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The Mullaly skatepark opened in 1989 and was the first public ramp park in New York City. And it continued to be the only public ramp option in the city's five boroughs for ten years. It's safe to say that, along with the Brooklyn Banks, Mullaly was one of the most prominent BMX spots in New York City during the 1990s. Over the course of the park's 23-year history, Mullaly and the locals who called it home exposed countless young kids to BMX and skating while providing a positive outlet to keep them out of trouble. The layout of the ramps has changed countless times and their condition fluctuates just as often. Prior to the Get It Together jam, the ramps were small and sparse. After witnessing the ramps firsthand a few months ago, Base Brooklyn's Enos Columbo felt compelled to organize a jam to bring all the riders in the New York scene (and from anywhere else) together to ride, chill, have a good time, and of course—to give some much needed love back to the Mullay park. The day of the jam saw riders from multiple generations, from all across the country, and with every riding background and style you can think of sessioing together and having fun—and that my friend, is exactly what "Get It Together" was meant to be.
The story behind the Get It Together jam with Enos Columbo…
What was your motivation to put together this jam?
The motivation came from a few different things. The first would be about getting everyone together—I've been involved in BMX since the 80's and within that time I have seen it change a lot—some for the better and some for the worse. What really stuck out is that BMX in NYC has lost its unity or brotherhood. So the main goal was getting everyone together. Another factor was that I grew up on jams and contests at Mullaly during the late 80's and early 90's. There where some great contests at Mullaly back then, just ask Dennis McCoy, Steve McCann, T.J. Lavin, Dave Voelker… I could go on for hours with this one. I went up to Mullaly a couple of months ago and saw what used to be a great and first NY public skatepark was in shambles. The City of New York just gave up or cut financial support for Mullaly. What used to be a park full of crazy ramps was cut down to three small prefab ramps. So the idea was born, lets do a jam about bringing everyone together and rebuild the park at the same time. The final motivation came from Ed Pollio, Brendan Vail, Glenn P.P. Milligan, Ralphy Ramos, Dexter, Rob Ramos, and Ralph Sinsi.
Will this be taking place of the Mayday jam?
No, Mayday started out as a Grand Opening Jam for The Port Jefferson Bike Doctor, and to get the word out about Base Brooklyn making a return. It turned out to be way bigger than we had planned, but at the end of the day it was so successful because it was about everyone getting together for a fun day of BMX. I did plan to do Mayday again this year, but The Port Jefferson Bike Doctor was such a hit they had to double their shop and didn't have the time to put into the event this year. It was a little disappointing, but I'm not sure if we could have matched what we pulled off last year.
What are your thoughts on how the New York scene has grown? It's not uncommon to see a growing scene eventually splinter off into smaller groups and the sense of community to get lost a little…
The New York scene rules—always has. Street riding really started in New York. There where no parks back in the day. You either raced or freestyled, but if you lived in New York City there was no room for parks, trails, or racetracks so you rode street. What I have seen really change is that back in the day everyone rode together, it was all one click. It didn't matter if you where good or where you came from, if you rode BMX you where down. Today everyone is off doing their own thing. That's where I saw a problem. I knew we could do more as a group working together for both the NYC BMX scene and ourselves. That's why I linked up with Animal, your-inn.com, Group Home Bikes, and the shops that support the BMX lifestyle in NYC for this event and together we really did a lot. We didn't have the financial support of any corporate sponsors and we pulled off the best BMX event NYC has seen in years.
The ramps at Mullaly have changed a lot over the years (sometimes for better or worse), what were they like last time you were there?
Mullaly was always know for it's crazy ramp setups. I have seen it change a lot. One thing for sure, you either loved or hated the Mullaly ramps, but every time you went to Mullaly a good time was had. The last time I was there was almost 10 years ago, when Snapple put up some money for prefabbed ramps. Two months ago, the three ramps that where there was all that was still standing from the Snapple ramps. Really sucks that between the Parks Department and Yankee Stadium across the street no one shows the park any support.
I remember back in the day guys like Lou Perez would help hold it down, is there anyone looking over the place today, you know… to help with the upkeep?
Rob Ramos, who was Lou Perez's right hand man. Brendan Vail is also stepping up, he has set up Mullaly as a non-profit group to obtain some grants for the park and riders in the Bronx. Can't forget Casio, he's like the Mayor of the park. As for Lou Perez, he called me a few weeks ago and said he wanted to move back to the Bronx, I told him his timing was perfect, so hopefully he will be helping Rob with the park.
How did you get funding for the new ramps?
The ramps came from Ed Pollio of Group Home Bikes and 50/50 Skatepark. Ed had the ramps in Staten Island. They where used for one of his clients for an event and then Ed got them back. Animal paid to have them trucked over to the Bronx, and with a grant from the Citizen's Committee For New York City we where able to buy the plywood to redo the ramps with some fresh layers. Also, all the riders who came out for the rebuild day at Mullaly played a real part too.
What other brands and/or shops have helped out in some way?
Animal helped out with getting the ramps, and any last minute expenses we ran into. Group Home Bikes /50/50 Skate Park got us the ramps and their ramp building crew really did a great job. SuperBMX printed the event shirts. Grove Street Bike shop paid for the DJ. The Port Jefferson Bike Doctor threw up and ran best trick over the box jump. Your-inn.com got the grant from the Citizen's Committee For New York City and made sure everyone knew about the work party. Dah-Shop donated a bunch of used bike parts for the local riders. Base Brooklyn gave away a ton of shirts, ran promotions and media coverage for the event. Glenn P.P. Milligan for making us look good with his video edits.
I would also like to thank the following people:
Ralph Sinsi, Ed Pollio, Glenn PP Milligan, Keith Mulligan, Jeff Z., Brendan Vail, Chris Moeller, Aaron Nardi, Keith Terra, Matt Brown, Kyle Meeker, Steve Schuchman, Dan Thrax, DJ Dome, CoCo, Rich Soper, Lamar Davis, ECD Crew, Corey Pronsky, Rob Mic, Anthony Torriero, Lou Perez, Tyrone Williams, Ralphy Ramos, Rob Dolecki, Mike Ick, the Joes, Rob Ramos, Chris Gulich, Dexter, Casio, Amie Z. and everyone that came out that day.


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Jan 20, 2012
Interview with Muppeteer Peter Linz and Walter
All of my thanks, both professionally and personally, go out to Peter Linz for his kind heart, his witty mind, and his friendship. I know you're probably reading, Peter, so thank you! (PS - Still no PS.)
The Muppet Mindset by Ryan Dosier, email@example.com
Tags: Interview, Peter Linz, The Muppets, Walter
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subset: commoncrawl


