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Jiddu Krishnamurti, (born 1895, Madanapalle, India—died 1986, Ojai, Calif., U.S.), Indian spiritual leader. He was educated in theosophy by the British social reformer Annie Besant, who proclaimed him the coming "World Teacher," a messianic figure who would bring about world enlightenment. He became a teacher and writer, and from the 1920s he spent much time in the United States and Europe. In 1929 he broke with the Theosophical Society, the international religious organization run by Besant, and renounced any claims to being the World Teacher. Proclaiming the necessity of total spiritual freedom (even from teachers) through unflinching self-awareness, he continued to be a popular lecturer. He established Krishnamurti foundations in the United States, Britain, India, Spain, and Canada to further his aims. His books include The Songs of Life (1931) and Commentaries on Living (1956–60).
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The past two weeks, nearly 60 Chargers players have attended the team's offseason workouts.
Beginning Monday, they'll have new company.
Coaches will be allowed on the practice field for the first time, instructing players and running drills. Neither helmets nor contact is permitted.
Under the NFL's new collective bargaining agreement (CBA), a team's nine-week offseason workout program is divided into three phases. This is the second.
In the first phase, only the strength and conditioning staff could observe or participate in activities. Coaches were limited to player meetings afterward.
"We've been getting things done, but it certainly turns the notch up a little bit," quarterback Philip Rivers said. "It takes the teaching from the classroom, and you're able to go out and slowly build. I think that's the biggest thing. We're not going to win a game on Monday, but it just continues to build."
Phase Two, as it's called, will last three weeks.
Player attendance remains voluntary, and on-field workouts can include offensive, defensive and special teams drills.
No direct competition, however, is possible. According to the CBA, the offense can't drill against the defense, and vice versa. On special teams, the Chargers can practice kickoff returns, for example, but not against a coverage team.
The Chargers can run "perfect play."
In the common football drill, an offense or defense will execute a specific play, testing each player on his assignment and technique.
"Offensively, we've got some new guys," Rivers said. "Speaking to our wide receivers, (tight end Dante) Rosario, guys on the offensive line, I know they're eager to get it down. They want to be where they need to be, but it's going to be gradual — just keep chipping away, day by day.
"But certainly Monday, it'll be a little change of pace. We've had two weeks where you know what to expect. Now we change up the pace a bit, and then three weeks from now, we get to go into OTAs. I think the schedule sets up well."
Getting acclimated
In 2012, wide receiver will look widely different.
Vincent Jackson, Patrick Crayton and Bryan Walters are out. Via free agency, Robert Meachem, Eddie Royal, Micheal Spurlock and Roscoe Parrish are in, joining veterans Malcom Floyd, Vincent Brown and Richard Goodman.
With four new wide receivers on the roster, not to mention newcomer tight end Rosario, Rivers says he's been throwing the ball around "quite a bit in the past two weeks."
"It's been good," Rivers said. "We're learning together, and that's the biggest thing: getting to know each other and grow each day. It could be little by little. There's not going to be that day where it's, 'Oh! We got it!' It's little by little, and so far, it's been good."
The Chargers also drafted a tight end, Ladarius Green of Louisiana-Lafayette, in the fourth round last month. Rookie minicamp will begin Friday.

subset: commoncrawl


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This Thursday, the Sun-Ray Cinema in Five Points is marking the opening of "Seven Psychopaths," a dark comedy, with a day's worth of movies featuring some of those titular psychopaths, in earlier roles.
The day is called "Playin' Hookie with Sun-Ray," and it looks a fine way to spend an otherwise sleepy Thursday.
So check out "Woody Harrelson in "A Scanner Darkly" (2 p.m.).; Christopher Walken in "The Dead Zone" (4 pm.); Colin Farrell in "In Bruges" (6 p.m.); Sam Rockwell in "Moon" (8 p.m.); and Tom Waits in "Down By Law" (10 p.m.).
That leads up to the first showing of "Psychopaths" (from the director of "In Bruges") at 11:55 p.m.
Tickets are $20 for all the films that play before "Psycopaths," whose ticket price is not included. Or you can separate tickets for any of them. "Seven Psychopaths," by the way, has been getting rave reviews, sitting at 96 percent favorable at Rotten Tomatoes as this is written.


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Acta Cryst. (2013). B69, 288-293 [ https://doi.org/10.1107/S2052519213008713 ]
Abstract: The absolute structures of R-(-)-2-methylpiperazine (rmpip), S-(+)-2-methylpiperazine (smpip), R-(-)-2-methylpiperazinediium dibromide (rmpipBr) and S-(+)-2-methylpiperazinediium dibromide (smpipBr) have been determined by anomalous dispersion employing the Parsons' Q and Hooft methods. The studies were undertaken to determine the limitations of the absolute structure determination of light element structures (C, H, N) employing routine single-crystal X-ray diffraction laboratory conditions. The structures of the neutral methylpiperazines were known from a priori non-crystallographic methods and were confirmed by the absolute structure determination of their dibromide salts. By employing the full data collection of Bijvoet pairs and minimizing systematic errors, the absolute structure parameters 0.09 (8) (Hooft) for R-(-)-2-methylpiperazine and 0.05 (8) (Hooft) for S-(+)-2-methylpiperazine were determined.
Keywords: absolute structure; Flack method; Parsons' Q method; Hooft method; anomalous dispersion.
Structure factor file (CIF format) (53.5 kbytes)
Structure factor file (CIF format) (54.7 kbytes)
Structure factor file (CIF format) (84.8 kbytes)
Structure factor file (CIF format) (83.2 kbytes)
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"The book includes a description of the main features found in English castles during the 13th century, some of the main castles, and a history of the major events they were involved... The illustrations cover several selected castles and sections of them with cutaways... The reader may find parts he is familiar with, or details he is not interested in, but the book makes a great compact reference for castles and their relationship to this part of English history." -JE Kaufmann, Site-O / siteo.net (January 2010)
"This book provides a clear, well written description of the varied characteristics of castles in the 13th century, with special attention to the Tower of London, which, while predating this period, was extensively remodeled during this time, as were many earlier castles... This interesting, informative book is recommended to anyone wishing a short introduction to English castle building near its peak." -Bolling Smith, Coast Defense Journal (March 2010)


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IT IS desperate times for the Blackbird Leys Choir. Rather than singing hallelujah, it is more likely to be delivering a dirge as it finds its numbers dwindling.
It will be a sorry affair if this wonderful group has to disband.
It has a great reputation, having been part of the Singing Estate television show and members even performing in front of the Queen at Buckingham Palace.
With this and other TV shows such as The Choir and Glee raising the profile of singing, there must be plenty of people out there with a song in their heart.
Singing has reportedly been proven to improve you mental health.
Experts have claimed that joining a choir could improve symptoms of Parkinson's, depression and even lung disease.
It is a great way to meet other people in the area and is a wonderful social exercise.
People need to find just two hours a week to help prevent this choir from being silenced.
As with most activities, those who give it a try might just fall in love with it.
And The Leys will be alive with the sound of music again.
So make a song and dance about the choir and let us help them to keep hitting the high notes.


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Mahoning Valley under tornado watch until midnight tonight; high wind advisory until 7 a.m. Monday
Staff report
The National Weather Service has issued a tornado watch for Mahoning, Trumbull and Columbiana counties as well as much of Ohio, until midnight tonight.
A wind advisory remains in effect until 7 a.m. in the Mahoning Valley.
A tornado watch means weather conditions are favorable for the development of severe thunderstorms that are capable of producing tornadoes.
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Get a FREE loom recommendation!
Not sure which Mirrix is right for you? We offer free, personalized loom recommendations to ensure you find the perfect loom for your needs.
On The Blog
Weave-Along 25: The Rosalie Bracelet
The Weaving Meditation Challenge
Why Choose a Loom with a Tensioning Device?
Warp tension, as every tapestry or bead weaver knows, is one of the most important factors in any weaving. Both the amount of tension and the evenness of tension is important and the difference between good tension and bad tension can make or break a piece. A few weeks ago a customer emailed me about a class she took where students had to use the looms provided. To warp, pieces of warp were cut and each thread was tied individually. She ended up quitting the class. Her explanation of her experience has stuck with me and I keep thinking about the difference that having a loom with a tensioning device built in (like a Mirrix) makes for bead or weft-faced/tapestry weaving.
I don't have a loom where you need to tie individual warp threads, but I do have a basic frame that I made a few years ago that I warped for tapestry alongside a Mirrix to test see what difference an on-loom tensioning device makes.
Choose a smaller Mirrix if you're looking for a loom that is light, portable and able to be used on a lap or on a table.
The 5" Mini Mirrix Loreli LoomLearn More
8" Lani LoomLearn More
12" Little Guy LoomLearn More
Select a larger Mirrix if you're looking to make bigger pieces or several smaller pieces at the same time.
16" Big Sister LoomLearn More
22" Zach LoomLearn More
28" McKinley LoomLearn More
32" Joni LoomLearn More
38" Zeus LoomLearn More
"I am so happy with my 16″ loom. Anyone who has trouble with tension on other loom, this is the one for you. The first time I loomed, I made a bracelet. When I took it off the loom, it looked perfect. The Mirrix Loom is the most wonderful invention."
"The Mirrix tapestry loom combines the simple elegance of a portable frame with a modern invention. Its clever solutions for warping, set control, and shedding are logical and precise. A graceful synthesis of traditional weaving methodologies, the Mirrix tapestry loom is itself an example of fine craftsmanship."
"Who knew that after many decades of trying every kind of hand work I could find, I found the perfect way to combine my two greatest loves; fiber and beads. The flexibility built into the Mirrix looms, along with the durability you'd expect, allows me to go anywhere the creative spirit moves me."
Download This Free Holiday Project: The Tapestry Tree Ornament
Make your own unique ornament with gorgeous silk and crystals on a Mirrix Loom.download this free project


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I have 6 fat quarters that go together
Can you suggest a pattern? One print is large. Thanks
Welcome to the Quilting Board!
I have 6 fat quarters that go together
Can you suggest a pattern? One print is large. Thanks
Pictures help.
The wickedly easy pattern is free online and uses fat quarters. I am making two right now. They are crib size but you could add to them. Good luck!
The Yellow Brick Road pattern by Atkinson is relatively easy. The 48 x 57 size takes 6 fat quarters. Slide Show is another easy one of theirs and would highlight your larger print piece.
Many of their patterns call for fat quarters.
Woven ribbons by M'Liss Rae Harley. (In the book, Fat Quarter Quilts)
It will look as though five ribbons are surrounding your larger print!
I've working on one myself! 6 FQ's, plus 1/3 yard of another fabric.
Top went together so fast and the quilt is SOOOO pretty. I'm not at home, so can't attach pictures at the moment.
I agree... this was the first pattern that came to mind. You can always make it bigger by adding more borders.Originally Posted by LaurieE


