thickness) serving as the barrier between the two chambers, an upper silicon layer 25 defining the lateral boundaries of the upper liquid chamber 26, and a cover consisting of an n+ poly-silicon layer (5,000 Å in thickness) 27 and a silicon nitride layer (1 micron in thickness) 28. A hole 29 in the silicon nitride barrier 24 serves as the opening, and a cell 30 (or cell mass) covers the hole. Channels extend through the acrylic base to serve as inlets and outlets for the liquids that pass through the upper and lower chambers, as shown by the arrows in the Figure. A further layer of n+ poly-silicon (5,000 Å) 31 resides above the acrylic base 21, and this layer, together with n+ poly-silicon layer 27 above the upper chamber 26 serve as the two electrodes. Each electrode is joined by electric leads to a printed circuit board 32 which controls the voltage applied between the electrodes and measures the current passing between them.
The microelectroporation apparatus shown in FIG. 2 can be fabricated by conventional microfabrication techniques, typically involving chemical vapor deposition, masking, etching and sputtering. The operation of the apparatus will be analogous to the operation of the microdiffusion apparatus of FIG. 1. The movement of biological cells through the apparatus is achieved by suspending the cells in the liquid used to fill the upper chamber, and cells are drawn to the opening, one at a time, by imposing a pressure differential between the chambers, which also holds a cell in place once the cell has been drawn to the opening. A convenient method of imposing such a pressure differential is to maintain atmospheric pressure in the upper chamber while lowering the pressure in the lower chamber below atmospheric by attaching a syringe to the lower chamber and pulling on the syringe plunger. Care should be taken to limit the pressure differential to one that will not damage the cell.
FIGS. 3a and 3b illustrate to a different apparatus and method within the scope of this invention. This apparatus and method involve a fluid suspension of biological cells flowing through a conduit or flow channel, in which the cells pass through a region between a pair of electrodes. The longitudinal cross section of FIG. 3a shows the walls 41 of the channel, and a biological cell 42 passing downward through the lumen of the channel (in the direction of the arrow). The transverse cross section of FIG. 3b shows that the channel is rectangular in cross section, although other cross-sectional geometries may be used. Electrodes 43, 44 are formed as coatings on two opposing walls of the channel. The electrodes are connected through leads to a printed circuit board 45 which measures the impedance and controls the voltage applied to the electrodes. The biological cell 42 is shown passing through the region between the two electrodes.
The area of the cross section of the channel is large enough to permit the cell to pass through essentially unimpeded by the channel walls, and yet small enough that only one cell can pass through the inter-electrode region at a time. In addition, each electrode 43, 44 is either approximately equal in length or slightly larger in length than the diameter of the biological cell, so that the cell upon entering the region causes a significant or measurable decrease in the current passing through the region due to the voltage applied across electrodes. The spacing of the electrodes, i.e., the distance between them, is likewise subject to the same considerations. The biological cells are suspended in a liquid solution of the species to be introduced into the cells, and the suspension is passed through the channel. A voltage is applied between the electrodes as suspension flows through the channel, and the current between the electrodes (or the impedance) is monitored. A significant drop in the current indicates the presence of a biological cell in the inter-electrode region. Once the cell is detected in this manner, an electroporation pulse can be applied to the electrodes while the cell is still in the inter-electrode region, and impedance can be observed further to detect the onset of electroporation. The species dissolved in the liquid solution will enter the cell as a result of the electroporation.
Variations on these structures and methods will be readily apparent to those skilled in the art. For example, the barriers described above can be minimized or avoided by using microelectrodes that are the same size as or smaller than the biological cells. Examples of such microelectrodes are carbon fiber microelectrodes (such as ProCFE, Axon Instruments, Foster City, Calif., USA) used in conjunction with high-graduation micromanipulators (such as those available from Narishige MWH-3, Tokyo, Japan). Microelectrodes can be used in place of the electrodes shown in FIG. 2 or in place of those shown in FIGS. 3a and 3b.
The following examples are offered for illustration, and are not intended to impose limits on the scope of the invention.
A series of experiments was performed using a microelectroporation system consisting of the microelectroporation device described above and shown in FIG. 2, combined with flow and pressure control units and pressure gauges for the liquids to be circulated through the upper and lower chambers, a variable DC power supply, a pulse generator and power amplifier for imposing voltage pulses across the device, a digital oscilloscope for monitoring the pulses, a fluorescent microscope, a CCD (charge coupled device) camera, and a computer with image processing and waveform processing software. Both chambers of the device were filled with physiological saline and cells were introduced into the upper chamber. Liquid motion in the top and bottom chambers was controlled by syringes. The pressure in the upper chamber was atmospheric while the pressure in the lower chamber was reduced below atmospheric by pulling on the barrel of the syringe connected to that chamber. The voltage was applied in single square pulses ranging from zero to 120V in magnitude and from 2 microseconds to 100 milliseconds in duration. The distance between the electrodes in the upper and lower chambers was 900 microns.