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Woodall's 'Top 10' Campground News Stories
December 15, 2010 by RVBusiness · Comments Off on Woodall's 'Top 10' Campground News Stories
These, of course, are not regular times. These are post-recessionary times — emphasis on recessionary — when the U.S. is digging out of a tough situation. And, as you know, we're still digging, more so in some areas of the country than others.
Yet, as sister publications RVBusiness's and Woodall's Campground Management's small staff sat down to review the year that was, we couldn't help but marvel at the top-ranked story in our list below — the fact that the industry had indeed outperformed many other American business sectors for the second year in a row.
Without looking a gift horse in the mouth, it's hard to figure in times like these.
But maybe that's the point: Maybe times like these in some cases bring out the best of those lucky enough to enjoy them. Maybe we'll just chalk it up to North America's drive — lust might be a better word — for affordable recreation, a habit that the average family has retained throughout the global recession.
We've seen it at the tollgates in large public parks like Yellowstone and at the registration desks of hundreds of private campgrounds and resorts enough to know that it's real. Having said that, here's a quick look at our Top Ten campground news stories for 2010:
(1): RV park and campground business again bucks recessionary pressure to post gains in 2010. "Our members are generally reporting a better year than 2009, with the exception of the Gulf Coast areas," Linda Profaizer, retiring president and CEO of the National Association of RV Parks and Campgrounds (ARVC), noted in her President's Message.
"It appears that snowbirds are returning in bigger numbers than last year as well," she added. "Most parks report an increase in rental accommodations and tenting is still a major part of the picture."
Indeed, in his remarks during ARVC's annual meeting, ARVC Chairman David L. Berg said the economic downturn has actually created new business opportunities for private parks as families and other travelers look for more affordable ways to enjoy weekend getaways and vacation time.
It's not just an isolated trend. Industry leader Kampgrounds of America Inc. (KOA) reported year-over-year gains in each of the first 10 months of 2010 with total revenues up 8.5 % over 2009. For the summer camping season, same store revenues were up 6% and camper nights up 4.5%.
Leisure Systems Inc. ((LSI), franchisors of the Yogi Bear's Jellystone Park Camp-Resorts, reported same park revenue up 5% in 2010. While site revenue was down 4%, rental income was up 15%, store revenues grew 15% and miscellaneous revenue was up 19%. And LSI looks for more of the same in 2011, thanks in part to the release in late 2010 of a new "Yogi Bear" Warner Brothers movie starring the voices of Dan Akroyd and Justin Timberlake.
(2) The RV Centennial, the focus of which was in June when the ARVC Business Forum ventured to Elkhart, Ind., along with much of the recreational vehicle industry for Recreation Vehicle Industry Association's (RVIA) joint Annual Meeting and Committee Week proceedings and a party at the RV/MH Hall of Fame.
RVIA determined that 2010 was the centennial year for the industry, and what a year it was. The Go RVing Coalition promoted events throughout the year in conjunction with the centennial. The Coalition's "Ambassadors of Affordability" cartoon characters appeared in Go RVing TV spots, and "Centennial Charlie," a stuffed bear, made a PR tour across the country. The highlight of the year came in early June with a big bash in Elkhart, but other events were held across the nation at campgrounds, RV dealerships and elsewhere. It was, by most accounts, a public relations extravaganza.
(3) Gulf Coast RV parks share with other commercial segments in negative oil spill spillover. An explosion on April 20 at a Deep Water Horizon oil rig in the Gulf of Mexico killed 11 men working on the rig and set off a massive oil leak that would ultimately send more than 250 million gallons of oil into the Gulf and threaten hundreds of miles of ocean frontage.
The oil fouled beaches from Florida to Louisiana, stifling tourism for months. Business was down significantly at campgrounds across the region, even where the beaches were never involved. It was a major PR problem for the tourism industry, which was still gradually recovering at year's end.
(4) Flash flood kills 20 campers in June at Arkansas campground. Heavy rains the night of June 11 sent a wall of water in the "Loop D" area of secluded publicly operated Albert Pike Campground nearly Langley in western Arkansas. In all, 20 campers at the 54-site park were killed in the disaster,
The Caddo and Little Missouri rivers — two normally gentle waterways — rose by 20 feet overnight, engulfing the hikers and campers who were spending the night in tents along the rivers in the isolated Ouachita Mountains. "Within ten minutes the water had rose and campers were floating down," a survivor told ABC News. "If they didn't get out of their camper within five, ten minutes, they weren't getting out."
(5) Succession at ARVC — Linda Profaizer retiring and Paul Bambei succeeding her. Profaizer announced in the spring that she would be retiring after 10 years at ARVC's helm and 40 years in the industry — a tenure that included her time as president of then Chicago-based Woodall Publications Corp.
ARVC looked both inside and outside the RV park and campground sector for her successor and recently hired Comcast Corp. veteran Paul Bambei. He never owned a campground, but he's an avid RVer and was touted as an expert marketing executive. He was introduced at ARVC's InSites Convention in December.
(6) Succession at KOA — Pat Hittmeier named president of KOA in February, succeeding Shane Ott. A 29-year veteran of the Billings, Mont.-based franchisor's front office, Hittmeier was named president on Feb. 28 after Shane Ott stepped down. Hittmeier held several positions at KOA before taking on the presidency of the 475-member campground chain, and so far, from all we can gather, his relatively quiet demeanor and astute business instincts are serving him well.
(7) Virtual Campground Expo breaks ground on new era of trade shows. Campground vendor Art Lieberman earned praise in 2010 for introducing the industry's first virtual online trade show, a "Virtual Outdoor Hospitality Expo" that kicked off in early November and continues today for those choosing to pay the site a visit.
While some argue that these online expos are the way of the future, Lieberman gets an "A" for effort, but a "C" for the site's actual performance due to software issues that soured some on this landmark event. Lieberman, owner of MCPS for Campgrounds, was up front about the problems and pledged to try again, if not in 2011, then in 2012.
(8) Best Parks in America expands membership to 71 by year's end. Best Parks in America, launched in 2004 by industry consultant and entrepreneur David Gorin as a marketing network, must be somewhere close to reaching critical mass by now, as the 71-park organization held its annual meeting and a slate of seminars Dec. 1-2 at the Rio All-Suites Hotel & Casino in Las Vegas on the brink of the ARVC InSites convention.
Now, having made some serious changes over the past year, Gorin says Best Parks is getting ready to grow to the next level as an organization that will provide more business tools yet remain "a system of independent unique parks."
(9) CalARVC takes lead on holding tank chemical ban in California, despite legislative headwinds. Based on the premise that formaldehyde-containing products used in RV holding tank waste treatments have troubled RV park and campground septic systems for years, the California Association of RV Parks and Campgrounds (CalARVC) decided earlier this year that it was time for a change.
So, CalARVC lobbied extensively to ban six specific chemicals from all holding tank treatment solutions utilized in the Golden State, and the California State Assembly passed landmark legislation in late summer. However, outgoing Gov. Arnold Schwarzenegger vetoed the bill to ban the chemicals and referred the matter to a state agency for remediation while CalARVC Executive Director Debbie Sipe has vowed to march on with an education program.
(10) ARVC's ongoing headquarter saga. The national trade association announced in late spring its decision to move from Larkspur, Colo., a rural location to which it had moved in 2009 from Washington D.C.'s suburban Virginia, to more urban Castle Rock in the Denver area on the premise that it would be a better place in which to do business.
Then, in December, after some internal debate a few weeks ago, the ARVC board voted unanimously not to move to Castle Rock and instead authorized new CEO Paul Bambei to look elsewhere for suitable space in the greater Denver area.


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April 1, 2006
SANTA BARBARA, Calif. - The UC Santa Barbara men's tennis team crushed Big West rival the University of Pacific 7-0 Saturday afternoon at UCSB's Recreation Center Courts. After rallying to win the doubles point, the Gauchos won all six in singles to remain perfect in conference play.
Resuming Big West action, the Gauchos improve to 10-7 overall and 2-0 in conference dual meet action this season, while the Tigers' record dropped to 11-5.
In doubles, Nick Brotman and Anders Dalskov defeated Jorgen Vestli and Tim Koniarski 8-6, at No. 1 and Carsten Thorstensen and Scott Hohenstein teamed to handle Austin Kakar and Ahad Ebrahim 8-0 at No. 3.
Pacific, who was without two of its top players, posted its only victory of the day when Mortiz Starke and Alan Kelly defeated Elad Stern and Grant Robertson 8-4 in the second spot.
In singles action, Brotman was outstanding once again at the No.1 spot, beating Vestli 6-7, 6-2, (10-3) for his second win over a nationally ranked player this season. Bijan Hejazi and Dalskov posted impressive victories at the No. 2 and No. 3 spots, respectively, and the Gauchos last three singles players all defeated their opponents in straight sets.
Next up for UCSB is another conference opponent in UC Davis on Sunday. The match will begin at 12:30 at the Rec Cen courts. The Gauchos finish league play against UC Irvine on the road April 15th.
UCSB 7 - Pacific 0
Doubles 1. Brotman/Dalskov (UCSB) def. Vestli/Koniarski (UOP)-8-6 2. Storke/Kelly (UOP) def. Stern/Robertson (UCSB)-8-4 3. Thorstensen/Hohenstein (UCSB) def. Kakar/Ebrahim (UOP)-8-0
Singles 1. Nick Brotman (UCSB) def. Jorgen Vestli (UOP)-6-7,6-2 (10-3) 2. Bijan Hejazi (UCSB) def. Tim Koniarski (UOP)-6-2,3-6 (10-7) 3. Anders Dalskov (UCSB) def. Moritz Storke (UOP)-2-6,6-3 (11-9) 4. Elad Stern (UCSB) def. Alan Kelly (UOP)-6-1,6-3 5. Carsten Thorstensen (UCSB) def. Austin Kakar (UOP)-6-1, 6-4 6. Max Taylor (UCSB) def. Ahad Ebrahim (UOP)-6-2,6-2