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Stanley Desisso, 35, Jackson-ville, was arrested Friday on an active warrant for fraud in Duval County.
Officer Ronald Bilyew was dispatched to a gas station on Ponce de Leon Boule-vard in reference to a suspicious person.
When the officer arrived, he made contact with Desisso, and a computer check revealed the warrant. Desisso was taken to the county jail.
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Underrail, formerly known as Timelapse Vertigo
View Single Post
August 6th, 2014, 20:45
Version 0.1.12.0 released
Hey guys, the new version is up. We're all very happy to have finally released this. It's the centerpiece of the game, our Athkatla/Sigil/Neverwinter. The main story still has places to go and we still plan on introducing more factions and sideplots, but expect the future updates to be a lot more focused and a lot less spacious.
We showed you bunch of screenshots already, so for this occassion we decided to make a short teaser trailer instead.
As before, we're releasing it to Steam first until we're sure it's stable and any potential major bugs that we missed during internal testing are fixed.
And because no dev log would be complete without a bullet list, here's an incomplete list of minor tweaks and stuff that were done since the last dev log:
EMP mine blueprint component requirements changed to be the same to that of the EMP grenade
Increased the amount of bear traps crafted when not applying poison to 10
Increased the amount of caltrops crafted when not applying poison to 10
Reduced the cost of metal plates and, by extension, some items that are crafted from them
Added an option to turn off automatic door opening
Removed the cooldown of Burst attack (don't know why it was there in the first place)
Hopefully finally fixed the bug(s) that caused the factions to get all messed up after the Junkyard showdown event
Merchants will no longer roam while in bartering mode
Game will no longer crash when saving while mining
Chemical pistols have weight now
Action point counter fixed to work with extreme amounts of action points
Adrenaline shot will now always properly grant 20 bonus AP
Execute will no longer deal extra damage even if the target is not stunned/incapacitated
Enjoy the update folks, report bugs and let us know how you liked it. Cheers.
- an indie roleplaying game
Click if you'd prefer to keep an eye on Underrail dev log through an RSS feed.
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(I debated whether this should go on the end of the Pilot thread or here, my apologies if I guessed wrong...)
It appears my children are becoming "bag connoisseurs" (addicts-in-training?), I went to the store today to get them each a bag for our Spring Break trip. While I was in the store, a Nordic/Solar Pilot decided to follow me home (along with a frightening number of other things - what's up with that??)
Since I know curiosity about Pilots is running rampant, and I feel privileged to live close enough to go the store regularly, I thought I would share a couple of photos about what fits in the bag. Since I have never had a co-pilot, I am not a terribly good packer (yet) of this style of bag, but it should give folks an idea... (apologies for the picture quality, its dark in Seattle at this points, so light is hard, and I am using my phone...)
First I did my normal (mostly) carry for work:
Here is everything that is inside (list at the bottom of the post)
and how it was arranged -
Front left pocket has my kindle in the soft pocket (pocket is slightly bigger than my Kindle) and my Side Effect (PLO)
Middle pocket has a can of soup (lunch) and a #1 stuff sack w/a million 3X5 cards in it
Right pocket has a #2 stuff sack with my power cord, and a bowl for my soup
Back has the heavy laptop, a plastic file folder to hold papers, snacks, kleenex and my sunglasses case (sticking up in the picture) were laying on top over the laptop when I zipped the bag.
(iPad is in the back slip pocket (not shown), as it gets added to my bag as I walk out the door in the mornings)
Here is what was inside:
Power cord in #2 stuff sack
Side Effect (Black/wasabi) with wallet, mini pouch, forum gift pouch, small pouch, pen, stylus, earbuds
3X5 cards and wallet thing for them
Protein bar
Sunglasses in case
Plastic folder for papers
Soup bowl
Stack of several hundred 3X5 cards w/2 pens in #1 stuff sack
It all fit, and wasn't too tight, but I probably wouldn't try to put much else in there. I think if I played around with it, I could probably figure out a better configuration of the stuff...
Since I can only do 5 photos, I will follow with another post showing an airplane trip packout w/o laptop.


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Have you checked your notifications here on Credit Karma? It shows you the most recent things that have happened to affect your credit. Usually if something changes it will show you why. It has for me anyway..
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Tee Boon Hock says Ronnie Liu has failed his constituents.
KLANG: Sacked DAP grassroots leader Tee Boon Hock plans to contest in Pandamaran as an independent candidate if the party fields incumbent Ronnie Liu Tian Khiew for that state constituency.
He referred to the Selangor executive councillor as an "opportunist" and "tainted candidate" who had failed to deliver anything worthwhile to his constituents since he was elected in 2008.
If the DAP were to nominate such a person as its candidate for Pandamaran, he told FMT, "it will ignite my morale and determination" to contest for the seat.
He alleged that Liu had been unable to uphold DAP's socialist ideals.
Tee, who joined DAP in 1984, was sacked in July 2010 for allegedly misusing Liu's official letterhead to secure contracts for his cronies, an accusation he rejects. He also lost his seat in the Klang Municipal Council.
He sought election in Pandamaran in 1999 and 2004 and lost on both occasions. He made way for Liu in the 2008 election. They were then close friends.
"Since I was the one who proposed him for Pandamaran, I admit and take full responsibility for the mistake," he said.
"I sincerely apologise to the people of Pandamaran for his shortcomings and failures."
Tee said he hoped that the DAP leadership would, for the coming polls, field candidates with "credibility, high integrity, dignity and honesty" and who were team players able to work closely with grassroots leaders to serve the public.
He said elected representatives must be familiar with issues affecting their constituents and have the ability to solve problems at the grassroots.
"A representative should work with, by and for his voters."
He said DAP leaders had deviated from the party's principles and that its CAT (competency, accountability and transparency) governance had become an empty boast.
He accused them of having distanced themselves from the public and working only for their own materialistic gains.
He described the running of the Selangor government as a "comedy of errors" and said the Shah Alam administration was infested with abuse of power, graft, cronyism and nepotism.
"The CAT has become a sham and a gimmick and DAP has lost its credibility," he said.


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Written on: 12/10/2010 by Kimbikermad (1 review written)
I brought a Urban 400 after having a racer before,I have to say I am very pleased with this bike,it's very light almost,as light infact as my racer was,and the bike is also capable riding very fast,the too smallest coges at the back are extremely tiny,so you can go really fast if you want to.
But unlike a racer it can handle the ruth as well as the smooth.
It is hard to think of any bad points to say about this bike,it is very good all-rounder,smooth light and fast,and comfortable to ride.
Fantastic bike!!
As rated by our community of reviewers
" Great Value Bike " Read More
Written by semintimus
" Turned off by Specialized " Read More
Written by don0017
" Poor hydraulic barkes " Read More
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Written by Laverdabru1116


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To The Ranch
How amazing, to be transported to New Mexico and the DH Lawrence Ranch through images and text [cover, March 20: "Ghost Ranch"]! Especially on this cold and snowy first day of spring in Wisconsin. I never dreamed I'd be able to see actual photos and learn so much! Thank you, heartfelt for this gift today!
Bravo for the northern Rio, bravo [SFReporter.com, March 22: "Pres. Obama Declares Rio Grande del Norte a National Monument"]! Congratulations to the people of the Rio Grande del Norte and many thanks to Sens. [Jeff] Bingaman, [Tom] Udall and [Martin] Heinrich, and to Congressman Ben Ray Luján. Efforts leading to this step actually started about 15 years before 2007, when then-Rep. Bill Richardson assembled a cross-section of private and public representatives to explore the natural, historical and cultural aspects of the area. Until President Obama designated this newest unit of the National Landscape Conservation System 20 years later, efforts focused on the legislative arena—where state boundaries and congressional delegations are paramount.
As a state official on that early task force, I think it's fair to say most of us knew that the real natural boundaries extended upriver into southern Colorado. It's also true that the history of National Monument designations includes many cases of boundary expansion, as more becomes known and understood about the areas. Perhaps the Rio Grande del Norte National Monument will one day reflect its geologic, cultural and scientific contours rather than those of a political map. Until then, from the canyons and its environs, we'll celebrate this important moment.
Geoff Webb
The Dirt, March 13: "Canine Assets"
Moral Progress
Laura Paskus has written another outstanding and sensitive article for SFR on animals/environment. Her knowledge (with her unique perspective of this major issue) offers incredible insight.
What always comes to mind in reading her articles on this important topic are the words by Gandhi: "The greatness of a nation and its moral progress can be judged by the way its animals are treated."
Mary R Dykton
Santa Fe
Fighting Chance
Laura Paskus' article on the disastrous [Fish & Wildlife Service] Mexican wolf program is a glaring example of the fallacy of wildlife "management." These wolves never had much of a chance to begin with: When they were designated "experimental nonessential" (a political move to appease ranchers), it opened the door for an ongoing slaughter.
Since the livestock industry rabidly fights any endangered/threatened wildlife status, or any legislation to protect native wild animals, wolf re-introduction was, and is, a program set up to fail.
Maybe it's time to try a new, more enlightened program? Why not designate the livestock industry "experimental nonessential" and get livestock off our public lands, say, for the next 100 years or so. Perhaps all of our native wild animals will fare better, and have a fighting chance, as their habitat deteriorates because of climate change—and because of the damage to the land due to livestock grazing. For more info: thepetitionsite.com/343/829/766/stop-public-lands-ranching.
Rosemary Lowe
Santa Fe
What a victory for the consumer rights of every citizen of Santa Fe [SFReporter.com, Feb. 7: "Santa Fe to Consider GMO Labeling"]! Whether you love or hate genetically engineered food is a discussion for another time. This is about labeling, plain and simple. It's about my right and your right to know what we feed our families. Period. If you love GE food with all your heart, then great, eat it. If you hate GE food, then great, don't eat it. But how will we know unless it's labeled? And regardless of how we feel about it, why wouldn't we want it labeled? Thank you, City Council of Santa Fe, for championing the people. The many supporters of this resolution across the city look forward to you continuing to champion our right to know what is in our food.
Carol Norris
Santa Fe
Mail letters to Letters, Santa Fe Reporter, PO Box 2306, Santa Fe, NM 87504, deliver to 132 E Marcy St., fax them to 988-5348 or email them to firstname.lastname@example.com.
SFR welcomes original, signed letters to the editor. Letters (no more than 200 words) should refer to specific articles in the Reporter.
They may be edited for clarity and space. Include address and phone number for verification purposes; these will not be published.
To The Ranch