The tests in this example were performed using ND-1 human prostate adenocarcinoma cells with a typical diameter of 20 microns. The opening in the microelectroporation device was 5 microns in diameter. A rectangular voltage pulse was applied with a duration of 60 milliseconds, and the pulse was applied at various amplitudes ranging from 10V to 60V in increments of 5 volts. With each pulse, the electric current passing through the opening was measured. Experiments were performed with the cells and were repeated both with the opening stopped by a glass bead and with no obstruction at all in the opening. The results in each case were expressed as microamperes of current vs. volts of pulse amplitude and are plotted in FIG. 4, in which the upper curve (data points represented by x's) represents the unobstructed opening, the lower curve (data points represented by asterisks) represents the data taken with the glass bead residing in the opening, and the three middle curves (open squares, open upright triangles, and open inverted triangles) represent data taken with three different ND-1 cells residing in the opening.
The upper curve shows that the current increases in a substantially steady manner as the voltage increases when there is no barrier to the passage of current through the opening. The lower curve also shows a substantially steady rise as the voltage increases, although at a much lower level. The current values shown in the lower curve represent stray currents through the device. The curves of data taken with the ND-1 cells across the opening show that at low voltages the current is close in value to that obtained when the opening is closed by the glass bead while at high voltages the current rises to the levels obtained with an unobstructed opening. The transition is a sharp increase which is indicative of the formation of pores in the cell membrane through which an electric current can pass, i.e., the onset of electroporation. In all three cells, the transition occurred at voltages between 30V and 40V. In two of the three cells (open squares and open upright triangles), the onset of electroporation occurred essentially at the same voltage, while in the third (inverted triangles), the onset occurred at a voltage that was lower than the other two by about 5V. This illustrates the value of controlling the process for individual cells to achieve optimal results.
After the data shown in FIG. 4 was generated, the pulses were reapplied in descending order of amplitude values; and the resulting curves displayed hysteresis, i.e., the curves obtained with descending amplitudes were higher in voltage than those obtained with ascending amplitudes. This indicated that the electroporation in these experiments was irreversible.
Using the same microelectroporation system used in Example 1, a series of tests were performed on rat hepatocytes (ATCC #CRL-1439), whose typical cell diameter was 20 microns, the microelectroporation apparatus having an opening that was 4 microns in diameter. Here as well, rectangular voltage pulses that were 60 milliseconds in duration were used, ranging in amplitude from 10V to 37.5V in increments of 5V in the portion from 10V to 30V and in increments of 2.5V in the portion from 30V to 37.5V. The experiments were performed in some cases only by increasing the amplitudes and in others by first increasing, then decreasing the amplitudes to evaluate reversibility. The results are plotted in the graphs shown in FIGS. 5a, 5b, 5c, and 5d. In each case, the upper curve (data points represented by circles) is the data taken with neither a cell nor a glass bead residing in the opening, the lower curve (data points represented by squares) is the data taken with a glass bead in the opening, and the middle curve (data points represented by triangles) is the data taken with a hepatocyte in the opening, using different hepatocytes for each or the four Figures.
In FIG. 5a, the amplitude was increased and not decreased, displaying an electroporation threshold voltage of between 25V and 30V. In FIGS. 5b and 5c, the amplitude was first increased and then decreased to produce the two middle curves. Although the ascending and descending curves are not differentiated, they are substantially identical in each Figure, indicating that the cell membrane in each of these two cases resealed after each voltage pulse and thus that the pore formation was reversible. In the test represented by FIG. 5d, the cell disintegrated once the applied voltage exceeded 37.5V, although this is not shown in the Figure. It is significant to note that despite the fact that the same cell types were used in each of FIGS. 3a, 5b, 5c, and 5d, the electroporation threshold voltage differed among the individual cells, although all were within the range of 20V to 35V. Adaptation of the procedure to individual cells is readily achieved by monitoring the current in this manner to note when the electroporation threshold occurs. Selection of the optimal exposure time, voltage, composition changes in the surrounding liquids, and other parameters of the system can then be made to achieve the desired treatment of the cell without destruction of the cell.
The methods described herein are useful tools in the laboratory for conducting fundamental research in the electroporation properties of biological cells, and useful tools in industry for processing large quantities of cells in a flow-through manner. By enabling one to observe and record the current flowing through individual cells, one can control the amplitude and duration of the voltage pulse to achieve optimal results. In addition, the devices described and shown herein for use in, practicing the invention can be constructed with transparent parts and of a size suitable for mounting on a microscope stage. This will permit one to correlate the electrical current measurements to visual observations and fluorescence measurements inside the cell. The device can be used to electrically detect, through the measurement of currents, the point in time when a cell becomes lodged in the opening as well as the point in time when pore formation is achieved in the cell membrane. For larger scale and industrial applications, large numbers of microelectroporation devices of the type described herein can be arranged in parallel. For each cell, electrical information indicating the trapping of a cell in the opening (such as a sharp drop in the current) can be used to generate a signal that will initiate an electroporation sequence, and further electrical information indicating the completion of electroporation (such as a sharp rise in current) will generate a signal that will release the cell (for example by eliminating or reversing the pressure differential) and permit the next cell to flow toward the opening.
Further implementations, applications, adaptations, and embodiments of the concepts, features and methods described herein that are within the scope of this invention will be readily apparent to those skilled in the art.