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Unions Make Ridiculous Arguments Against Patent Reform
from the a-little-economics-would-be-good-here dept
The latest news is that Riley has convinced the AFL-CIO to come out against the latest attempt at patent reform. Now there's a lot to dislike in the latest attempt at patent reform, and we'd be upset if it passed as is. But the two specific things that the unions are complaining about are the two most reasonable things in the reform package. The first would change how damages are calculated, so that if the infringing component is only a small piece of a larger product, the damages shouldn't be based on the value of the larger product, but the value of that small piece. That seems completely fair. Why shouldn't the damages be reflective of the actual value? It's hard to see why that's controversial, but it is if you hold completely irrelevant patents and you want to hold up those companies that are actually making useful technologies. The second complaint is with making it easier to contest a patent after it's been issued. This is also a no-brainer. Given how little review goes into a current patent, along with the fact that patent examiners are given incentives to approve, rather than reject -- combined with the length of time it currently takes to get a patent reviewed, the incredibly arcane rules that everyone is required to go through to contest a patent and the quick draw of some courts who refuse to wait for the patent office to review patents, it makes sense to have a better system to make sure a patent is valid. Why would anyone be opposed to improving the quality of patents... unless they hold questionable patents?
Unfortunately, Riley's organization appears to have blinded the AFL-CIO to what's really happening. In convincing them that this is about stopping theft, he apparently left out all the economic research showing that it would actually do plenty of harm to the industries that most employ AFL-CIO workers. That's because it would limit their ability to innovate, make it more expensive to do research, and open up opportunities for foreign companies to do a much better job innovating and beating us in the market. Of course, given the history of the AFL-CIO, they must be used to that kind of effect, because that's been the result of previous policies in previous decades. In the meantime, Riley is hoping that by getting the support of unions, Democrats will feel compelled to vote against patent reform -- and, in fact, a Wall Street Journal article on this same subject suggests that the AFL-CIO's letter has been effective in slowing the reform effort. Hopefully, though, someone will explain to both the unions and the politicians the basic economics of monopolies and how they slow competition and innovation. If the folks at the AFL-CIO would like a detailed explanation for why their letter actually goes against the best interests of the people they supposedly represent, they should give us a call.


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NASHVILLE, Tenn. - The Lipscomb women's cross country squad knows what the top of the Atlantic Sun Conference feels like. The new goal, to learn what running at the NCAA National meet feels like.
Returning the top-seven performers from a year ago, Taylor thinks the goal is attainable.
"It gives us a huge advantage," Bill Taylor, director of cross country and track and field, said. "We clearly won last year, we didn't win huge but it wasn't close. Returning those seven makes us a year stronger and a year smarter which is a huge advantage."
The Lady Bisons are led by All A-Sun athletes Dani Walker and Tessa Hoefle on the first team and Kendra Lobley, Madi Talbert, Maggie Lawrence and Minna Fields on the second team.
Walker is returning from an injury suffered during the track season but Taylor isn't worried about the injury or having the team leader out there.
"Dani had a freak injury but is back," Taylor said. "She has been our leader. We are hitching our wagon to her but she won't be alone. We have Madi and she has the confidence from nationals last year. She is ultra-talented and is just figuring things out. Tessa has experience and has shown that she is capable of being up there.
"It is going to be a really good group. Going into the season we really don't have any idea of the order everyone will be and every week it could be completely different."
With the ability of only being able to run nine girls per meet, Taylor thinks there will be fierce competition within the team each week for those nine spots.
"We could have 19, 20, 21 women that have the ability to be in the top nine," Taylor said. "We won't have any drama though. The girls see this as a team and love each other. They accept where their position is. We have a healthy competitive environment."
Making the competition even stiffer is the stellar recruiting class Taylor has brought in this year. It is a class that he calls historical for Lipscomb.
The Purple and Gold landed Wake Forest transfer Meredtih Kilburn. The sophomore gained valuable experience at Wake Forest but comes to Lipscomb with the potential to be even better.
"She was a great high school runner and ran a sub-11," Taylor said. "Her Wake Forest career was good but not as good as she is capable of. She made their top seven, ran at conference and ran at the regional championships. She is talented and comes in with big race experience already."
Lipscomb also added two Footlocker National Finalists in Katie Bianchini and Kendra Weitz.
"Katie was on a team where she was the number two runner," Taylor said. "The top runner was one of the best in the nation. I think Katie was a top runner as well but was always number two. She was part of a really great program at Glacier Peak High School and has a chance to make the top group.
"Kendra was a finalist two years ago and ran a 10:20 which is incredibly fast for a girl. She has had some health issues in the past, so we are not going to rush her. If she contributes this year, great, but we want to give her every opportunity to have an amazing career here. We will error on the side of caution."
The Lady Bisons also added Lipscomb Academy graduate Barbara Lee Ball who was a three-time Tennessee State Cross Country Champion.
"We have gotten to see her quite a bit," Taylor said. "She is tough as nails with high desire and pretty good range. She will fight for her spot. She is going to do well. She will improve and will make other people better as well."
Although Taylor is excited about his talented group of freshmen, he cautiously points out that the success of this season does not hinge on their performance.
"Traditionally freshman have done very well for us," Taylor said. "However, none of these girls have to be in the top seven for us to do well. We return our top seven. There is no pressure on them to perform.
"We are going to be a really good team no matter how you look at it. The freshmen are just really free to run and see how good they can be."
Looking to reach their third consecutive A-Sun Championship the Lady Bisons will not be under pressure to do so. In fact, Taylor thinks there will be even less pressure than the previous two years, allowing the women to just run.
"The first year we were trying to do it for the first time," Taylor said. "North Florida was a rival for us, they looked a lot better most of the year and had beaten us the previous year. Last year trying to repeat, people were trying to psychologically get under our skin.
"This year we feel a sense of relief. There is so much confidence. We have other goals regarding conference now. We still have to take care of conference, but we are gearing toward the South Region.
With the goals now towards the region and national meet, Taylor thinks he has the team to do it.
"Our goal is to go to nationals," Taylor said. "Our goal is to win nationals. These girls buy into that. They are 100 percent committed to that."
Lipscomb gets its season underway Aug. 30 at the site of the 2013 A-Sun Championship at Percy Warner Park. The race is scheduled to begin at 4 p.m.