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DURBAN, South Africa (Thursday, 5 July, 2007)
Durban's prodigal surfing son Jordy Smith (Umbilo), suffered a shock elimination from the 6-Star ASP World Qualifying Series Mr Price Pro this morning, when he was defeated in his first heat during the Men's Round of 96.
Currently leading the WQS ratings, Smith found himself at the disposal of a becalmed ocean, caused by the high tide, which simply failed to deliver for the expectant ASP World Junior Champion.
While Russel Winter (UK) and ASP Africa wildcard Gavin Roberts (Scottburgh) snagged the few decent waves that pulsed between the piers to advance in 1st and 2nd respectively, Smith was left scratching for leftovers, taking his worst result of the year so far, an equal 73rd placing.
"It was a tough one for Jordy, but being so inconsistent it really was just about getting the right waves, and he couldn't find them. On the whole it was a very low scoring heat, but that's just the way it goes sometimes. I'm stoked to make it through because I'm coming back from a knee injury. I think that's all behind me and I'm feeling 100% for the rest of the year," Russel said.
With four of the seven Saffas' competing eliminated from the event, it was World No. 9 Travis Logie (Durban) who lifted the spirits of the South African supporters. Winning the final heat of the Round of 96, Logie advanced ahead of Aritz Aranburu (Spain), Shaun Ward (USA) and wildcard entrant Clinton Gravett (East London).
As conditions worsened due to the arrival of a stiff north easterly wind, the diminutive New Pier local unleashed his devastating backhand attack, scoring an excellent 8.00 (out of a possible 10.00), followed by a 6.50 for a combined heat total of 14.50 (out of a possible 20.00)
"The conditions are really bad out there with this onshore; it's making it very difficult. It's been a tough contest for the South Africans this year so obviously I'm stoked to make it through, and hopefully I can go on to a good result. I've been really consistent on the Foster's ASP World Tour this year, so thing's are going great," Logie said.
Former ASP World Tour campaigner Kirk Flintoff (Aus) was in a league of his own, defeating 2005 World Junior Champion Pablo Paulino (BRA), 2006 World Junior Champion Kekoa Bacalso (HAW) and South African Damien Fahrenfort (Durban). Posting a series of fast, vertical off the tops, back to back re-entries and a controlled floater; Flintoff earned an outstanding 9.33 to match Alain Riou's (Tahiti) highest individual wave score of the event so far.
WQS No. 22 Michel Bourez (Tahiti) emerged as another of the day's standouts, working his powerful frame across the New Pier bowl to score a near perfect 9.17, for a series of lightening snaps and a radical floater. With a 7.83 back up ride, Bourez earned the highest combined heat total of the day, a 17.00 (out of a possible 20.00).
WQS No. 2 Tiago Pires (from Portugal) laid claim to the WQS No. 1 spot, taking a definitive victory over World No. 12 Jeremy Flores (FRA), South African professional coach Quintin Jones (Cape Town) and World No. 8 Kai Otton (AUS).
The Men's Round of 48 kicks off at 08h50 tomorrow and will see all 12 heats completed.
The Men's final takes place at approximately 14h15 on Sunday 8 July 2007.
The Mr Price Pro forms part of the Durban Beach Festival and is being broadcast live on the webcast at www.mrpricepro.com.


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You cannot have 2 of the same wad...the second one will overwrite the first.
is it possible to install the same wad twice i wannaetween my flash dfrive and sd card from the loader menu install a usb loader to the wii system menu twice so i can use one loader for my flash drive and the same loader for my sd card with out having to switch between the flash drive and sd card via the loader menu if its possible let me know
You cannot have 2 of the same wad...the second one will overwrite the first.
ok then can you recomend me a second usb loader that i can use to run games off my sd card
Here's a thought: why don't you buy a USB HD?
im poor all i could afford was a 32 gig flashdrive for my retail games im b acking up and an SD card
You can use USB Loader GX for one, CFG Loader for the other...though, as nightstah has stated, you're better off saving for a USB HDD.
Last edited by SNES_Master; 05-17-2013 at 10:29 PM.
i cant get usb loader gx to see my sd card i dont see a drive selection in the gx options menu
But you said in your first post that you also have a USB flash drive, right?
I am posting from my iPhone but that ones all me lol. I'm having a very stressful day and ended up combining 2 trains of thoughtOriginally Posted by nightstah
Last edited by SNES_Master; 05-17-2013 at 09:09 PM.


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New Rabbi Darkside Songs, Mixtapes & Videos
The Band Called FUSE to Hold "Impossible Dream" Release Party at B.K.'s Littlefield [Feature ]
New York, N.Y. -- On Friday, October 25, Jersey emcee Silent Knight and NYC hip-hop/soul ensemble The Band Called FUSE will be unleashing their latest collaborative full-length, Impossible Dream. To celebrate the occasion,... Read More
Rabbi Darkside - See Something Say Something [Stream]
Ever since 2002, the MTA has been encouraging subway riders to report suspicious behavior with the slogan, "If you See Something, Say Something." On his latest single, Rabbi Darkside does his duty as a New Yorker,... Read More
Rabbi Darkside - True Lies [Stream & Download]
Realness, thy name is Rabbi Darkside. On new single True Lies, his first DJBooth feature, the Brooklyn-based rapper, DJ and hip-hop educator (who, in the interest of keeping it real, is not an actual rabbi) shares a few... Read More


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Battle of the Badasses0 Comments
June 27, 2005
X-Men Legends N-Gage Demo0 Comments
February 20, 2005
Help us solve the toughest (or most puzzling) game puzzles and challenges.
Our attempt to answer issues which most commonly occur on IGN's wikis.
About This Game
- Summary
- Specifications
- Features
- Game Editions
Based on the console editions of the same title, X-Men Legends is the first RPG game in the comicbook franchise. As members of the famous superhero group, you build your party from 11 available characters and travel the land looking for baddies. The story is that government crony General Kincaid is trying to stir up anti-mutant sentiments, so he lets loose Magneto with the help of the Brotherhood of Evil Mutants to wreak havoc on the city and give muties a bad name again. It's up to the player to organize the X-Men and beat these evil mutants into submission. The game can be played in single-player or up to 4-player co-op mode (via Bluetooth). There are 22 episodes and 50 missions, which total in at about 40 hours of gameplay time. In addition to fighting, you can customize your players on the fly and change your team's lineup at the end of each level. The game also features full motion video cutscenes.
- Supported Functions
- Number Of Players: 1-4
- Memory Card
- Multi-cartridge Multiplayer
- System Link / LAN
- The largest selection of X-Men ever! Select your squad of four mutants from the vast array of heroes that have been members of the X-Men in its 40-year history
- Replay the same mission using different characters
- Unique action-team-based fighting system
- Single player or team-based using the unique mutant powers of each X-Men character
- Up to 4 player cooperative multiplayer via Bluetooth wireless technology
X-Men LegendsPublisher: ActivisionRelease Date: February 07, 2005MSRP: 24.99 USD


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About This Activity
Riders should carry an extra tube, patch kit, tools, phone money, and identification.
The price of parking is included, but we strongly recommend carpooling because space is limited.
Please look for event direction signs.
See our website www.bikehikers.com for updated news and information on the event.Registration closes when 1,200 entries have been received, but not later than March 20th.Help Keep Party Pardee Alive
As an avid cyclist and participant in Party Pardee, WE NEED YOUR HELP to keep the event on the cycling calendar. The California Highway Patrol (CHP) and local law enforcement agencies will have an increased presence during Party Pardee. You will be on the road with other vehicle traffic. For your safety and the safety of others, PLEASE obey traffic laws.
Do not interfere with traffic. Ride single-file, as close to the right side of the road as practicable.
Stop as required at all controlled intersections (stop signs, traffic lights, etc.)
Yield to emergency vehicles.
Respond to the directives of law enforcement officers.
In addition to wearing your wristband, be sure to carry your identification and emergency contact information.
The number of riders is restricted under our permits with local government and Cal-Trans, and will be monitored by law enforcement. Pirating the ride (riding along as an unregistered rider) jeopardizes the future of this wonderful and historic event! If you know of someone who plans to ride along, please ask them not to unless they are a paid and registered participant.
We know that if you follow these simple rules, you can help keep the Party Pardee on the cycling calendar for years to come!
Organizer Info


text: In 2013, the UNH-IOL will enter its 25th year. In 1988, the needs of two competitive companies to work together to their mutual benefit helped form the InterOperability Laboratory. Since then, the thousands of companies partnering with the UNH-IOL all have benefited from this collaborative experiment between industry and academia.


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Figure 6:
(a) 3D structure of falcipain-2-cystatin complex. Cystatin is colored in orange, and falcipain-2 protease is colored in green. Refolding domain and hemoglobin binding domain highlighted in pink and salmon, respectively. The shaded area in the figure corresponds to the size of human cathepsin H, superimposed on falcipain-2 in light blue. (b) The catalytic Cys, His, and Asn are depicted in stick mode in green, and substrate binding pockets are labeled P3 to P1′. Ile-68 at P2 is depicted in stick mode to illustrate a slight protrusion in the S2 pocket of falcipain-2.


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I am the type of person who really loves having goals and something to look forward to. When I was in the process of paying off my credit card debt, I was so incredibly motivated. It was actually fun to throw money at it every month. I loved seeing the numbers dwindle and eventually go to zero. It was almost like a game, like the ultimate challenge to myself.
Right now, I am involved in the Yakezie Challenge, and I am keeping a spreadsheet of my ranking and the changes it has made over the past few weeks. (I am seriously obsessed with spreadsheets. It's an illness!) Once again, I feel excited and motivated. I know the goal is in sight, and I am looking forward to reaching it. It's not the same as becoming debt free, but it has the same feeling and the same momentum of getting inspired and excited about something and watching it come to fruition.
I can tell you that when I finally paid of that last credit card, I was euphoric! I remember feeling like I could do absolutely anything. I couldn't believe that my budget spreadsheet said ZERO in the credit card category. I gained back $500 a month in my budget that I could do whatever I wanted with! Of course, I knew the right thing to do was to snowball it and start working on my student loans.
Yet, when I started to pay off my student loans, a funny thing happened. I thought I would have incredible momentum to conquer them. After all, I'm a super motivated person, especially when I put my mind to something. Yet, because my husband has six figures worth of student loans, it feels like I'm trying to climb a mountain without any gear. As many blog friends around me knock out their debt left and right, I feel like blogs wont even exist anymore (blasphemy!) by the time I pay off all of my husband's medical school loans.
So, this time around, I am really lacking that drive and that motivation to get going, because it seems like the end goal is so very far away. I know that every little bit helps, but since my husband is still in school, the debt keeps growing.
Ultimately, I do remember what it felt like to be credit card debt free, but it seems like a distant memory now that I am focused on our student loans. Perhaps I can break it into smaller loans and work on my momentum that way instead of viewing it as one giant current I'm trying to swim against (lots of nature references today!) Either way, I'm hoping something will inspire me soon, so I can have that great debt-free feeling once again!
Are you debt free? Come on tell us: how does it feel? Is it everything you hoped it would be or do you feel kind of lost now that your goal has been reached?
**Lance's Two Cents** We have a long way to go on Tori's student loans so I definitely know how you feel with the huge student loan debt. We hope it feels pretty awesome, but more importantly it'll free up cash flow to enjoy on other things!