text:
On Wednesday, the New York Jets schedule was released for the 2014 season along with the rest of the NFL.
The Jets' schedule includes three primetime games, consisting of Monday Night Games against the Chicago Bears and Miami Dolphins, along with a Thursday night game against the New England Patriots. Aside from those, some other marquee match-ups include a game against the Titans, a game where Chris Johnson will play his former team for the first time, and a game against the Detroit Lions in which young Dee Milliner will be pitted against Calvin Johnson in a David vs. Goliath type matchup.
Note that the Jets' Week 17 game is against the division rival Dolphins, so if they are fighting for a playoff spot a win against Miami will likely be a must.
And with that, here is the New York Jets' schedule for the 2014 season:
**Note that home games are italicized and primetime games are in bold.
Week 1: New York Jets vs. Oakland Raiders 1:00
Week 2: New York Jets @ Green Bay Packers 4:25
Week 3: New York Jets vs. Chicago Bears 8:30 (Monday)
Week 4: New York Jets vs. Detroit Lions 1:00
Week 5: New York Jets @ San Diego Chargers 4:25
Week 6: New York Jets vs. Denver Broncos 1:00
Week 7: New York Jets @ New England Patriots 8:25 (Thursday)
Week 8: New York Jets vs. Buffalo Bills 1:00
Week 9: New York Jets @ Kansas City Chiefs 1:00
Week 10: New York Jets vs. Pittsburgh Steelers 1:00
Week 11: BYE WEEK
Week 12: New York Jets @ Buffalo Bills 1:00
Week 13: New York Jets vs. Miami Dolphins 8:30 (Monday)
Week 14: New York Jets @ Minnesota Vikings 1:00
Week 15: New York Jets @ Tennessee Titans 4:05
Week 16: New York Jets vs. New England Patriots 1:00
Week 17: New York Jets @ Miami Dolphins 1:00
Tags: New York Jets Nfl Schedule