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8:00 am
Dialogue "Naomi Shihab Nye: Conversations from the Sun Valley Writers' Conference" The Palestinian-American poet, essayist, novelist and teacher talks with host Marcia Franklin. In her writing, she is inspired by her experiences as an Arab-American, as well as the richness and diversity of people and places she has encountered in the U.S. and abroad.G
8:30 am
Outdoor Idaho "Chasing The Light" Some of Idaho's professional landscape photographers share their secrets for positioning themselves to capture a special scene in a photograph, often waiting hours, even days, for the natural lighting they seek to preserve. OUTDOOR IDAHO travels with them to locations that encapsulate Idaho's treasured scenery.G
9:00 am
Oregon Field Guide "Tillamook Railroad, No Ivy League, Missoula Floods Erratics" NULL
9:30 am
Roadtrip Nation "Santa Fe - New Orleans" The team drives east and stops in Santa Fe, New Mexico to interview Glenna Goodacre, a renowned sculptor, before heading into Roswell, the UFO Capital of the World, to speak with actor Malcolm McDowell, who starred in Stanley Kubrick's A Clockwork Orange. In New Orleans, the team munches on beignets and spends a long afternoon with Matthew Nolan, poet and author of Crumpled Paper Dolls. The artists they visit during this period have a significant impact on Autumn, who wants to be a writer. D
10:00 am
Secrets of the Dead "The Man Who Saved The World" Little known events unfold inside a nuclear-armed Soviet submarine during the Cuban Missile Crisis. This program combines tense drama with eyewitness accounts and expert testimony to reveal how Vasili Arkhipov, an officer aboard the submarine, refuses to fire a nuclear torpedo and averts disaster.G
11:00 am
Richard Bangs' Adventures With Purpose New Zealand: Quest for Kaitiakitanga The adventurer pursues an age-old Maori tradition - the origins of "kaitiakitanga," the responsibility of human beings to protect the natural world. He begins at Mt. Aspiring National Park, strikes north to the Franz Joseph Glacier, heads east to the coast at Kaikoura, then crosses the Cook Strait to the North Island and the capital city of Wellington. His journey culminates at Cape Reinga on the upper tip of New Zealand.G
12:00 pm
Wild! "In The Company of Moose" Biologist Vic VanBallenberghe has spent more time living with wild moose than anyone in the world. So much time in fact that he was absent for much of the childhood of his only son. Determined to learn more about the father he hardly knows, Vic's son, documentary filmmaker Jonathan VanBallenberghe, chronicles a season of his father's research at Denali National Park in Alaska and tells the story through his film. While capturing the most extensive, amazing, and up-close HD footage of moose ever caught on film and learning about this majestic animal, Jonathan recognizes the significance of Vic's life work, and a gap is bridged between father and son.G
1:00 pm
Globe Trekker "Spanish Islands" Alex Riley explores the Balearics and the Canary Islands.G
2:00 pm
Need to Know MARIA HINOJOSA ANCHORS. This week, Need to Know's Rick Carr reports on controversial changes to election rules in the battleground state of Florida- will they root out voter fraud or keep legitimate voters from the polls? Also, a conversation with James Carter, the man behind the infamous '47'%' video, who, like other amateur opposition researchers, is harnessing the power of the internet to upend campaigns. And from our "American Voices" series, comedian Dean Obeidallah on one group of voters that no one seems to be courting. D
2:30 pm
McLaughlin Group "Homestretch Face-Off (The Third Debate)" Pat Buchanan, Author & Columnist; Tim Carney, The Washington Examiner; Eleanor Clift, Newsweek; Mort Zuckerman, US News & World Report. D
3:00 pm
Biz Kid$ "What's in the Books?" Poor recordkeeping adds up to lost money. This episode examines bookkeeping and accounting, income and expenses, profit and loss, and gives tips on how to get over math anxiety (in case you have it). Meet the youngest Harlem Globetrotter who does a slam dunk with his books. D
3:30 pm
Oregon Field Guide "Prescribed Natural Fires, Raptor Recovery" NULL
4:00 pm
Cook's Country from America's Test Kitchen "Retro Desserts" Bridget uncovers the secrets to perfect Banana Pudding. Then, Adam reveals his top picks for finemesh strainers. And finally, Erin updates a lost recipe and shows Chris how to make the ultimate Chiffon Cake. D
4:30 pm
Rick Steves' Europe "France's Dordogne" Rick takes a canoe ride, visits a goose farm and savors the foie gras, wanders through a lamp-lit castle, enjoys a country market, and visits the Sistine Chapel of the prehistoric world.G
5:00 pm
NOVA "Iceman Murder Mystery" He's been dead for more than 5,000 years. He's been poked, prodded and probed by scientists for the last 20. And yet today, Otzi the Iceman, the famous mummified corpse pulled from a glacier in the Italian Alps nearly two decades ago, continues to keep many secrets.G
6:00 pm
My Vietnam Your Iraq The film tells stories from eight Vietnam War veterans who have children that have served in Iraq, including an Idaho man whose son was killed in action while serving in Iraq. The program showcases the pride, fear and myriad of emotions and challenges that parents and their children face during deployment.G
7:00 pm
Voces On PBS "Escaramuza: Riding from the Heart" A team of first-generation Mexican American girls construct their identities through a romanticized equestrian tradition, while facing challenges at home, and escalating violence in Mexico. D
8:00 pm
Cuban Missile Crisis: Three Men Go to War In October 1962, the world teetered on the brink of nuclear holocaust. The film brings to life the three central characters - Kennedy, Castro and Krushchev - and explores how these powerful men fell into an abyss of their own making and outlines the courage and luck it took to climb out again.G
9:00 pm
Antiques Roadshow "Milwaukee, WI - Part 3" Experts discover a valuable Persian rug the owner used to cover furniture in the rain and a stained glass figurine of horse racing legend Dan Patch. Two milestone game baseballs autographed by Milwaukee Braves legend Warren Spahn are valued up to $14,000. Part 3 of 3G
10:00 pm
My Vietnam Your Iraq The film tells stories from eight Vietnam War veterans who have children that have served in Iraq, including an Idaho man whose son was killed in action while serving in Iraq. The program showcases the pride, fear and myriad of emotions and challenges that parents and their children face during deployment.G
11:00 pm
Voces On PBS "Escaramuza: Riding from the Heart" A team of first-generation Mexican American girls construct their identities through a romanticized equestrian tradition, while facing challenges at home, and escalating violence in Mexico. D


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The Bluffton High School marching band is on a meteoric rise by winning a second division championship Saturday in the Garden City Band Classic. The band moves on to the Lower State championships on Saturday at West Ashley High School.
Under fourth-year director Anthony Granquist, the band is having its most successful season to date. The band has entered three competitions this year and came away with two division titles.
Its first competition was at the Carolina Low Country Tournament of Bands in Bamberg. Bluffton placed first in Class AA, defeating Hilton Head Island and Barnwell and receiving awards for best color guard and best percussion. At its second competition, the Walterboro Band Classic, the band placed second in Class AA, finishing ahead of Beaufort and Berkeley. Dan Brown received the award for best drum major in the class.
At the Garden City Band Classic in Orangeburg, Bluffton received its first ever rating of "superior" and placed first in Class AAA and second overall. The other bands in Class AAA were Hilton Head Island Lakewood. The band also won the awards for best color guard and best drum major.
Brown received a drum major score of 96 out of 100, the highest released score this year in South Carolina.
The Bobcats will compete Satuday at Lower State and have the opportunity to become the first Beaufort County school to reach the Class AAAA finals.


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Farmers in east Tennessee are harvesting 723 acres of switchgrass this fall which a new cellulosic ethanol plant will turn into 'grassoline.'
The plant, which is under construction at the Niles Ferry Industrial Park in Vonore, Tenn., 32 miles southeast of Knoxville, is part of the University of Tennessee Biofuels Initiative, a state-sponsored plan whose objective is to advance a biofuels economy in the state.
"Our goal is to go from the farm to the filling station," said Kelly Tiller, director of external operations, Office of Bioenergy Programs, University of Tennessee. "We want to demonstrate farm production, conversion to ethanol and end use." Tiller spoke at the West Tennessee Alternative Crop Conference at the Fogelman Executive Conference Center at the University of Memphis.
UT created a for-profit company called Genera Energy to construct and operate the new facility with a private company called DuPont Danisco Cellulosic Ethanol. Ground was broken on the Vonore plant Oct. 14. When completed, the principle product of the biorefinery will be "grassoline," ethanol fuel derived from plant material such as switchgrass, wood chips and other forest and agricultural biomass. Expected capacity is 250,000 gallons of cellulosic ethanol annually.
"We're doing this in phases," Tiller said. "Switchgrass was planted on 723 acres in 2008, and we're in the process of harvesting that now. We're going to contract for another 2,000 acres this coming spring, and 3,000 acres for the spring of 2010."
The acreage is expected to produce more switchgrass than the biorefinery can process, according to Tiller, "but we think this is a good opportunity for farmers to get the biomass growing in advance of there being lots of cellulosic ethanol opportunities."
Meanwhile the excess switchgrass will be pelletized "for use as green power, for example being co-fired in coal-burning power plants."
Almost 1.5 million acres of land in Tennessee has been identified as having a good fit for growing a dedicated energy crop "in a sustainable way that does not influence the food, feed, fuel, fiber balance," Tiller said.
West Tennessee farmer Willie German attended the conference to gauge the potential value of alternative crops to his cotton and grain operation in Somerville, Tenn. "Looking at what has happened to our cotton price recently has us wondering if cotton is going to be a viable option for us. We lost our Step 2 program, and it doesn't look like we can get the price up high enough to grow cotton profitably.
"I farm in an area where there is very little irrigation, so some years, even with good soybean and corn prices, we don't always produce the yield we need to make a profit. We need other options.
"I'm here to grasp a little bit of what's coming. It's good to see that this much money is being spent to develop an alternative to these fossil fuels. I want to stay on top of it, and when the price they want to offer us to grow some of these crops looks good, I want to be able to put my name in the hat."
R.D. James, a farmer and cotton ginner from New Madrid, Mo., is already familiar with switchgrass, having grown it in strips for the Conservation Security Program.
James attended the conference "to learn more about where the experts think this is going. Is it just going to be a bubble — farmers put a lot of money into new crops, lose the relationships in the crops they were growing, then three years later, the new crops go away?
"I think what I'm hearing here today is that is not the case. The way the experts see it, switchgrass is going to be a very useful crop."
The plight of cotton, with its high input costs and low prices relative to grains and soybeans, is another reason why James attended the conference. "We've been in the cotton ginning business since 1937. We've been down before, like the 1970s, but it always seems to come back. But the U.S. cotton industry has lost its spinning mills to China and India and most of the cotton we produce in the United States is exported.
"Our cotton industry has become an afterthought to the world economy. If that continues downward, then I have to figure out some other way to make a living. I don't want to wait until it happens before I do something about it."
Tiller says the pellet mill and biorefinery are expected to come on line by the end of 2009 in time for the second year of switchgrass harvest. Tiller noted that all of the signed up acreage will be within a 50-mile radius of the biorefinery. All of the farmers sign three-year contracts paying $450 per acre per year, with UT providing seed and technical expertise.
Tiller noted switchgrass harvested in 2008 will be stored, which will present UT with the opportunity to study various storage options for the feedstock. "We need to know what happens to a bale of switchgrass if it sits out in the open for a length of time."
Some farmers have reported switchgrass yields approaching 5 tons per acre this fall, which is surprising for an establishment year. "But we've had some fields that didn't do as well," Tiller said.
West Tennessee has the potential to produce nearly 7 million dry tons of switchgrass per year.
The Tennessee legislature approved funding of about $70 million for UTBI in June 2007 to develop the biorefinery project. Tiller says UT hopes to move toward a commercial scale biorefinery by 2012.
The conference was sponsored by the Tennessee Department of Agriculture and Memphis Bioworks Foundation.
e-mail: firstname.lastname@example.com