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Saturday, March 3, 2012
My parents took the boys on Saturday for an overnighter so FireDad and I could have a date night. We've been under a lot of stress as of late with house issues, work and two little boys who can't be in the same room without pushing each others' buttons. They had been missing Papau and YiaYia and I had been missing my husband. It was win-win.
We met at a city that rests almost exactly between here and there. I got out of our vehicle and my Dad exclaimed, "What's with all the green! It's not St. Patrick's Day yet!" So I reminded him of my love for green and explained #17daysofgreen. He laughed; he's used to my "unique" ideas after all. He's my Dad! He's been around for all of them.
They drove off in one direction with the boys while FireDad and I drove off in another.
Sunday, March 4
I met my parents for dinner tonight to pick up the boys. They were already getting seated when I walked into the restaurant. My Dad took a look at me and said, "What's up? You're not wearing any green!" I pointed at my pants. "Yes I am! It's not all green, all the time. You can wear all kinds of colors with green!" He laughed his laugh. I hugged my boys, chatted through dinner and did-not-enjoy a shamrock smiley cookie.
Eat 'n Park smiley cookies are cute — but gross.
The boys seem refreshed and happy. FireDad and I are refreshed and happy. Another week of green awaits… and I feel absolutely ready to tackle it head on.


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I want to manage the custom fields through the API, I have already done add and remove functionality but I haven't found a way to update the custom fields details (name and options).
Is there a function to do that?
Thanks in advance.
Hey Sylvain, you can do this using the Subscirber.AddWithCustomFields method. if the subscriber already exists (based on their email address), and new custom fields information will be used to update their record.
More details on the method here:
Hey Dave,
Thanks for your answer, but that's not what I'm looking for. It looks like I didn't explain properly what I want to do.
Actually, I don't want to set the customer's custom fields values. I would like to update the custom fields of the list.
For example, my list has a custom field call "Favourite sports", which is a MultiSelectMany. The options of this field are "Soccer", "Surf", and "Base Ball".
I want to add another option to this field: "Ice skating". Can I do this through the API?
Thanks in advance.
Hi Sylvain,
At the moment, it isn't actually possible to add a new possible option to a multiple option field, via the API. You'd either need to login through the app and add that field, or get the existing data for everyone, remove the old field, add the new one, and re update.
Ok, that's what I thought.
Thank you Mathew for your answer.
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I was so inspired by the design board thread I went to Home Depot and bought 2 not just 1 but 2 4x8 foot foam insulation boards. My car is not 8 feet - so two feet had to be cut off each one, my poor Lulu dog was totally traumatized by all this stuff in her car. Of course I have 9 million yards of fabric and there was a whole bunch of white flannel on the shelf - wrong _ I have to go buy some. How can one have literally 10 tons of fabric, but no white flannel. Yes I am laughing, the dog is napping. I will have 3 design boards when I am done 1 small one and 2 6 by 4- ought to keep me busy.
Oh, I was telling my daughter about the design board thread and how nice it would be to have one - as I am looking around my studio - there is a small one - it has been so covered with works in progress behind the quilting machine, impossible to get to for so long I forgot I had it.
I got out the scraps the other day and this is the city scapes I am working on and I think I like it.


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Synaptics Announces Groundbreaking InterTouch TouchPad™ Interface
Next-generation Interface Expands Data Bandwidth
Optimizes Synaptics' Image Sensing TouchPad Portfolio
May 31, 2011, 13:07 ET from Synaptics Inc.
TAIPEI, Taiwan, May 31, 2011 /PRNewswire/ -- COMPUTEX -- Synaptics Inc. (NASDAQ: SYNA), the leading developer of human interface solutions for mobile computing, communications and entertainment devices, is unveiling InterTouch at Computex. InterTouch is an open high speed interface standard that expands the capabilities of Image Sensing technology inside TouchPad™ and ClickPad™ equipped notebook PCs by enabling more sophisticated multi-touch gestures and highly accurate, multi-finger touch tracking, as well as providing enhanced suppression of inadvertent touch activations. The combination of InterTouch with Image Sensor-based TouchPads or ClickPads delivers a premium end user experience on x86 Intel and AMD platforms and will support current and future versions of Microsoft Windows, as well as Google Android and ChromeOS.
"Synaptics InterTouch raises the bar on notebook PC interactivity and user experience," said Tim Bajarin, president and principal analyst at Creative Strategies. "InterTouch is a game-changing interface enabling multi-touch capabilities and performance that OEMs can implement in today's notebook PCs to enhance the end user experience. Offering a faster pipe from the TouchPad to the CPU is analogous to moving from dial-up to broadband and delivering an enhanced gesture experience on the TouchPad."
Because Synaptics' powerful InterTouch solution delivers higher performance by providing more bandwidth and retains legacy support for operating systems and applications, it is the ideal transition technology for OEMs to future proof their motherboard designs. Legacy PS/2 interfaces create data bottlenecks that limit speed and restrain interactivity in a multi-touch world. InterTouch eliminates the restrictions imposed by existing PS/2 communications path, allowing transmission of multi-touch full-time finger tracking information. InterTouch leverages the existing I2C high speed PC interface (common in industry standard notebook PCs) and increases data processing from 10 to 40 times, allowing users to take full advantage of Image Sensor-based TouchPads and ClickPads from Synaptics. InterTouch also leverages the open Register Mapped Interface (RMI) 4.0 communications protocol over standard PC interfaces. It can operate at 100kHz in standard mode, 400kHz in fast mode and provides an open support path to even higher speed on future generation motherboards. InterTouch also enables high speed TouchPad and ClickPad end-user firmware upgrades, from 15 minutes to less than a minute.
"Synaptics InterTouch will open the door to new and more immersive user experiences for notebook users," said Mark Vena, senior vice president and general manager of Synaptics' PC Division. "InterTouch is a universal, open interface that can be used with all mainstream operating systems and CPU architectures that will allow PC OEMs to differentiate their notebook PCs through enhanced multi-touch experiences. InterTouch, combined with Synaptics TouchPads and ClickPads that utilize Image Sensing technology, will usher in a new age of end user productivity with notebook users that is simply not achievable with legacy PS/2-based solutions."
With as few as two additional pins from the motherboard to the TouchPad, BIOS and/or KBC-EC firmware modifications that enable the standards-based "I2C+" protocol, InterTouch supports multi-touch full-time tracking touch operating systems and applications. InterTouch supports both current and future motherboards and offers legacy support to interoperate with PS/2 protocols for true legacy/backwards compatibility.
To accentuate its open standard orientation, InterTouch can operate with the leading motherboards, BIOS, EC/KBCs. Synaptics has prototyped InterTouch with Phoenix Technologies and other PC ecosystem partners to ensure compatibility and integration at the motherboard system level. "InterTouch is a flexible solution that allows OEMs/ODMs to provide higher bandwidth by enabling the BIOS and connections directly to the PC Southbridge or KBC," said David Everett, senior vice president of Worldwide Field Operations at Phoenix Technologies. "As a leading enabler of these key components in the PC ecosystem, Phoenix offers flexibility in the design, reduced time to market, and the best possible end-user experience, fulfilling our vision of a connected, simple-to-use lifestyle for all-day computing and fun."
Synaptics is now engaged with leading PC OEMs on notebook designs that feature both TouchPad and ClickPad solutions using InterTouch support. Synaptics anticipates that these notebook designs will be available in the general market in early 2012.
To arrange a briefing of Synaptics' InterTouch technology solutions at Computex, partners may contact email@example.com and media may contact email@example.com, 650-346-1054 for more information.
To find out more about InterTouch, TouchPad Series 3, and Synaptics Gesture Suite, visit www.synaptics.com/go/InterTouch.
For the latest Synaptics news, follow @SynaCorp on Twitter.
About Synaptics
Synaptics (NASDAQ: SYNA) is a leading developer of human interface solutions for the mobile computing, communications, and entertainment industries. The company creates interface solutions for a variety of devices including notebook PCs, PC peripherals, digital music players, and mobile phones. The TouchPad™, Synaptics' flagship product, is integrated into a majority of today's notebook computers. Consumer electronics and computing manufacturers use Synaptics' solutions to enrich the interaction between humans and intelligent devices through improved usability, functionality, and industrial design. The company is headquartered in Santa Clara, Calif. www.synaptics.com.
Forward-Looking Statements
This press release contains "forward-looking" statements about Synaptics, as that term is defined under the federal securities laws. Synaptics intends such forward-looking statements to be subject to the safe harbor created by those laws. Such forward-looking statements include, but are not limited to, Synaptics' expectations that the combination of InterTouch with Image Sensor-based TouchPads or ClickPads will deliver a premium end user experience and result in end user productivity that is not achievable with legacy PS/2-based solutions; and anticipation that notebook designs that feature both TouchPad and ClickPad solutions using InterTouch support will be available in the general market in early 2012. Synaptics cautions that these statements are qualified by important factors that could cause actual results to differ materially from those reflected by the forward-looking statements contained herein. Such factors include, but are not limited to, (a) demand for Synaptics' products, (b) market demand for OEMs' products using Synaptics' solutions, (c) changing market demand trends in the markets Synaptics serves, (d) the success of customers' products that utilize Synaptics' product solutions, (e) the development and launch cycles of customers' products, (f) market pressures on selling prices, (g) changes in product mix, (h) the market acceptance of Synaptics' product solutions compared with competitors solutions, (i) general economic conditions, including consumer confidence and demand, and (j) other risks as identified from time to time in Synaptics' reports filed with the Securities and Exchange Commission, including Quarterly Reports on Form 10-Q and the Annual Report on Form 10-K for the fiscal year ended June 30, 2010. All forward-looking statements are based on information available to Synaptics on the date hereof, and Synaptics assumes no obligation to update such statements.
Synaptics, TouchPad, ClickPad and the Synaptics logo are trademarks of Synaptics in the United States and/or other countries. All other marks are the property of their respective owners.
For further information, please contact:
Nick Rottler Synaptics 408-454-5388 firstname.lastname@example.com
Katie Mech Edelman for Synaptics 650-762-2824 firstname.lastname@example.com
SOURCE Synaptics Inc.