text: HE Jan. I really didn't think that I would be the one to say this but Middleford is not by any means getting it together the split is worse than ever. Gilli is on a rampage with several other people at Burkhaven. The rest are against them and others are in the middle and getting hit by both ends. The seneschal has no control at populas meetings. He needs to control his wife. She is starting to get out of hand against Gilli. You should here some of the stuff she wanted to do on the list to gilli. and vice versa. Estaban's posting was totally out of line and it had nothing to do with burkhaven. She did this little event all her own and Gilli offered his place is all. The garb thing at the last populas was also another misunderstanding. Liam had his hand over his mouth and I didn't even here the answer like I said gilli asked again and Moira blasted him for nothing and then Louise got into it. Like I said There is no cohesion within the group what so ever. The preasure is building again and there is going to be another eruption like in March. This is from standing in the back and watching. We need help! HL Keyna


text:
October 23 2014 Latest news:
Jon Dean
Saturday, November 17, 2012
A man thought to have burgled a house during last summers riots is wanted by police.
Daniel Malinowski, 29, who also goes by the alias Aleks Sudej, is wanted for aggravated burglary in Enfield and failing to appear at Enfield Magistrates Court for common assault.
Anyone with any information can contact DC Simon Johnston of Operation Withern on 020 7275 3305 or email: firstname.lastname@example.com quoting reference 2325619/11.
0 comments


text: We've discussed using clay pots before. This new book might be of interest to some of us, It's by Paula Wolfert who has written extensively on Mediterranean foods and cookery. It's titled Mediterranean Clay Pot Cooking: Traditional and Modern Recipes to Savor and Share. "Wolfert shares her inimitable passion for detail and insatiable curiosity about cultural traditions and innovations, with Mediterranean Clay Pot Cooking. Here, the self-confessed clay pot "junkie"-having collected in her travels ceramic pots of all sorts: cazuelas, tagines, baking dishes, bean pots, Romertopf baking dishes, French diablos, ordinary casseroles, even Crockpots, which have a ceramic liner-shares recipes as vibrant as the Mediterranean itself along with the delightful stories behind the earthy pots, irresistible dishes, and outstanding cooks she has met along the way." Includes a A Clay Pot Primer. http://www.wiley.com/WileyCDA/WileyTitle/productCd-076457633X.html The website has some recipes in the ,pdf sections of the site. Browse all the pages. Johnnae