text:
Relaxed Fit Check Trench Coat
€1,790.00 €1,790.00
€900.00 €900.00
All orders will be fulfilled by Burberry Limited and shipped from the UK. When you place your order, you accept Burberry's Terms and Conditions and confirm that you have read our Privacy Policy.
Size Guide
Use the Size Guide to choose the best fit for your measurements.
In this window, you can find out the shipping and returns options for the selected item.
In this window, you can contact customer service for further assistance.
- A lightweight trench coat in a small-scale Italian check. Cut in our Westminster fit, the relaxed style is made for easy layering.
- Coat length: 105cm/41.3in. This is based on a size UK 10 as proportions change slightly according to size.
- Model's height: 178cm/5ft 10in
- Outer: 100% viscose
- Lining: 100% viscose
- Trim: 100% leather
- Buckles: 100% leather
- Double-breasted closure
- Button-through welt pockets
- Signature details: epaulettes, hook-and-eye collar closure, gun flap, belted cuffs, D-ring belt, check undercollar, storm shield
- Specialist dry clean
- Imported
Item 80023461
Wear this with
The Patent Leather Rope Stiletto
€260.00 €520.00


text:
Product Instructions
Download our Product Instructions and Activity Guide:
View now:
Aligned to:
L.4.4. Determine or clarify the meaning of unknown and multiple-meaning words and phrases based on grade 4 reading and content, choosing flexibly from a range of strategies.
L.4.5. Demonstrate understanding of figurative language, word relationships, and nuances in word meanings.
L.4.6. Acquire and use accurately grade-appropriate general academic and domain-specific words and phrases, including those that signal precise actions, emotions, or states of being and that are basic to a particular topic.


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keppy33 needed a new stationery design and created a contest on 99designs.
A winner was selected from 62 designs submitted by 30 freelance designers.
Teh Mafia Hits
We are a young company that is trying to convey both energy and ability. We do want to have a classy appearance that shows we are professional.
We are trying to attract Weddings, Proms/Dances, and graduation parties younger crowd most likely 22-37 year olds
two sided would be ideal front has logo back has what we do. Weddings, Proms, graduation parties, and dances.
Background dark
a blue or black
maybe a picture of speakers and lights
a white or maybe florescent color
Name : Teh Mafia Hits ( that is not a misprint)
Number : 515 290 0562


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I totally love Ellen too!!! She is so funny!! And I loved Portia when she was on Ally McBeal. I thought she was soooo pretty. I only just found out like a year ago she was a lesbian and was like, "Really!?!!" My gay-dar sucks by the way..A few years back, I was totally oblivious to Elton John being gay. I said, "I thought it just liked wearing really colorful, and flamboyant looking attire.." So yeahs, Ellen is totally awesome. I miss the Ellen Show..not the talk show but the sitcom she did.akire wrote:
Also Ellen makes me LOL. I want to move in with her & Portia. Do you think they will adopt me? can eEllen move into the e-house? and ePortia? We can make them e-veggie bacon and borrow their e-shoes. And eEllen will e-host the e-dance parties!! And ePortia will just walk into e-rooms and take down her hair dramatically.
I wouldn't mind them being my e-mums.


text:
Despite our best efforts to save a tooth, sometimes extraction is the only alternative. Many factors can contribute to this – extensive trauma, decay, gum disease or badly cracked or broken teeth. We here at Hillview Family Dental are committed to providing a comfortable, pain-free extraction procedure.
No one likes the idea of loosing a tooth, however there are options we can provide to fill the empty space or replace the tooth. One choice is adding implant retained dentures, which are dental implants that are attached directly into your gums securely, without sliding or slipping. These implants also look and feel like ordinary teeth. If numerous teeth need replaced, traditional and premium denture options are available as well.
If you have any questions or concerns regarding extractions or to learn more about restorative dental options, please contact Hillview Family Dental at 941-365-4500 today.


text:
This is a place to talk about motorcycle insurance. Discuss motorcycle insurance policies, quotes, companies, and more. Please do not talk about car insurance in this forum. Keep all posts related to motorcycles.
Use this control to limit the display of threads to those newer than the specified time frame.
Allows you to choose the data by which the thread list will be sorted.
Order threads in...
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text:
I had to include a sugar cookie on my last recipe for the 12 Weeks of Christmas Cookies. As much as I love cut out sugar cookies, it is very time consuming. So, I went with an easy drop sugar cookie with a unique cracked top. A little crunchy with a light sugary coating on the outside makes for a really good sugar cookie.
Cracked Sugar Cookies
Recipe slightly adapted from Allrecipes & Savory Sweet Life
Yield: 24 cookies
Ingredients1 cup butter, softened
1 1/4 cups white sugar
1 1/4 cups white sugar
3 egg yolks
1 teaspoon vanilla extract
2 1/2 cups all-purpose flour
1 teaspoon baking soda
1/2 teaspoon cream of tartar
Turbinado (coarse) sugar or granulated sugar
1. Preheat oven to 350 degrees F. Lightly grease 2 cookie sheets or use parchment paper.
2. Cream together sugar and butter. Beat in egg yolks and vanilla.
3. Add flour, baking soda, and cream of tartar. Stir.
4. Form dough into walnut size balls and roll in sugar of choice. Place 2 inches apart on cookie sheet. Don't flatten. Bake 12 to 15 minutes, until tops are cracked and just turning color. Remove from oven and allow to cool slightly before moving to a cooling rack.
Note: I rolled half the sugar cookie dough in coarse sugar and the other half in granulated sugar. The ones with the coarse sugar had a little extra crunch to them.
It's been alot of fun participating in these cookie posts the last 12 weeks. With the linky blog hop attached each week, I now have a ton of cookie recipes to try. And so do you! Check out the other cookies/treats I made the last 12 weeks:
Cracked Sugar Cookies
Mom's Cooking Club Lesson: Gift/Packaging Idea - These cookies are for Santa. So, place on a cookie plate and leave out with a tall glass of milk. Merry Christmas everyone!


text:
Met Office Datapoint API - Sample Source Code
The service provides access to weather and climate datasets, including 3-hour and daily forecasts for 5000 UK locations and actual recorded observations of weather events from the previous 24 hours. The service can provide data for individual sites or for all sites for which observations and forecasts are made. Additional functions provide listings of sites and reporting capabilities. API methods support requests for either 3-hour or daily forecasts by site or for all sites during specified time periods. Methods also accept requests for weather observations with similar site and time parameters.


text:
Workspace Refresh: Alphabet Art
Depending on where you live, school is back in session or will be in the coming weeks. So it's the perfect time to freshen up a child's workspace or bedroom with some alphabet art. Here are ten more to add to our growing collection in a range of style and themes.
1. Illustrated Letters, Set 1 by Chris Rushing ($15 - 48, Society6)
2. ABC Print by JooJoo ($39, JooJoo on Etsy)
3. ABC Ologies Wall Decal by Aaron Christensen ($39, Land of Nod)
4. ABC Poster 1 by Erin Althea ($22, Erin Althea on Etsy)
5. "Shadow" by The Head Light Hotel ($25, Tiny Showcase)
6. London A-Z by Helen Lang ($95.42, The Calm Gallery)
7. Screenprinted Alphabet by Bombina Studios ($87.60, Bombina Studios on Etsy)
8. Alphabet in Motion by Erica Krystek ($108 - 510, Minted)
9. Safari Alphabet Print by Small Fry ($13 - 27 AUD, Small Fry)
10. Animals - Black by Neighborhood Studio ($40, Neighborhood Studio)
Want MORE alphabet art? We have a whole Pinterest board to look at with over 120 prints and posters.
(Images: as linked)


text:
WELLESLEY, Mass. – Seniors Zach Kabelac (Potomac, Md.) and Max Stein-Golenbock (Boston, Mass.) along with junior Ben Lewis (West Hartford, Conn.) were voted to the first team of the NEWMAC Men's Soccer All-Conference Team on Tuesday. For the second year in a row MIT had three first team placements and extended its streak to 13 consecutive seasons with multiple honors.
Kabelac became the third player in program history to collect first team accolades all four seasons, joining Christian Therkelsen '10 and Nick Nestle '04 on the list. He graduates tied for second in goals (40), third in points (97), and fifth in assists (17). Kabelac ranked sixth in the NEWMAC in goals (eight) and seventh in points (19) while tying for first in game-winning goals (four).
Lewis collected his third league award and second straight first team honor after totaling two goals, both which were game-winners, and two assists for six points. After a pair of second team awards, Stein-Golenbock was tabbed to the first team for the first time in his career. A steadying presence in the midfield, he posted one goal and two assists for four points.