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As an international College we believe that studying abroad is an incomparable experience. It is a once-in-a-lifetime chance to live the life of another country, learn the customs of another culture and adapt to new surroundings. The success of a student's experience depends to a large extent upon their own efforts to acclimatise to living and studying in a foreign country.
Life here for students is unlike that of city college campuses. At BCA students fully integrate with village life, becoming recognised members of the local community and often forging strong bonds and friendships that last beyond graduation. Students coming from large cities may be struck by the stillness and unhurried pace of the area. It can be amazing how much of our lives is taken up in traffic, noise and bustle. At BCA it takes 5 minutes to get from housing to your studio - allowing for an undisturbed creative environment.
BCA has a maximum enrolment of 55 students at any one time and because of our small scale students quickly become acquainted with each other. Undergraduate and graduate students work together to programme social events such as film screenings, daytrips or overnight trips to other parts of Ireland and the Activity Fee supports these events. Doing without the distractions and therefore some of the amenities of a large city is part of the experience at BCA.
Those who thrive here learn to be resourceful. Most appreciate this and are thankful for the break from the materialistic nature of modern living. The Food of the Arts Cafe is at the heart of the campus. Home-made, largely vegetarian lunches, using local ingredients are freshly cooked each day. As all student housing is on a self-catering basis, most students prepare dinner for themselves and we provide a regular bus service to larger towns and cities to access larger supermarkets there.
The college is situated a little over a mile from Ballyvaughan, a quiet rural harbour village with a population of 400, which increases considerably during the summer months. This small village boasts some wonderful pubs and restaurants and visitors to Ballyvaughan will be impressed at the continuous calendar of events the village hosts, from music and walking festivals to conferences and guided tours of the Burren. Drama, choral, walking and cycling clubs provide activities throughout the year and the wonderful farmers market showcases the best of local produce in season. Rock-climbing, horse-riding, swimming and surfing are all available locally with some of the finest beaches in Co. Clare located close to the college.
Independent Travel
Students are encouraged to use Ireland as a gateway to Europe, and the growth of low-cost airlines has made this ever more possible. If you do intend to travel, it would be difficult to over-emphasise the value of the ISIC card (International Student Identity Card) To assist students in getting to know Ireland, the College organises a number of optional trips at weekends. The Student Liaison Officer will discuss this at the beginning of the Semester - popular options include Belfast, Cork, The Aran Islands, Dingle etc