text:
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text:
Library Staff Resources
Happy Holidays and Happy New Year from NC LIVE!
NC LIVE is based at NC State University Libraries. NC State University is closed from December 24th-January 1st. The NC LIVE Help Desk will have limited hours on December 26th, 27th, and 30th to assist member library staff who encounter technical issues.
NC LIVE is happy to announce that Gale InfoTrac Newsstand is now available to all member libraries.
Be the first in your library to learn more about NC LIVE's two newest resources: Gale InfoTrac Newsstand and Mergent Intellect!
Mergent Intellect webinars for NC LIVE staff: learn about the features of the full version if Intellect, coming to NC LIVE member libraries beginning January 1, 2014:
Are you interested in learning more about data-driven decision-making in libraries? In April 2012, NC LIVE began a benchmarking study of electronic resource usage among member libraries. On Wednesday, December 11th from 3:00-4:00 PM NC LIVE staff will be presenting on the project for the Library Journal webinar "The Evolution of Usage: Analyzing and Benchmarking Use," a part of the "Data-Driven Academic Libraries" webcast series.
You can register for the free webinar to watch live on Wednesday, or to receive access to the archived webinar for on-demand viewing at your convenience.
NC LIVE experienced a technical issue in a mail handler update performed at 10:00am today. This issue has since been resolved. This issue may have disrupted messages to firstname.lastname@example.com, email@example.com or submitted through the NC LIVE Help Desk form between 10:00am and 1:15pm today (Tuesday, December 3rd).
If you submitted a Help Desk form or sent an email to firstname.lastname@example.com or firstname.lastname@example.com between 10:00am and 1:15pm today, please re-send the message or call the NC LIVE Help Desk at 919-513-0451. We apologize for the inconvenience.
NC LIVE will be closed Thursday, November 28 and Friday, November 29 for the Thanksgiving holiday.
Happy Thanksgiving!
At its recent meeting on November 15, 2013, the NC LIVE Librarians Council voted to approve recommendations of our Resources Advisory Committee to make a few changes to the content line-up. As of January 1, 2014, the consortium will be offering Gale's Infotrac Newsstand and Mergent's Intellect products.
Beginning in August 2013, NC LIVE usage reports may show large increases for Searches and Sessions among libraries that use the NC LIVE website. These increases are related to the new search boxes on nclive.org, which are powered by EBSCO Discovery Service (EDS). NC LIVE started using the EDS search boxes in late July 2013. When discovery services are implemented, usage reports often show an increase in Searches and Sessions because many resources are being automatically searched each time the search box is used. If you have any questions about your library's usage reports, please contact the NC LIVE Help Desk.
Downloaded eBooks borrowed from the eBooks on EBSCOhost collection can now be returned before the checkout period has expired. Returning early will make the eBook available to other users. Patrons can return the eBooks within the same software they used to download the eBook-- Adobe Digital Editions or the Bluefire Reader app.
The directions for returning eBooks can be found in this FAQ, and are included below.
To return an eBook from Adobe Digital Editions 2.0 before the checkout period has expired:
On November 15, 2013, EBSCO is scheduled to complete the final phase of a systems upgrade that will result in changes to the domain name for some areas of EBSCOhost. The intent is to provide additional access points to improve response times for EBSCOhost users. EBSCO has distributed their recommended settings and libraries using proxy servers should update their settings accordingly.
For libraries using EZproxy, please check to ensure that your settings match those outlined here for EBSCO: http://nclive.org/librarystaff/proxyinfo
If your library uses a proxy authentication method other than EZproxy, please check with your local IT department or proxy provider to ensure that the recommended settings are in place.
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text:
So I know you're going pretty soon
Radiation sore throat got your tongue
Magic markers tattoo you and show it where to aim
And strangers break their promises
"You won't feel any, you won't feel any pain…"
Dead of Winter by Eels
Everett's father (lyricist for Eels), Hugh, died in 1982. His sister, Elizabeth, long troubled by schizophrenia, committed suicide in 1996, and in 1998 his mother, Nancy Everett née Gore, died of lung cancer. Following these tragedies, Everett and the Eels released Electro-Shock Blues in 1998. The release, whose sound alternates between furious indefatigableness, black humor, and strikingly authentic desperation, gained almost unanimous critical praise.
This particular song is obviously about Everett's mother. Radiation causes the person who receives it (if around the face/throat area) to have an extremely sore throat, to the point that they can't talk and everything tastes like metal. Doctors use magic markers to pin-point where to aim the radiation when they do the first radiation session.
Electro-Shock Blues is my favorite album of all time. If you ever listen to it, and you don't even feel a twinge of sadness, then fuck you. You aren't human.
My heart goes out to Everett. I'm so glad that he uses his sadness and morbid past to create beautiful music. Mr. E is a gorgeous writer with terrible inspriation. It'd be very hard to try to convey these things if you hadn't gone through some of the same shit.
It makes you wonder what poetry or lyrics hasn't he published that he's holding on to? Is there something that's too personal for him to share? What songs didn't they record and produce?
9 Notes/ Hide
- ignoble-crook reblogged this from pre-postmortem
- nettlemind reblogged this from pre-postmortem and added:
Dead of winter by Eels
- foreverficticious reblogged this from pre-postmortem
- perturbed reblogged this from pre-postmortem
- bozly likes this
- blushingsiren reblogged this from pre-postmortem
- whatihavetooffer reblogged this from pre-postmortem
- okfuckit reblogged this from pre-postmortem and added:
eels- dead of winter
- pre-postmortem posted this


text:
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text:
"I would rather have a mind opened by wonder than one closed by belief.
Find a translation for this quote in other languages:
Select another language:
Discuss this Gerry Spence quote with the community:
Use the citation below to add this quote to your bibliography:
"Gerry Spence Quotes." Quotes.net. STANDS4 LLC, 2014. Web. 24 Oct. 2014. <http://www.quotes.net/quote/21429>.