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Difference between revisions of "Talk:20.109(S08):Protein-level analysis (Day6)"
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Revision as of 10:46, 9 May 2008
- 1 Tuesday/Thursday Red
- 2 Tuesday/Thursday Yellow
- 3 Tuesday/Thursday Green
- 4 Tuesday/Thursday Blue
- 5 Tuesday/Thursday Purple (Violet!)
- 6 Tuesday/Thursday Pink
- 7 Wednesday/Friday Red
- 8 Wednesday/Friday Yellow
- 9 Wednesday/Friday Green
- 10 Wednesday/Friday Blue
- 11 Wednesday/Friday Purple
- 12 Wednesday/Friday Pink
Tuesday/Thursday Red
We chose to vary the viscosity of our alginate beads. We used 500,000 cells for our 2-D cultures and a density of 5,000,000 cells/mL in our 3-D samples. For our low-viscosity 3-D sample, we used Sigma-Aldrich alginate, while for our high-viscosity sample, we used FMC 10/60; both alginates were used at 2%. In addition, we added ascorbate to our 3-D samples only to enhance the contrast between our 2-D and 3-D culture results. Our light microscope cell count showed very few cells in our 3-D samples; however, a microscope examination of our alginate beads showed extensive cell populations. This discrepancy was possibly due to errors made in the cell isolation process. As a result, our cell viability assay was not very informative, since so few cells were present for analysis. To analyze the differentiation state of our cells, we performed RT-PCR on our cells to isolate cDNA for collagens I and II. RNA extraction was very successful for all of our samples, especially the 3D-High sample. Analysis of the cDNA gel showed a sizeable presence of cDNA in each sample. The 2D sample yielded the highest Collagen II : Collagen I cDNA ratio, followed closely by our 3D-High sample. Our 3D-Low sample yielded the least cDNA, and also had almost a 1:1 ratio of Collage II : I. These transcript results seem to indicate that our cells were relatively healthy and maintained chondrocyte differentiation in 2D culture and high viscosity alginate, but were not so in low viscosity alginate. Unfortunately, our ELISA data yielded the exact opposite results. Only our 3D-Low sample yielded significant amounts of Collagen II, but all samples produced a sizeable amount of collagen I. This would suggest that the low viscosity 3D alginate is best for maintaining chondrocyte differentiation. The extreme discrepancy in our results is most likely due to imprecise execution of the ELISA protocols: using our standards curve, our fitted data gave significantly negative (less than 0) collagen II concentrations for most of our samples.
ELISA Results
|Collagen II
|Collagen I
|2D - Sup
|2D - Cells
|3D Lo - Sup
|3D Lo - Cells
|3D Hi - Sup
|3D Hi - Cells
|Cell count per mL
|3D Low Viscosity
|3D High Viscosity
|RNA Concentration (ug/mL)
|3D Low Viscosity
|3D High Viscosity
Cell volumes for ELISA
|Volume Used(uL)
|3D Low Viscosity
|3D High Viscosity
|RT-PCR Sample
|3D Culture 1
|Low-viscosity alginates
|3D Culture 2
|High-viscosity alginates
We used the standard 200ng of RNA per sample for the RT-PCR.
Tuesday/Thursday Yellow
In our experiments we added Collagen II to one of our 3D 1% alginate samples. Our other 3D and 2D samples were cultured normally. Our hypothesis was that the presence of collagen II would reduce the amount of dedifferentiation of the chondrocytes to fibroblasts. When we observed the cells after they were grown for a week, the morphology of the cells in our 3D cultures retained a round phenotype, suggestive of a chondrocyte phenotype. However, the cells in our 2D cultures appeared more spread out, suggesting dedifferentiation info fibroblasts. For each of our cultures, we recovered about 100,000 cells/mL but recovered slightly more (about 155,000 cells/mL) for the 3D culture with collagen. We noticed that the cells in the 3D cultures were very small, but rounded, and we found it difficult to distinguish between simply debris or a very small cell. However, few cells, live or dead, were observed under microscopy in the LIVE/DEAD assay. This may be due to the fact that it was difficult for us to observe cell pellets before aspirating our supernantants and we could have lost cells at this step in the LIVE/DEAD assay. Despite our concerns with having low cell yield and lack of results for the LIVE/DEAD assay, isolation of the RNA for our RT-PCR reactions showed mRNA concentrations of 2D: 6.8ug/mL, 3D+coll: 14 ug/mL, and 3D+none: 8.4 ug/mL. We split our sample for RT-PCR and ELISA, using 1 mL for each. We used 100 ng of RNA for each PCR reaction. We ran the PCR fragments from our RT-PCR on an agarose gel with the intention of comparing the relative intensities of Collagen I and Collagen II cDNA. The samples were run as the protocol described, with 3D-1 corresponding to none and 3D-2 corresponding to with collagen added. Unfortunately, faint bands in many of the lanes could not be photographed and therefore full image analysis of the gel could not be performed. Even so, our image analysis of our 2D sample surprisingly suggested that our 2D samples produced more collagen II than collagen I despite their previously observed fibrobast phenotype. No 3D collagen ratios could be calculated by detection by the computer since no 3D bands were visible on the photo. In general, our collagen II bands, though faint, were stronger than the collagen I bands (many of the collagen I bands were completely missing all together). Our calculated 2D collagen II to collagen II ratio was 1.30, which we didn't expect as we believed that the 2D culture would promote dedifferentiation of chondrocytes. After performing ELISA, we calculated standard curves. After removing outliers for our standard curve data, we were able to calculate measurable quantities of both collagen I (both initial and overnight) and II. All collagen II/I(overnight) ratios were ~.06, except the 3D collagen supernatant which was ~.13. One possible explanation for the large difference between our calculated ratios by transcript analysis and ELISA analysis may have been due to cross-reactivity of our collagen I primary antibodies. This would explain the much higher collagen I concentration obtained through ELISA analysis.
Tuesday/Thursday Green
In our experiment, we chose to test the effect of cell density on cell viability in 1% 3D Sigma alginate scaffolds. Our respective densities were 5x10^6/ml (3D1) and 20x10^6 cells/ml (3D2), versus our 2D T25 sample of 2.5x10^5 cells. Cell counting for the 3D1 sample revealed 4 times as many dead cells for a concentration of 2222 cells/ml, whereas the 3D2 sample revealed approximately 2 times as many live cells per dead cell for a cell count of 3.56x10^5 cells/ml. The 2D sample had a cell count of approximately 3x10^5 cells/ml. Observation under the light microscope also revealed small cell clusters in the 3D2 beads but not in the 3D1 sample beads. 2D cells were slightly round but were starting to exhibit a fibroblast-like phenotype with projections attached to the plastic.
The RNA was isolated, and the total RNA concentrations were 0.0467 ug/ml for the 2D sample and 4.678 ug/ml for the 3D2 sample. The 3D1 sample had a low cell count and unusable (negative) OD value. Therefore, RT-PCR was not performed on this sample. RT-PCR was performed on 7.5ul (50ng) of 2D RNA and "0.1754ul" (50.25ng) of 3D2 RNA. For our RT-PCR gel, the lanes were as follows: (1) ladder, (2) ColI 2D, (3) ColI 3D2, (4) ColII 2D, (5) ColII 3D2, (6) Col I control, (7) Col II control. Analysis of the gel indicated that the ratio of collagen II to collagen I was 2:1 in general for both 2D and 3D2.
For the ELISA sample prep, we obtained a pellet from 1ml of each sample after cell counting. Our ELISA results, interestingly, indicated that for at least the 3D2 sample, the colII:colI ratio was 1:6. This difference in ratios may be due to the direct measurement of proteins versus measuring expression "indirectly" from the supernatant or cells themselves. All collagen I samples had measurable amounts of protein, but for collagen II, only the 3D2 sample had a non-negative number.
Tuesday/Thursday Blue