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Lung endothelial cells respond to changes in vascular pressure through mechanotransduction pathways that alter barrier function via non-Starling mechanism(s). Components of the endothelial glycocalyx have been shown to participate in mechanotransduction in vitro and in systemic vessels, but the glycocalyx's role in mechanosensing and pulmonary barrier function has not been characterized. Mechanotransduction pathways may represent novel targets for therapeutic intervention during states of elevated pulmonary pressure such as acute heart failure, fluid overload, and mechanical ventilation. Our objective was to assess the effects of increasing vascular pressure on whole lung filtration coefficient (Kfc) and characterize the role of endothelial heparan sulfates in mediating mechanotransduction and associated increases in Kfc. Isolated perfused rat lung preparation was used to measure Kfc in response to changes in vascular pressure in combination with superimposed changes in airway pressure. The roles of heparan sulfates, nitric oxide, and reactive oxygen species were investigated. Increases in capillary pressure altered Kfc in a nonlinear relationship, suggesting non-Starling mechanism(s). nitro-l-arginine methyl ester and heparanase III attenuated the effects of increased capillary pressure on Kfc, demonstrating active mechanotransduction leading to barrier dysfunction. The nitric oxide (NO) donor S-nitrosoglutathione exacerbated pressure-mediated increase in Kfc. Ventilation strategies altered lung NO concentration and the Kfc response to increases in vascular pressure. This is the first study to demonstrate a role for the glycocalyx in whole lung mechanotransduction and has important implications in understanding the regulation of vascular permeability in the context of vascular pressure, fluid status, and ventilation strategies.
- endothelium
- pulmonary edema
- permeability
the endothelial glycocalyx has been hypothesized to play a role in vascular barrier regulation through both passive and active mechanisms. Passive properties include the formation of a molecular filter overlying the cell-cell junction that limits water flux (2, 14) and protein flux (3, 38, 41) into the cell junction. Active barrier regulation occurs through mechanotransduction that alters junctional integrity via nitric oxide (NO) and reactive oxygen species (ROS) (15, 16, 18). Observations regarding glycocalyx-mediated signal transduction, however, have come from cultured endothelial cells and not from in vivo or ex vivo whole organ studies that would validate a physiological role for the glycocalyx in a more complex as well as clinically relevant model.
Recently, we reported that lung capillary endothelial cells, in vitro, respond to increases in hydrostatic pressure by production of intracellular NO and ROS that mediate barrier dysfunction manifested by an increase in hydraulic conductivity (15, 16, 18). Pressure-induced production of NO is a response characteristic of whole lung microvessels (24), clearly demonstrating that cultured endothelial cells and in situ pulmonary vessels share common responses to hemodynamic forces. We demonstrated that heparan sulfates (HS) were crucial for both the activation of endothelial nitric oxide synthase (eNOS) and pressure-induced increase in permeability. The present study extends our observations from cell culture models (15, 16, 18, 24) to a physiologically and clinically relevant model; our results provide the first evidence for a role of the glycocalyx, specifically HS, in mechanotransduction and barrier regulation in the intact lung.
Ex Vivo Lung Preparation
All animal experiments were approved by the University of Utah's Institutional Animal Care and Use Committee and in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council).
Adult male Sprague-Dawley rats (300–400 g) were anesthetized with ketamine-xylazine (90:10 mg/kg), a tracheotomy was performed and they were mechanically ventilated with a pressure-controlled ventilator (Kent Scientific, Torrington, CT) at a respiratory rate of 60/min, FiO2 = 0.21 and positive end-expiratory pressure (PEEP) of 3.0 cmH2O. The chest and pericardial sac were sequentially opened and ligatures were placed around the aorta and pulmonary artery. Heparin (200 U) was injected into the pulmonary artery and allowed to circulate for 2 min. The rat was exsanguinated via transection of the abdominal aorta; the left atrium was cannulated via a left ventriculostomy. The pulmonary artery was cannulated via the right ventricle and lungs were perfused with Krebs-Ringer-bicarbonate solution containing 3% bovine serum albumin.
Pulmonary arterial (Ppa) and left atrial pressures (PLA) were measured continuously via in-line pressure transducers (P-75, Harvard Apparatus, Natick, MA) connected to an analog-to-digital board. Solenoids (20 PSI, 12 V DC, Cole Parmer; Mount Vernon, IL) were placed in-line on both the arterial and venous tubing and could be closed simultaneously for measuring double-occlusion pressures (Pdo). An in-line ultrasonic flow probe (Transonic, Ithaca, NY) was placed in the pulmonary artery cannula and flow data was recorded in real time. Lungs were suspended from a force transducer (Radnoti, Monrovia, CA) and lung weight was zeroed; lung weight, vascular pressures and flow were recorded using a custom-written program (LabVIEW, National Instruments, Austin, TX).
Heparanase III (E.C. 126.96.36.199) was from Ibex Technologies (Montreal, QC, Canada); Krebs-Ringer buffer, nitro-l-arginine methyl ester (l-NAME), nitro-d-arginine methyl ester (d-NAME), S-nitrosoglutathione (GSNO), and diethyl pyrocarbonate (DEPC) were purchased from Sigma Chemical (St. Louis, MO); Mn(III)-tetra(4-benzoic acid) porphyrin chloride (TBAP) was purchased from Biomol (Plymouth Meeting, PA). Bovine serum albumin fraction V was purchased from Proliant (Ankeny, IA). Anti-nitrotyrosine was from Abcam (Cambridge, MA) and HSS-1 and 3G10 from US Biological (Swampscott, MA).
Calculation of Kfc
The change in lung weight expressed as milliliters of filtered fluid during the time interval from 18 to 20 min was divided by time (2 min) and then by the capillary pressure (Ppc), where Ppc = (Ppa + Pla)/2, yielding milliliters per minute per centimeter H2O (32); this value was normalized to 100 g of predicted lung weight (PLW), which was calculated from the equation PLW = 0.0053 (rat weight) − 0.48 (32).
Pressure-Volume Curves
Tidal volumes (Vt) in isolated perfused rat lungs during pressure-controlled ventilation were measured with a differential pressure transducer (DP-45, Validyne, Northridge, CA), driven by a Carrier Frequency Bridge Amplifier (type 677, Hugo Sachs Elektronik-Harvard Apparatus). Airflow (ml/s) was recorded with a custom-written program (LabVIEW 8.5, National Instruments, Austin, TX) at 200 Hz sampling rate, and each respiratory cycle was integrated to obtain Vt. A calibration curve was created by applying gas flow (air) (air pressure = 1 bar) from negative 16.16 ml/s (vacuum) to positive flow at 16.16 ml/s, through the DP-45 and recording the corresponding voltage output. This flow was regulated with a calibrated flowmeter (Dwyer, model UA1049) (accuracy ±2%) connected to a pressure regulator. The calibration curve was entered in a custom scale before recording of Vt. The lungs were ventilated with different peak inspiratory pressures (PIPs) and positive end-expiratory pressures (PEEPs) (ΔP = PIP−PEEP) to record the resulting Vt values. Flow/Vt was recorded for 1 min for each ΔP. We used inflation pressures that produced Vts identical to previous literature values (20, 33) during standard tidal volume (ST Vt). Low tidal volume ventilation (Low Vt) inflation pressures were chosen to recreate clinically relevant lung-protective strategies.
Whole-lung protein preparations were assayed in triplicate for concentrations of nitrate/nitrite (NOx) using a chemiluminescence analyzer for NO detection (NOA 280i; Sievers Instruments, Boulder, CO) as previously described (27). Briefly, 50 μl of sample was injected into the reaction chamber containing a saturated solution of 0.05 M vanadium (III) chloride in 1 M HCl heated to 95°C to reduce NOx to NO. The liberated NO was carried in gas phase into the analyzer by a constant flow of helium gas. The analyzer was calibrated by using a standard curve derived from serial dilutions of a 0.1 M stock solution of sodium nitrite (NaNO2). Individual results for tissue NOx are expressed as micromoles of NO per micrograms total protein per Δ wet weight; the normalization to Δ wet weight is to correct for the dilution of NOx by increased extravascular lung water.
Lungs were perfused with neutral-buffered formalin via the pulmonary artery while Pla was held at 5 cmH2O; formalin was also instilled into the trachea at a pressure of 25 cmH2O. Lungs were processed by standard histological methods. Anti-NO-tyrosine, HSS-1, and 3G10 antibodies were conjugated to a biotin-labeled secondary antibody (IgM) and then incubated with streptavidin-horseradish peroxidase.
Experimental Protocols
Protocol 1: Low Vt + double pressure step.
Lungs were ventilated at Low Vt and perfused for 20 min at isogravimetric pressure. Baseline Kfc (Kfc1) was determined by increasing left atrial pressure (Pla1) to 7.5 cmH2O for 20 min followed by a return to isogravimetric conditions for 20 min. A second pressure step (Pla2) to 7.5, 10, 12, and 15 cmH2O was performed for 20 min and Kfc2 was measured. The ratio Kfc2/Kfc1 was used to assess the influence of Pla on the filtration coefficient. Double-occlusion pressures (Pdo1, Pdo2, Pdo3) were measured before and after each pressure step to ensure stability of the individual preparation. The general scheme for the double-step protocol is shown in Fig. 1A, top and middle.
The double pressure step protocol was used for the following groups: control, l-NAME, TBAP, heparanase III, and GSNO. Test reagents were infused into pulmonary artery beginning 10 min into the second isogravimetric period and were present in the recirculating media for the remainder of the experiment. Test reagents were used at the following concentrations: l-NAME (200 μM), TBAP (200 μM), heparanase III (75 and 150 mIU/ml), and GSNO (500 μM).
Protocol 2: ST Vt + double pressure step protocol.
Lungs were ventilated with ST Vt, and Kfc was measured at baseline Pla = 7.5 cmH2O and after a second step of Pla to 15 or 17 cmH2O, as described above. Reagents were added to the perfusate beginning 10 min into isogravimetric period 2 and were present in the circulating media for the remainder of the experiment.
Protocol 3: low Vt + single-step protocols.
To examine the influence of the baseline Kfc measurement (initial pressure pulse) on subsequent Kfc measurements, we evaluated Kfc following a step from isogravimetric conditions to Pla = 7.5 or 15 cmH2O. Pdo values were measured before and after each pressure step. The Kfc obtained from ΔPla = 0 to 15 cmH2O was compared with Kfc2 obtained from the double pressure step protocol when Pla = 15 cmH2O. l-NAME (final concentration, 200 μM) was added to the media 10 min into the isogravimetric period and was present for the step increase in Pla to either 7.5 or 15 cmH2O. The general scheme for the single-step protocol is shown in Fig. 1A, bottom.
All data are presented as box plots showing sample minimum and maximum, 25th and 75th percentile, and median. Differences between groups were assessed by ANOVA followed by either Tukey's honestly significant difference or Scheffé's post hoc test.
All statistical analyses were performed with Kaleidagraph for Windows (version 4.0.3), Synergy Software (Reading, PA); probability levels (P < 0.05) were taken to indicate statistical significance.
Pulmonary Hemodynamics
We assessed pulmonary hemodynamics in all lungs (Table 1). As can be seen in Table 1, at any given Pla, measured Ppa, Ppc, and Pdo did not vary between groups. As expected, Ppa increased linearly as a function of elevated Pla. Ppa was not altered by l-NAME, d-NAME, TBAP, or heparanase III at any given Pla. Pulmonary capillary pressure, Ppc, [Ppc = (Ppa + Pla)/2], was not different across treatment groups when Pla = 15 or 17 cmH2O. Pulmonary artery Pdo1, Pdo2, and Pdo3 were not significantly different across groups at Pla = 15 or 17 cmH2O.
Lung Pressure-Volume Measurements
Pressure-controlled ventilation was used in the present experimental protocols; to quantify Vt delivered at net inflation pressure, we built a custom-made system to integrated airflow per unit time to derive Vt. The resulting pressure-Vt curve presents ΔP (PIP−PEEP) vs. Vt, normalized to body weight (kg) (Fig. 1B). In our Low Vt group, average Vts were in the range of 2.0–2.5 ml/breath; in the ST Vt group, Vt were 3.0–3.5 ml/breath. The Low Vt group was ventilated with 7–8 cmH2O providing a Vt per body weight of ∼4–6 ml/kg. The ST Vt groups were ventilated with 10–12 cmH2O yielding Vt per body weight of ∼6–8 ml/kg.
Low Vt Studies
Baseline Kfc.
All lungs in the double-step groups had a baseline measurement (Kfc1) at Pla1 = 7.5 cmH2O; thus Kfc1 for all groups was pooled and yielded an average Kfc1 = 0.081 ± 0.04 ml·min−1·cmH2O−1 per 100 g (n = 112). Subgroup analysis of baseline Kfc1 was compared between all control groups and no differences were noted. This rules out time- and technique-dependent factors that may have confounded subsequent Kfc comparisons. Pulmonary artery pressure (Ppa1), double-occlusion pressure (Pdo1), and capillary pressure (Ppc1) are denoted by a subscript "1" to indicate the baseline pressure step from which the measurement was derived.
Protocol 1: double pressure step.