text:
This Tumblr has been sitting here for quite some time just gathering dust. I think it's been close to a year since I was truly active.
At first I was hoping things would get better, and then I was stalling and avoiding change. I didn't want to make any rash decisions sprung from stress.
There's a new game coming out. That's very exciting, but it hasn't really been any inspiration to me. I realized recently that I just can't picture myself coming back to this variation of the character.
So, I'm going to close up shop.
You have been supportive during the hard times, and I appreciate that. I wish you all well.


text:
Machu Picchu!
Trip started with a fancy train ride to Aguas Calientes. I visited the hot springs there to relax a little. Then the next morning I got up at 4:30 to catch the first bus up to Machu Picchu.
Upon arrival, the ruins were covered in clouds, but by 10am the clouds had dissipated and the anticipation was well worth it. Pictures cannot even remotely capture it.
The most interesting thing I learned today at Machu Picchu: The Incans had public restrooms and dealt with human waste kind of like how we deal with our cats. They would take the ashes from the fires used to cook their food and put them in the designated potty area. Anyone who needed to use the bathroom would dig themselves a little hole, do their business, and then cover it up again. Every once in a while, someone would have the unfortunate duty of having to go through that shit (ha) with what I assume was probably a human sized pooper scooper. They would then use the excrement as an offering to the pachamama or mother earth (in other words their food was fertilized using their own shit).
These people were brilliant.


text:
Definition of disadvantageous in English:
disadvantageousLine breaks: dis|ad¦van|ta¦geous
Pronunciation: /ˌdɪsadv(ə)nˈteɪdʒəs /
Involving or creating unfavourable circumstances that reduce the chances of success or effectiveness: the new employment scheme is disadvantageous to women
More example sentences
- Michael had nearly been killed in that battle, possibly because of the disadvantageous circumstances under which the battle was fought.
- Reduced fruit set after a hurricane could be especially disadvantageous because hurricanes can create sites for plant recruitment.
- However, they have long been in a disadvantageous and vulnerable position due to their status and restrictive regulations.
detrimental, prejudicial, deleterious, harmful, damaging, injurious, hurtful, destructive;
inopportune, ill-timed, untimely, inexpedient
disadvantageously adverb
- More example sentences
- This is discriminatory: to discriminate against members of a group of people just is to treat them disadvantageously compared to members of other groups, when there's no adequate justification for doing so.
- But we had no evidence that a scheme like that described in clause 3, 'Overview', will actually change anything in New Zealand advantageously, and there is every likelihood that it will change things disadvantageously.
- The plight of gallant, Catholic little Belgium, invaded by a militaristic aggressor, was disadvantageously compared with Ireland, achieving freedom (so Redmond argued) within the British Empire, rather like Canada or Australia.
Definition of disadvantageous in:
- The US English dictionary
- The English Synonyms
- The US English Synonyms
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Word of the day oleaginous
Pronunciation: ˌəʊlɪˈadʒɪnəs
rich in, covered with, or producing oil; oily


text:
main index
This is the tendency for space states not to have an actual identification beyond a generic term for a given political system. Often this is specified a little by equally generic terms such as "of planets". But there is no identity with any local culture. Writers can justify this by explaining that past conquerors decided that it was bad politics to hurt their subjects' feelings more than necessary. This is partly a result of Good Republic, Evil Empire: The Republic or The Kingdom are generic goodness, empires are simply evil, and that's all that needs to be said.
Examples:Anime and Manga