For our experiment, we chose to vary the cell density added to the 1% alginate beads. All other conditions were held standard. Additionally we prepared a 2D sample with 1 million cells, in the interest of comparing to other groups who prepared 2D samples with varying cell counts. Our two 3D samples were .5 million cells/mL (3D-1) and 10 million cells/mL (3D-2). The results of our light microscope cell count showed no cell recovery for 3D-1. However, 2D and 3D-2 showed comparable cell counts of 310,000 and 270,000 cells/mL, respectively. In all cases the cell count was lower than expected. This most likely due to poor recovery of cells from media and not cell death, since we did not observe dead cells. Our live-dead assay showed no cells for 2D and 3D-1, which could be due to poor staining, dye bleaching, or, again, poor cell recovery from the media. For 3D-2, we observed one live cell (fluoresced green but not red). We then ran RT-PCR on the cDNA from the samples to test for collagen 1 and collagen 2 production. We first purified RNA from the three samples and obtained concentrations of 2 ug/mL, 40 ug/mL, and 45.6 ug/mL, for samples 2D, 3D-1, and 3D-2, respectively. After running RT-PCR on the RNA samples and analyzing 60 ng RNA total from each on agarose gel (using the standard lane schematic), the gel showed no collagen of either type for our 2D sample. This could be due to any number of problems, from low cell viability rate, to poor purification of mRNAs from the cells, or inefficient reverse transcription of the RNA to form cDNA (perhaps because of issues with the primer), or even problems with the PCR step itself. However, for 3D-1, the RT-PCR gel shows that a small amount of collagen 2 was produced, and that for 3D-2, a small amount of collagen 1 and a relatively large amount of collagen 2 were produced. The presence of collagen 2 in these two samples seems to indicate that the cells mostly retained chondrocytic phenotypes, as it is in this phenotype that higher amounts of collagen 2 are produced. These results could also indicate that growing cells in a 3D alginate setting causes increased production of both collagen types, since our 2D culture did have a significant cell count, and it seems unlikely that the RT-PCR failed to work properly only in both 2D samples. From this analysis, we were only able to determine the CNII:CNI ration for 3D-2 sample, which was approximately 3.25:1. We also ran ELISA as a secondary protein-level assay of the ratio of collagen types; however the standards we used were underdeveloped and we did not run our actual samples, so we were unable to determine any ratios (or really, any data at all) from the ELISA experiment. Overall, we were able to determine that for our high cell density culture, collagen II was produced approximately 3X as much as collagen I, which suggests that this sample retained the chondrocyte phenotype, as it is in this phenotype that collagen II is predominately produced.
Tuesday/Thursday Purple (Violet!)
Experimental Background and Set-Up After researching the various external and internal influences that affect the chondrocyte phenotype of cells, we decided to target the viscosity of our alignate beads along with the Red group. We targeted the G/M ratio of the alginate in order to attempt to influence the structure of the polymer formed. For the 3D sample, we altered the cross-linking of the polymer. In one sample we used Sigma Aldrich alginate, this serves as our control. We then decided to use a high cross-linking subject of FMC Biopolymer (Protanal 10/60) to increase the stiffness of the alginate polymer. It has an increased cross-linking due to its high G/M ratio. We suspect that there will be an increase in the production of collagen I and an increase in the cell dedifferentiation with the high cross-linking samples. The Sigma Aldrich sample has low cross-linking and we hypothesize that there will be an increased production of collagen II. We also added ascorbate to the 3D samples to attempt decrease dedifferentiation where we did not add any to the 2D samples. The lack of ascorbate in the 2D samples will cause the cells to dedifferentiate faster. Overall, we hypothesized that the 3D sample with higher cross-linking factor will have chondrocytes become fibroblasts more quickly. Also, the 2D samples not containing ascorbate will also make fibroblasts faster.
For the 3D samples, we used a density of 5 x 10^6 cells/mL and only 5 x 10^5 cells for our 2D samples. For our low viscosity level assay, we used 1% Sigma ALdrich alignate while for the high-level we used 1% FMC Biopolymer. In addition, we added ascorbate to our 3D samples only to create a contrast between our 2D and 3D culture results, as explained above. The 2D sample was split at first confluence at 1:20 after 5-6 days and split at second confluence at 1:10 after day 10-11.
Cell Observations After one week of growth, we observed the cell count of our three samples. Number of Cells per mL: For 2D Sample:
211, 111
For 3D Sample: Sigma Aldrich:
1,111 with 5 dead cells per 4X4 square
133,333 with 2 dead cells per 4X4 square
These results seem strange as we expected our FMC sample to have less alive cells because of its high G/M ratio (stiffness of alignate). This could be due to any number of labratory errors with culturing.
Cell Viability Results On Day 3 of the Module, we prepared harvested cells from out three samples to test for cell viability in a Live/Dead© (Invitrogen) cell count assay. Dead cells appeared to be red due to their uptake of ethidium while the live cells appeared green due to the uptake of SYTO10. Conclusions drawn are that the 2D sample had the most amount of live cells, however, most were in cluster formation, showing that they were not separated well. The 3D samples had around the same viability as each other. The images can be seen on the "Talk" page of Day 3. Below are our results:
2D Sample:
approximately 120 live (green) cells---mostly in clusters ~4 dead (red) cells
3D Sample: Sigma Aldrich:
~4 live cells ~2 dead cells
~5 live cells ~3 dead cells
These results are different from what was predicted. We thought that the 2D sample would have the most fibroblasts, however they had the most alive cells. Also, we expected the FMC sample to have less alive cells, however, we cannot really accept these results seeing as we could not see many cells. This could be due to labratory errors or leaving our samples in the light for too long.
Cell Counts for Preparing Cell Lysates We prepared cells to test if the cultured chondrocytes have secreted some collagen protein into the surrounding medium. We created two samples; one to test for protein and one to isolate RNA to later test for collagen message. Cell Counts: 2D Sample:
22 cells --- 244,444 cells/mL
3D Sample: Sigma Aldrich:
143 cells --- 1,588,888 cells/mL (cells were in clumps, round)
2 cells --- 22,222 cells/mL
These cell counts make more sense than those previously recorded (above). This is because, after a longer period of time, more cells grew in the Sigma Aldrich alginate than the FMC alginate. This has potential to prove our hypothesis as cells cannot grow on a more tense alginate.
RT-PCR Results We isolated RNA from the samples to conduct RT-PCR. We desired to perform RT-PCR in order to obtain amplified cDNA that would be later run on an agarose gel to compare collagen ratios. The collagen II:I ratio shows the ratio of cells with chondrocyte phenotype. Unfortunately, we could only see our 2D sample and the DNA ladder. This means that the 3D samples did not appear due to not having the correct amount of DNA and due to labratory errors. RNA Concentrations: Sample RNA conc. (ug/mL) 2D 11.2 3D Sigma 540 3D FMC 0 It is interesting to note that there is a higher RNA concentration in the Sigma Aldrich sample than the other two samples. This could be due to the fact that there was more cells in the Sigma sample. We ulitimately ran 168 ng of RNA. The gel was run with the lanes 1- DNA Ladder and 2- 2D sample collagen I. No 3D FMC (with collagen I or collagen II) samples were run due to not having any resulting RNA concentration. The results with image analysis: Sample --- Mean/Min/Max: Background --- 58.4/28/124 2D Collagen I --- 50.047/9/87 2D Collagen II --- 145/67/196 The intensity ratio of the 2D sample was determined to be 3:1 (collagen II : collagen I). The image can be seen on "Talk" page of Module 3, Day 5. Therefore, the ratio implies that a 3:1 ratio of cells are chondrocytes:fibroblasts (because chondrocytes express more collagen II than fibroblasts).
Indirect ELISA Results We used the protein samples taken after the second cell observation in order to perform indirect ELISA. We desired to perform ELISA to quanitify specific proteins (collagen I and collagen II) in order to determine the chondrocyte phenotype of our samples.