The second step increase in Pla is designated Pla2, and the measured Kfc is denoted as Kfc2. Pulmonary artery pressure (Ppa2), double-occlusion pressure (Pdo2), and capillary pressure (Ppc2) are denoted by a subscript "2" to indicate the second pressure step from which the measurement was derived. The ratio of Kfc2/Kfc1 derived when Pla = 7.5 cmH2O for both pressure steps (C7–7) is considered "control" for the remainder of the studies. The control Kfc2/Kfc1 = 1.43 ± 0.78 (n = 8).
To characterize the influence of increased capillary pressure on Kfc, lungs were exposed to a second step increase in Pla2 to 10, 12, 15, or 17 cmH2O for 20 min. There was no significant difference in Kfc1, Kfc2, or Kfc2/Kfc1 when Pla = 7.5, 10, or 12 cmH2O. When Pla2 = 15 or 17 cmH2O, Kfc2 increased significantly compared with the baseline (Fig. 2A). At Pla2 = 15 cmH2O, Kfc2 = 0.65 ± 0.35 ml·min−1·cmH2O−1 per 100 g and the Kfc2/Kfc1 ratio increased to 7.9 ± 3.27 (P < 0.01). At Pla = 17 cmH2O, Kfc2/Kfc1 = 9.86 ± 1.50 ml·min−1·cmH2O−1 per 100 g. The Kfc2/Kfc1 vs. Pla relationship is presented in Fig. 2A and shows nonlinear dynamics of the pressure vs. permeability relationship that cannot be explained by a simple Starling mechanism.
Inhibition of endothelial NO synthase with l-NAME (200 μM) had no effect on pulmonary hemodynamics (Table 1) or on baseline Kfc2/Kfc1 (i.e., when Pla2 = 7.5 cmH2O) where l-NAME-treated lungs had a Kfc ratio of 1.68 ± 1.35 ml·min−1·cmH2O−1 per 100 g. l-NAME attenuated the increase in Kfc2 when Pla2 = 15 and 17 cmH2O (Fig. 2B). The Kfc2/Kfc1 ratio at Pla2 = 15 cmH2O was 3.49 ± 1.40; when Pla = 17 cmH2O, Kfc2/Kfc1 was 3.70+0.88. These values are significantly different than untreated control (P < 0.05) as shown in Figs. 2 and 3. d-NAME (200 μM), the inactive enantiomer of l-NAME, had no effect on pressure-induced increase in Kfc2 (data not shown). GSNO increased the mean Kfc2/Kfc1 ratio when Pla = 15 cmH2O to 9.1 (P < 0.0001) (Fig. 3).
We tested two concentrations of heparanase III on pressure-mediated increases in Kfc. Heparanase III at 75 mIU/ml had no effect on Kfc relative to controls (data not shown). At a concentration of 150 mIU/ml, heparanase III had no effect on Kfc2 when Pla = 7.5 cmH2O, indicating that the enzyme did not alter baseline permeability, but heparanase III significantly attenuated the increase in Kfc2 when Pla2 was increased to 15 cmH2O (Hep7–15; Fig. 3). Heparanase III significantly reduced Kfc2 at Pla2 = 15 cmH2O to 0.134 ± 0.065 vs. 0.65 ± 0.36 in untreated lungs; the Kfc2/Kfc1 ratio for heparanase III-treated lung was 3.10 ± 1.59 vs. 7.91 ± 3.28 in untreated lungs (n = 8, P < 0.0001).
The intracellular superoxide dismutase mimetic, TBAP (200 μM), had no effect on pressure-induced increase in Kfc ratio when assessed at Pla = 15 cmH2O. TBAP at 200 μM is a high concentration and the duration of exposure was appropriate based on similar uses (25, 30). Kfc2 after a step to Pla2 = 15 cmH2O in TBAP-treated lungs was 0.63 ± 0.19 vs. control Kfc2 = 0.65 ± 0.36 ml·min−1·cmH2O−1 per 100 g; Kfc2/Kfc1 = 6.74 ± 1.58 was not significantly different than untreated lungs at Pla2 = 15 cmH2O (n = 9; p > 0.05) (Fig. 3).
Kfc measurements are dependent on vascular surface area that can be increased by vascular recruitment; zonal characteristics (ZCs) were derived from ΔPpa/ΔPla as described by Brower et al. (10, 11) and Anglade et al. (7) and indicate the percentage of zone 2 and 3 conditions at each Pla. In control lungs, ZC significantly increased with the increase in Pla from 7.5 to 10 cmH2O (0.44 vs. 0.58; P < 0.05); additional increases in Pla above 12 cmH2O did not result in statistically significant changes in ZC (Fig. 4A).
ZCs were derived for l-NAME-treated groups at Pla2 at 7.5, 10, 12, 15, and 17 cmH2O (Fig. 4A); increases in Pla from 7.5 to 10 cmH2O resulted in the only statistical difference in ZC. A comparison of ZC for control, heparanase III-treated, and l-NAME-treated lung at Pla2 = 15 cmH2O is shown in Fig. 4B. There were no significant differences in ZC between treatment groups at Pla = 15 cmH2O, suggesting that recruitment was not responsible for observed differences in Kfc when Pla2 = 15 cmH2O with heparanase and l-NAME.
To assess the effect of increased Pla2 and treatments (heparanase, l-NAME) on interstitial volume, we measured the retained lung weight after the second pressure steps (ΔVi) and normalized this value to predicted lung weight (PLW). A plot of ΔVi/PLW for each group demonstrates that heparanase III and l-NAME significantly reduced interstitial volume, in direct correlation with their effects the Kfc2/Kfc1 ratio (Fig. 5).
Protocol 2: Standard Vt studies (ST Vt).
In this series of experiments, lungs were ventilated with PIP = 10–12 cmH2O, an increase in ∼30–40% increase relative to Low Vt experiments; the effect of Pla2 and test reagents (heparanase III, heparanase III + DEPC, l-NAME, d-NAME, GSNO) on Kfc were measured at Pla = 7.5, 15, and 17 cmH2O.
All lungs in this group had baseline Kfc measured at Pla1 = 7. 5 cmH2O, followed by a second step increase to Pla2 = 15 or 17 cmH2O. At ST Vt baseline Kfc1 values were 0.078 ± 0.03 ml·min−1·cmH2O−1 per 100 g, which was not different from Kfc1 during Low Vt (Fig. 6A). At Pla2 = 15 cmH2O, the Kfc2/Kfc1 ratio = 7.1 ± 2.71 (n = 12), which is significantly higher than the ratio obtained during Low Vt and Pla2 = 15 cmH2O. When Pla2 = 17 cmH2O, Kfc2/Kfc1 = 15.80 ± 1.20.
Heparanase III (150 mIU/ml) significantly reduced Kfc2 when Pla = 15 cmH2O; mean Kfc2 was 0.35 ± 0.21 and Kfc2/Kfc1 = 3.12 ± 1.93 (n = 12) (Fig. 6B). DEPC inhibits heparanase by binding to the histidine residue within the active site and renders the enzyme inactive (35a). This approach mitigates changes in enzyme structure that would be altered by boiling as a means of inactivation. Heparanase III was incubated with DEPC (100 nM; 20 min), then added to the circulating perfusate. In contrast to active enzyme, heparanase-DEPC had no effect on pressure-induced increase in Kfc2/Kfc1 (Fig. 6B).
When Pla was increased to 17 cmH2O, Kfc2/Kfc1 increased to 15.8 from 4.08 (Fig. 6C).
Heparanase III significantly attenuated the increase in Kfc at Pla = 17 cmH2O (P < 0.01) (Fig. 6C).
We tested the effect of l-NAME (200 μM) and d-NAME (200 μM) on Kfc when Pla2 = 15 or 17 cmH2O. l-NAME significantly decreased Kfc2 when Pla2 = 15 and 17 cmH2O (Fig. 6, B and C). d-Name had no effect on Kfc (data not shown). GSNO significantly increased Kfc2/Kfc1, from 7.1 to 11.0 when Pla2 was increased to 15 cmH2O (P < 0.01, Fig. 6B).
With ST Vt, ZC was 0.68, 0.79 and 0.79 at Pla2 = 7.5, 15, and 17 cmH2O, respectively; heparanase III and l-NAME had no effect on ZC (Fig. 7A and B).
To assess the effect of increased Pla2 and treatments (heparanase III, l-NAME) on interstitial mechanics, we measured the retained lung weight (ΔVi) after the second pressure step and normalized this value to PLW. Plots of ΔVi/PLW closely parallel the relationship of Kfc2/Kfc1 ratio between groups. There were no significant differences when Pla2 = 15 cmH2O between control, heparanase III-treated, or l-NAME-treated lungs (Fig. 8A). When Pla2 = 17 cmH2O, heparanase III and l-NAME significantly reduced ΔVi/PLW (Fig. 8B).
Protocol 3: single pressure step.
To assess the effect of the baseline pressure step (Pla1) on subsequent Kfc measurements (i.e., priming effect), we performed a single step from isogravimetric pressure (0–2 cmH2O) to Pla = 15 cmH2O. This group of experiments was performed in Low Vt lungs only. Kfc during the single pressure step to Pla = 15 cmH2O was 0.33 ± 0.16 ml·min−1·cmH2O−1 per 100 g and was significantly less than Kfc2 at Pla2 = 15 cmH2O in the double pressure pulse experiments (0.34 ± 0.16 vs. 0.65 ± 0.35 ml·min−1·cmH2O−1 per 100 g), Fig. 9.
Heparanase III reduced Kfc during the single pressure step to 0.29 ± 0.15 ml·min−1·cmH2O−1 per 100 g, compared with controls (0.33 ± 0.16 ml·min−1·cmH2O−1 per 100 g), although the effect did not reach statistical significance (Fig. 9).
l-NAME lower Kfc by 50%, compared with controls (in the single pressure step protocol. 0.13 ± 0.09 vs. 0.33 ± 0.16 ml·min−1·cmH2O−1 per 100 g; p = 0.11) (Fig. 9). In fact, Kfc measured from l-NAME-treated lungs using the single pressure protocol at Pla = 15 cmH2O, was reduced by almost 50% compared with the corresponding Kfc2 from l-NAME-treated lungs during the double pressure pulse protocol (0.13 ± 0.09 vs. 0.23 ± 0.12, ml·min−1·cmH2O−1 per 100 g), respectively.
To validate the effects of heparanase III on removing lung HS, anti-HS antibody (HSS-1) was used to stain for HS. Control lungs demonstrate substantial capillary staining for HS within the alveolar septa (Fig. 10A); heparanase III completely abolished HS staining (Fig. 10B). An alternative approach to validate the efficacy of heparanase III was achieved by using 3G10, an antibody that recognizes the neoepitope on the HS chain generated by the activity of heparanase III. Control lungs showed no 3G10 staining whereas heparanase III -treated lungs showed significant staining (Fig. 10, C and D). Collectively, these two complementary approaches demonstrate significant removal of HS from the lung vasculature.
Staining for nitrotyrosine was increased in lungs exposed to increased vascular pressure (Pla2 7.5 vs. 15 cmH2O) as shown in Fig. 11, A vs. B, respectively. During low vascular pressure (Pla2 = 7.5 cmH2O), small regions of positive staining are seen at low power (×20) and shown as a close-up (inset, ×60) (Fig. 11A). Following exposure to Pla2 = 15 cmH2O, an increase in nitrotyrosine staining is observed in both low- and high-powered images (Fig. 11B). Heparanase-treatment before increasing vascular pressure significantly reduced nitrotyrosine staining (Fig. 11C).
We directly measured lung tissue NOx concentrations. The relationship between NO concentration vs. Kfc2/Kfc1 for pooled Low Vt and ST Vt are presented in Fig. 12A. The Low Vt group shows a statistically significant linear correlation between NOx vs. Kfc2/Kfc1 (r2 = 0.59, n = 29) and the Low Vt group is left shifted compared with the ST Vt group (inset). The combination of increased vascular pressure and ST Vt resulted in significantly higher tissue NOx concentrations; the NO vs. Kfc2/Kfc1 relationship was fitted to an exponential curve (r2 = 0.92); the permeability response was flat above an NOx concentration of ∼4 μmol.
Tissue NOx levels are presented in Fig. 12, B and C. During Low Vt and baseline Pla (7.5 cmH2O), tissue NOx was low (0.028 ± 0.026 μmol) and increased almost 12-fold when vascular pressure was increased to 15 cmH2O (C7–15; mean NOx = 0.35 μmol; P < 0.01). GSNO increased tissue NOx levels to 0.70 μmol. Heparanase III treatment had no effect on NOx levels during baseline Pla (Hep7–7); during elevated vascular pressure, heparanase (Hep7–15) decreased the tissue NOx from 0.35 to 0.23 μmol (P > 0.05). NOx could not be measured in the l-NAME groups because l-NAME liberates NOx, which interferes with the assay.
During ST Vt, increasing vascular pressure resulted in an exponential increase in tissue NOx. Baseline NOx at Pla = 7.5 cmH2O (C7–7) was 0.03 μmol, which is identical to that of the Low Vt group. However, the combination of higher Vt and increasing in vascular pressure to 15 and 17 cmH2O resulted in marked increased NOx concentrations to 0.7 and 8.4 μmol, respectively (Fig. 12C). GSNO was synergistic with increasing Pla and increased NOx levels to 2.6 μmol. Heparanase III significantly attenuated the increase in NOx when Pla = 15 cmH2O to 0.5 μmol (P < 0.01, Fig. 12C).
The major findings of this study were 1) Kfc increased in a nonlinear relationship to vascular pressure, over the range of 7.5 to 17 cmH2O; 2) heparanase III significantly attenuated the pressure-induced increase in Kfc2 and inactivation of heparanase III by DEPC abolished its inhibitory effect; 3) l-NAME (200 μM) significantly attenuated the pressure-induced increase in Kfc2, whereas the NO donor GSNO increased permeability; 4) TBAP had no effect on pressure-induced increase in Kfc2; and, finally, 5) the first step increase in Pla increased the sensitivity of the vasculature to the second step in Pla, a phenomenon we call pressure conditioning.
Nonlinear Relationship of Kfc vs. Pla
For our initial studies, we performed a detailed characterization of increasing Pla on Kfc. In these studies, Kfc1 was measured in all lungs at Pla1 = 7.5 cmH2O. After a second isogravimetric period, Pla2 was increased to 7.5, 10, 12, 15, or 17 cmH2O and Kfc2 was derived. In each set of experiments, the ratio of Kfc2/Kfc1 was calculated to establish the influence of Pla2 on whole lung permeability; the Kfc2/Kfc1 ratio did not significantly change until Pla2 = 15 cmH2O. The Kfc2/Kfc1 ratio following double pressure steps of 7.5/7.5, 7.5/10, 7.5/12, 7.5/15, and 7.5/17 was 1.43, 1.43, 2.83, 7.5, and 9.86, respectively. The nonlinear dynamics of the pressure vs. permeability relationship mimics what was observed in a cell culture model using lung capillary endothelial cells (15) and suggests a non-Starling mechanism. Kfc is the transvascular water flux normalized to capillary pressure and, therefore, assuming vascular permeability remained unaltered by pressure, the Kfc vs. Pla relationship should have a slope of zero. The nonlinear relationship, which has been reported in vitro, and now in a whole organ model, suggests a non-Starling mechanism consistent with active vascular mechanotransduction.