text: Lady Catrin The site for Kings College is at TCU where Round Table was held last July. Fiona -------------------------------------------------- From: "Kathy" <dkv250 at sbcglobal.net> Sent: Tuesday, May 17, 2011 6:17 AM To: "Local SCA" <Loch-Ruadh at lists.ansteorra.org> Subject: [Loch-Ruadh] King's College help > Greetings one and all from your friendly neighborhood A&S Minister, > > As you know we are hosting King's College next month at UT Arlington. In > preparation for that I am asking for volunteers to assist our scheduled > Teachers/Trainers in hauling and toting their equipment and supplies for > their classes. > If you will be available to be on site 30 minutes early and/or stay after > by the same and are willing to assist in this manner, please let me know. > In most cases this won't require much heavy lifting, just helping with > bags, boxes, and stacks of the various supplies needed by Teachers to use > in their demonstrations or handouts for lectures. > This is a great way to meet others in the Kingdom and help out the Shire > at the same time. > > > > > > Catrin > Loch Ruadh MoAS > _______________________________________________ > Loch-Ruadh mailing list > Loch-Ruadh at lists.ansteorra.org > http://lists.ansteorra.org/listinfo.cgi/loch-ruadh-ansteorra.org


text: There has been an increase in crime on the Georgia Tech campus these past few months, including sexual assaults and rape. There's an interesting thread on the Tech subreddit about trying to make the campus a safer place. I found this image in one of the comments, the idea being for society to stop teaching people not to get raped, and instead to teach people not to rape.


text:
© Depositphotos.com/ Aquir014b
Bangkok| 8 year old Mai Li Fay, from Bangkok, is far from living the usual life of a girl her age. Everyday, thousands of people gather to the front of her family's house to get a chance of seeing and possibly touching the young girl, a gesture which both buddhists and hindu pilgrims seem to perceive as a garantee of good fortune.
According to the country's top medical expert, Dr Ping Lao, the young girl suffers a very rare syndrome known as the Serpentosis Malianorcis or Jing Jing's disease, which gives her lower body a distinct reptilian form and aspect. Only a handful of such cases have been recorded throughout history, so the scientific knowledge accumulated about the pathology is rather limited and there is no cure in sight at the moment.
The Fay family have welcomed the situation brought by Mai Li's situation with a mix of anguish and excitement, as the never ending lines of tourists and pilgrims have brought the badly impoverished family some substantial revenues and given them an important raise in their social status but cost them there privacy and intimacy.
According to the report, "snake girl", Mai Li Fay from Bangkok, Thailand, suffers from a very rare condition called "Serpentosis Malianorcis" or Jing Jing's disease. The report claims that one Dr Ping Lao, who is billed as Thailand's top medical expert, diagnosed the rare syndrome, which gives the lower body a "distinct reptilian form and aspect".
Supposedly, the girl has become a major attraction with thousands of pilgrims and tourists gathering outside her house each day.
But, of course, the claims in the report are nonsense and the picture is clearly the result of digital manipulation. I would have hoped that common sense alone would have been enough for people to recognize that the image is just a silly hoax. But, apparently, "common" sense is misnamed. Reports and submissions suggest that quite a few people around the world do actually believe the story. And, in fact, the story has even been picked up by some fringe news sources.
There are no credible medical reports about a condition known as "Serpentosis Malianorcis". Nor are there any believable references to the supposedly renowned Dr Ping Lao.
The article and image originates from the World News Daily Report website, a publication that specializes in churning out unmitigated nonsense. The site features all manner of odd tales, including a horse with a duck's head, gigantic home invading lobsters, and a circus lion who has shamefully confessed to being a vegetarian. In fact, all of the articles on the site are fictional are not meant to be taken seriously. They are clearly intended to entertain and amuse.
Last updated: January 13, 2014
Thailand: Snakegirl Attracts Crowds of Pilgrims and Tourists
World News Daily Report


text:
|22 Mar 2013
Windows 7 premium Home edition
Picture but no Sound through HDMI and No HDMI icon in sound settings
Had to replace motherboard from authorized acer parts dealer now I get no sound through HDMI port. I did through the original board until it got fried by electrical surge. Can any one help or send me a picture of the inside the same model to make sure all jumpers on the motherboard are in the right spot. Or if any one else had this problem tell me how you fix it. It does not have HDMI sound icon in the mixer. I do have a picture.
What I have done:
1. I have looked for disabled devices.
2. Tried looking in sound mixer settings for the default audio.
3. New install and drivers.
4. Tried a system utility software RegInOut. Looking for serious replies on fix even if it was not an Acer.
AMD Athlon II X4 Quad-Core Processor 640,
Windows 7 Home Premium,
NVIDIA GeForce 9200 Chipset, NVIDIA GeForce 9200 Graphics Integrated ,
4096MB DDR3 Memory,
1TB SATA Hard Drive (Green Product variable RPM),
16X DVD±R/RW SuperMulti Drive,
Multi-in-1 Digital Media Card Reader,
802.11b/g/n Wireless,
High Definition Audio with 5.1-Channel Audio Support,
Any help appreciated
|My System Specs
|Thread Tools
|Similar help and support threads for2: Picture but no Sound through HDMI and No HDMI icon in sound settings
|Sound Control panel takes 2 minutes to open; HDMI sound inconsistent
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text: R29's Fash Cab Takes On Some Of Fashion's Big Shots 2 comments Sep 13, 2013 12:15 PM Rachel Besser 107 SHARES While P'Trique had us barreled over laughing in a memorable Fash Cab debut (in which his answers were quite creative but not very correct), we knew it was time to test more insiders to see how they'd fare. So, we took a joyride with some of fashion's best, including executive editor at Harper's Bazaar, Laura Brown, and fashion market director at Cosmopolitan, Shiona Turini. Watch the video and see the pros get it done (kind of). SHARE THIS STORY TWEET THIS STORY TV / Refinery29 Fash Cab - Laura Brown Back to header Back to navigation Back to footer


text:
Ross, to his son Rhys, 4 years old
Marcella, to her daughter Pearl
Pearl gets a big kick out of rubbing the cats bellies and looking for the nipples. So she sits there petting them and then shouts "I found a nipple!!" And then a minute goes by and again "I found a nipple!" She was doing this when she was supposed to be cleaning up her room before her bath.
To Cole, 8:14am
He is excited about his school's pajama day.
I'm just saying, we don't usually sleep in our clothes and then wear them to school the next day.
To Olivia, 7:56am


text:
StarShooter is a Shoot 'em up game, send your comments. "This free app is ad supported by advertisement." "Sponsored by AppGovideo.com (http://www.appgovideo.com/)" Ships (...)
Link To This App At AndroidPIT
Thank you for your interest in putting a link to this app at AndroidPIT on your website. The HTML code below creates a button that will lead your visitors directly to this app's details page.
Option 1 (green button):
Option 2 (black button):


text:
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text:
All Items > Knives > Butterfly Knives > Under $16
Bravado Blackwood Balisong Butterfly Knife - Satin Plain
- Overall Length: 8.875"
- Blade Length: 4.10"
- Cutting Edge: 3.75"
- Blade Thickness: 0.11"
- Blade Material: Stainless Steel
- Blade Finish: Satin
- Edge Type: Plain
- Handle Length: 4.80"
- Handle Material: Blackwood
- Handle Color: Black
- Handle Thickness: 0.36"
- Weight: 4.60 oz.
- Knife Type: Butterfly
- Best Use: Recreational
The Bravado butterfly knife has a satin finished 440 stainless steel clip point blade with swedge and blood groove channel. The handles feature steel liners with stainless bolsters and blackwood onlays. The Bravado knife has Torx screws for easy adjustment.
Please Note: As with all butterfly knives, please be sure to maintain and tighten the screws, especially the pivot screws. We recommend using a threadlocker (Click HERE!) for best results. Adjust with care, we do not warrant stripped screws!
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text:
Intro-Tech Designer Floor Mats 1038-4-CA-112
- Our Price: $104.69
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- Part Number: 1038-4-CA-112
$104.69Free Shipping
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Ships between 11/11/14 - 11/14/14
Intro-Tech 1038-4-CA-112 fits:
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