l-NAME had no effect on baseline Kfc1 but significantly attenuated the increase in Kfc2 when Pla2 = 15 and 17 cmH2O. These findings are similar to results obtained from monolayers of bovine lung capillary endothelial cells (15, 19, 43). The increase in hydrostatic pressure appears to activate eNOS, leading to an increase in endothelial permeability. The role of hydrostatic pressure and increased transendothelial flow on NO production has been reported by Burns et al. (12), using human umbilical vein endothelial cells as a model system. Thus there is a consistent finding both in vitro and now in an ex vivo lung preparation where pressure-mediated mechanotransduction involves eNOS activation and subsequent barrier dysfunction. GSNO, an NO donor, exacerbated pressure induced increase in Kfc during both Low Vt and ST Vt protocols, supporting the conclusion that NO is a mediator of lung endothelial barrier dysfunction.
Heparanase Experiments
This is the first study to demonstrate a role for vascular HS on mechanotransduction using a whole lung model. In the present study, heparanase III (E.C. 188.8.131.52) had no effect on baseline Kfc1 yet significantly attenuated the increase in Kfc2 when Pla2 was increased to 15 and 17 cmH2O (Figs. 3 and 6). These results are similar to our observations in vitro (15) strongly supporting the conclusion of glycocalyx-mediated mechanotransduction (26, 36, 42). We tested DEPC-inactivated heparanase III and found that it had no effect on reducing Kfc, during elevated vascular pressure; thus heparanase's enzymatic activity was required to attenuate mechanotransduction. To validate the removed of HS by heparanase, we performed immunohistochemistry and demonstrated nearly complete removal of vascular heparan staining after heparanase treatment. Likewise, the staining of heparanase-treated lungs by 3G10, an antibody that recognizes the neoepitope created by heparanase, demonstrated robust staining.
To determine whether heparanase's reduction in Kfc was actually due to a reduction in permeability and not due to altered interstitial pressure, we present the following line of reasoning. Negrini et al. (28) used intravascular heparanase III to induce lung injury in rabbits and reported an increase in interstitial pressure due to the accumulation of interstitial fluid. In their experiments, heparanase III-treated lungs had an increase in wet-to-dry weight ratio consistent with an increase in interstitial fluid. As interstitial pressure increased, the gradient for fluid flux decreased and, therefore, could reduce the apparent value of Kfc. In the present study, the increase in lung weight following each pressure step was taken as a marker for ΔVi (45); heparanase III-treated lungs had a significantly lower ΔVi/PLW at Pla = 15 and 17 cmH2O compared with controls (Figs. 5 and 8), consistent with a reduction in endothelial permeability and reduced interstitial edema. The significantly lower values for both Kfc and ΔVi/PLW in heparanase-treated lungs support the conclusion that mechanotransduction and increased permeability were attenuated by removal of vascular HS.
TBAP is a cell-permeable superoxide dismutase mimetic that scavenges free radicals. We previously reported (15) that TBAP attenuated pressure-induced increases in endothelial permeability, suggesting that ROS were partially involved in barrier alterations. In the present study, TBAP had no effect on pressure-induced increase in Kfc. We conclude that ROS are not the cause of pressure-mediated barrier dysfunction in this ex vivo lung model.
Technical Considerations
The relationship between Pla, Ppc, and Kfc is complex and requires assumptions that have been addressed elsewhere (9). To rule out factors other than mechanotransduction that could have influenced measured Kfc, we address the following technical issues.
Pulmonary hemodynamics.
Ppa, pulmonary artery flow, and Pdo1, Pdo2, and Pdo3 were measured throughout the experiment. There were no significant differences in pulmonary hemodynamic variable that could explain the observed differences in Kfc2 when Pla2 = 15 or 17 cmH2O (Table 1).
Fluid flux is dependent on vascular surface area; if step increases in Pla increased recruitment, our measured Kfc values may reflect only the increasing surface area and not an actual change in endothelial permeability. Short et al. (40) reported that, in rat lungs, recruitment of subpleural capillaries accurately reflected recruitment of interior capillaries; using similar methods, Presson et al. (35) showed that lung capillary recruitment was 90% complete when capillary pressure equaled 10 cmH2O and was nearly 100% complete when capillary pressure was 12 cmH2O. Pulmonary artery pressure is an indicator of downstream resistance and should decrease with increasing vascular recruitment. In the present study Ppa was stable after 3–5 min following the step increase in Pla, suggesting that recruitment and downstream resistance was not changing.
Evaluation of ZC (= ΔPpa/ΔPla) was undertaken to further evaluate the effects of increased Pla on vascular recruitment and to quantify the relative degree of Zone 2 and Zone 3 conditions throughout the experiments. During Low Vt experiments when Pla2 = 7.5 cmH2O, all lungs were primarily in Zone 2 as evidenced by ZC = 0.44. Increases in Pla2 to 10, 12, 15, and 17 cmH2O increased ZC to 0.58, 0.64, 0.71, and 0.76, respectively. According to ZC analysis, recruitment was completed when Pla = 12 cmH2O consistent with the finding of Presson et al. (35). In summary, Kfc continued to increase after ZC had plateaued, suggesting that changes in endothelial permeability as the cause for increased Kfc.
Mechanism of Pressure-Induced Increase in Kfc
Our results, using an intact lung model, demonstrate that permeability changes as a function of vascular pressure and are similar to mechanotransduction pathways elucidated with use of cultured endothelial cells (12, 15, 16, 18, 31). These findings challenge the Starling principle and demonstrate non-Starling mechanism(s) to account for the development of pulmonary edema. Enhanced staining of NO-tyrosine during increased vascular pressure and the reduction of NO-tyrosine after heparanase treatment strongly supports our hypothesis for a HS-eNOS mechanotransduction pathway. In fact, direct measurement of tissue NOx showed increasing tissue NOx with increased vascular pressure. Heparanase III reduced tissue NOx concentration by 50% when Pla = 15 cmH2O in the Low Vt groups and reduced NOx by eightfold, when Pla = 17 cmH2O, during ST Vt. Lastly, GSNO increased tissue NOx and significantly increased Kfc during elevated vascular pressure. Taken together, these results support our conclusion that NOx is involved in barrier dysfunction. A schematic for the proposed mechanism of glycocalyx mechanotransduction is shown in Fig. 13.
Kuebler (24) has shown that an increase in Pla increases lung capillary NO formation, consistent with the findings of our study. In fact, high vascular pressure has been shown to activate cytochrome P-450 (CYP450)-dependent pathways in the lung resulting in increased Kfc and metabolites of the CYP450 pathway, including 20-HETE, can activate eNOS in pulmonary endothelial cells (13). Experiments to elucidate the mechanism(s) linking the glycocalyx to eNOS activation and alterations in barrier function are currently underway in our laboratory. Stretch-activated transient receptor potential (TRP) channels likely participate in lung vascular mechanotransduction and have been shown to modulate both CYP450 pathways and eNOS activation (5, 21, 44).
Airway Pressures and Kfc
A major finding of the present study was the combined influence of airway pressure and Vt on tissue NOx and Kfc when vascular pressure was increased. At ST VT, baseline Kfc was not different than baseline Kfc at Low Vt (Fig. 6A), thus endothelial permeability is not altered by the changes in Vt when Pla < 15 cmH2O.
When lungs were ventilated with ST Vt and Pla was elevated, Kfc increased to a greater extent than when lungs were ventilated with Low Vt at elevated Pla. For example, at Low Vt and Pla = 17 cmH2O, Kfc2 increased ∼5-fold (Fig. 2) whereas at ST Vt and Pla = 17 cmH2O, Kfc2 increased ∼15-fold. Thus inflation pressure had a significant effect on the permeability response to increased vascular pressure.
Heparanase and l-NAME had a greater effect in the reduction of Kfc during ST Vt compared with Low Vt. These results cannot be explained by changes in vascular recruitment, since ZC did not change with heparanase or l-NAME treatments.
Yin et al. (47) demonstrated that in statically inflated lungs NO participates in a negative feedback loop, via a cGMP mechanism, that attenuates hydrostatic pulmonary edema. They suggest that elevated vascular pressure activates stretch-induced calcium channel opening (presumably TRPV4) resulting in eNOS activation, NO-stimulated soluble guanylate cyclase activity, and cGMP-mediated inactivation of TRPV4 channels. In their model, l-NAME exacerbated pressure-induced Kfc and NO donors attenuated pressure-induced increase in Kfc. The major difference that explains the results of Yin et al. Compared with the present work is the role of cyclic ventilation in maintaining endothelial cGMP levels. Lung cGMP levels are positively influenced by mechanical ventilation and nonventilated lungs show a significant reduction in cGMP levels (8, 22, 34). A complex interplay between endothelial cell cGMP concentrations and barrier function has recently been described by Pearse and colleagues (34, 37) and Kuebler (23), who demonstrated that cGMP concentrations increased in lung endothelial cells in a Vt- and time-dependent manner. This increase in cGMP stimulates the activity of PDE2A, an enzyme that degrades cyclic AMP, leading to barrier dysfunction and elevated Kfc. Support for this idea can be inferred from the findings of Parker and Ivey (32), who reported that high vascular pressure-induced permeability was attenuated with isoproterenol, a α-adrenergic agonist that increases intracellular cAMP.
Direct measurements of tissue NOx in lung samples from our study revealed differences in the NOx concentrations vs. Kfc2/Kfc1 relationship (Fig. 12) depending on Vt; the slope of NOx vs. Kfc2/Kfc1 was steeper and left shifted in the Low Vt group compared with the ST Vt group. This means that during low Vt, vascular permeability was greater at a given NOx concentration. During ST Vt there is a large increase in tissue NOx in response to elevated vascular pressure; this supports our conclusion that the combined effects of alveolar stretch and endothelial mechanotransduction result in more severe edema development.
NO can contribute to barrier dysfunction through mechanisms other than altering cGMP-to-cAMP ratios. For example, NO and its reactive metabolites such as peroxynitrite can directly nitrosylate tyrosine residues on endothelial proteins, resulting in barrier dysfunction. NO• and ONOO•-induced nitrosylation of β-actin are principal mechanisms of TNF-induced barrier dysfunction (29). Our immunohistochemistry data demonstrated an increase in NO-tyrosine staining in lungs subjected to increased Pla and, conversely, a reduction in NO-tyrosine in heparanase-treated lungs. Superoxide anions, produced via the activation of NAD(P)H oxidase (17) and mitochondrial oxidases (4, 39), can react with endogenous NO to generate peroxynitrites, leading to oxidative barrier dysfunction. The lack of effect of TBAP, an intracellular superoxide dismutase mimetic, suggests that ROS are not the primary mechanism underlying pressure-induced increase in Kfc.
NO can also nitrosylate and activate conductance of the TRP-family ion channels that allow calcium influx and sustain the calcium-dependent activation of eNOS (48). Cytoplasmic cys553 and cys558 can be S-nitrosylated and appear to account for the direct NO sensitivity of TRPC5, TRPV1, TRPV3, and TRPV4 channel gating. Other TRP family members including TRPC1 and TRPC4 possess conserved cys residues on the putative pore region that predicts NO-sensitive activation. The balance between cGMP gating of stretch activated channels as proposed by Yin et al. (47) and NO activation of TRP channel (48) has yet to be reconciled in the lung vasculature. Nitrosylation of TRP channels and subsequent calcium influx may lead to continued activation of eNOS and resultant barrier dysfunction.
Clinical Relevance
The Acute Respiratory Distress Syndrome Network data has demonstrated that low-Vt ventilation improves outcomes in patients with existing lung injury (1, 6), whereas conservative fluid strategies improve pulmonary function and reduce ventilator-free days (46). From a clinical perspective, lung vascular mechanotransduction may contribute to worsening extravascular lung water resulting from overaggressive fluid resuscitation in combination with elevated airway pressure and may help explain the beneficial mechanism(s) of conservative fluid management and low-Vt ventilation.
This work was supported by National Heart, Lung, and Blood Institute Grant 5R01HL085255-04 and the Presidential Endowed Chair in Anesthesiology to R. O. Dull.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: R.O.D. and D.T.M. conception and design of research; R.O.D. performed experiments; R.O.D., M.C., J.K., D.H., H.C., S.H., D.T.M., and R.K. analyzed data; R.O.D. interpreted results of experiments; R.O.D. drafted manuscript; R.O.D. and J.K. edited and revised manuscript; R.O.D. approved final version of manuscript; M.C., D.H., H.C., and S.H. prepared figures.
We are indebted to Ibex Technologies (Montreal, Quebec, Canada) for the generous gift of high-quality heparanase III for use in this study.
A special thanks to Drs. Kurt Albertine, Robert Lane, and Lisa Joss-Moore for helpful discussions during this work and manuscript preparation.
- Copyright © 2012 the American Physiological Society