(S = supernatant, C = Cell) Collagen I (y = 13.767x - 0.5706) Collagen II (y = 0.672x - 0.0389)
Our Collagen Ratio Table:
|Collagen I (in ug/mL)
|Collagen II (in ug/mL)
|CI : CII ratio
|2D Supernatant
|2D cell
|3D Sigma S.
|3D Sigma C.
For ELISA, we took unequal amounts of cells. We took from 2D 1 ml (~250,000 cells), 3D FMC 1 mL (~22,000 cells), and 3D sigma .15 mL (~250,000 cells). As the results show, the FMC collagen I (fibroblasts): collagen II(chondocytes) ratio was the highest, proving that there was a larger amount of fibroblasts than chondrocytes due to the more tense alginate. The 2D sample also had a higher collagen I: collagen II ratio because of the lack of ascorbate (nutrients). The 3D sigma had the lowest ratio (supernatant), meaning that it did not de-differentiate quickly. The 3D sigma cells had a higher ratio than the 2D cells, but this could be due to labratory errors or the lack of protein expression. We would like to believe that our hypothesis was proven correctly, but we would need to perform this experiment again in order to verify the results and also to obtain results when using the RT-PCR/agarose gel assay. Since we only got a bands for the 2D sample, we cannot compare the results.
Overall, we proved our hypothesis that the 3D sigma alignate allowed for higher chondrocyte phenotype due to analyzing our ELISA results.
Tuesday/Thursday Pink
In our experiment we tested 2D plating with rich medium versus 3D plating in poor medium in the presence or absence of an actin inhibitor toxin. Our toxin was solubilized in DMSO, also toxic to cells; the negative control received DMSO without the toxin. Our LIVE/ DEAD Assay yielded no results as our cells were photobleached because we neglected to wrap the samples in tin foil. We recovered many cells from the 2D sample and the 3D sample with toxin. We were unable to amplify the 3D without toxin sample in our RT PCR reaction. The bright bands on our gel were as follows: 2D Collagen II, 2D Collagen I at both 5 and 10 μL loading volumes. Image J analysis of the bands showed that the Collagen II to Collagen I ratio in the 2D sample was ~2.7. ELISA Analysis was performed on the cell lysates, and standard curves were generated with Bovine Serum Albumin (BSA). The results of ELISA yielded a 2D Collagen II to 2D Collagen I ratio of .18.
Day 3:
- 2D Morphology and Density Observations:
- Sample- Round + dense
- 3D Morphology and Density Observations:
- (-)Toxin: Cells were clumped together alginate funky shaped – perhaps created by droplet too quickly.
- (+) Toxin: Cells more homogenous, alginate round.
- 2D Cell Culture Counts:
- 3.77 *10^5 cells/mL
- Viability = 100%
- 3D Cell Culture Counts:
- (+) Toxin: 450 cells/mL, viability ~10%
- (-) Toxin: 900 Cells/mL, viability ~20%.
- Concern: Resuspension for 3D not homogenous.
- LIVE/DEAD Assay:
- Very few cells seen on either. Did not cover with aluminum foil.
- Went back to look at 2D samples, observed many cells.
Day 4:
- Cell Counts:
- 2D: 5.5*10^5 cell/mL, added 0.5 mL protein, rest of solution was used for RNA.
- 3D (+) Toxin: 2*10^5 cells/mL, added 1mL protein, rest of solution was used for RNA.
- 3D(-) Toxin: 1*10^4 cells/mL with lots of specks of dead cells, added 1mL protein, rest of solution was used for RNA.
- Normalized cells concentrations to 3D(+).
- RNA concentrations: used 200ng RNA per reaction for RT-PCR
- 2D: 198 μg/mL. Amount added (per reaction): 1.01 uL
- 3D(-): ~0. Amount added: 0 uL
- 3D(+): 12 μg/mL. Amount added (per reaction): 16.7 uL
- Hypothesis: DMSO kills cells.
Day 5:
|11 uL
|2D Collagen I 5uL
|18 uL
|dim, low on gel
|3D Collagen I 5uL
|18 uL
|not visible
|2D Collagen II 5uL
|18 uL
|strong, middle of gel
|3D Collagen II 5uL
|18 uL
|not visible
|2D Collagen I 10uL
|18 uL
|strong, low on gel
|3D Collagen I 10uL
|18 uL
|not visible
|2D Collagen II 10uL
|18 uL
|very bright, middle of gel
|3D Collagen II 5uL
|18 uL
|not visible
|Control I 5uL
|18 uL
|not visible
|Control II 5uL
|18 uL
|dim, low on gel
Day 6/7: Elisa Protein Analysis
- Collagen I plate had bubbles, so we did not analyze that data and looked at the Collagen I O/N data instead.
- All the samples of Collagen II and Collagen I O/N were able to get results with protein concentrations in ng/mL
Wednesday/Friday Red
We, along with the pink group, are testing the effects of mechanical stress on the maintenance of chondrocyte phenotype. We are comparing our 2D and 3D control samples to a set of cells in 3D culture that are under constant mechanical stress in the form of two washers (49.2 g total) placed on top of our petri dish apparatus (Figure 1). Our 3D control chondrocyte-seeded beads were placed in the apparatus without a filter and any weights.
We seeded our 1% Sigma alginate beads with a cell density of 5 million/mL and used normal medium. For 2D culture, we used 600,000 cells per flask (total 1.2 million) in normal medium, split them every 4 days at 1:10.
Cell Morphology
As expected, the 2D cells appeared de-differentiated and exhibited fibroblast phenotype (strand-like) under the light microscope while our 3D control cells maintained a chondrocyte phenotype (round). We were unable to observe our 3D weighted cells because the beads were covered by the filter.
Cell Viability
In terms of cell count, we had very few cells in both of our 3D cultures. There were approximately 455,555 cells/mL in 2D, 33,333 cells/mL in 3D control, and 11,111 cells/mL in 3D weighted. In both 3D cultures, the lower counts may reflect the limited exposure to nutrients and oxygen to cells in the alginate beads. Moreover, 3D culture under mechanical stress had even fewer cells possibly because the filter further limited the flow of nutrients and oxygen to cells. In the live/dead fluorescence assay, the cells in 2D and both 3D cultures were similar in the low number of live and of dead cells, perhaps due to low recovery and incomplete staining.
RT-PCR analysis
Following RNA isolation of our three samples, we performed RT-PCR using about 270 ng of RNA for each reaction with collagen I (CN I) and collagen II (CN II) primers. In the visualization of our RT-PCR fragments on agarose gel with CN I and CN II controls, the band intensities were normalized by using the same band area and subtracting off backround intensity, and are indicated in Table 1. Our data indicated that the weighted 3D sample expressed more CN II than unweighted 3D and on comparable levels with the CN II control. For CN I, unweighted 3D control had the most expression than all other samples. With regard to the CN II:CN I ratio, the 3D weighted sample had a much higher ratio (21.7) than all other samples including the collagen controls (5.52). As predicted, this demonstrates that the 3D weighted cells exhibit chondrocyte phenotype, while the 2D and 3D controls do not. For CN I, the control and 3D control had similar intensities and similar amounts of DNA (area of band on gel). For CN II, control and 3D weighted have similar intensities and amounts of DNA. Yet for the 2D sample, it was about half as intense and the band area was about 1/2 to 1/3 of the other two samples. These results confirmed our hypothesis that applying mechanical stress promotes chondrocyte phenotype.
Table 1. Band intensities and areas on agarose gel electrophoresis of RT-PCR fragments and CN I and CN II controls. Intensity values are normalized by band area (of 0.002 using ImageJ from NIH) and area values reflect the total area of the band.
|~0 (band only visible by eye)
|~0 (band only visible by eye)
|3D control
|3D weight
|3D control
|~0 (band only visible by eye)
|~ 0 (band only visible by eye)
|3D weight
ELISA results
Wednesday/Friday Yellow
Wednesday/Friday Green
We chose to vary the physical culture conditions. We originally planned to have four culture conditions: the 2D culture in a T25 flask, a 3D culture in 2% Sigma-brand low-viscosity alginate beads, a 3D culture in PEG-APGL-PEG-RGD and a 3D culture in a modified hollow-fiber bioreactor with 100 kDa cutoff cellulosic fibers. The PEG-RGD polymer contains the RGD peptide, which is an integrin binding sequence. We hoped that the cells would adhere to the polymer better with that peptide present. The bioreactor continuously circulates media and gas at a rate of 120 mL per minute and 27.5 mL per minute, respectively. It also puts shear stress on the cells at an adjustable quantity, which we hoped would stimulate the cells to produce collagen. Unfortunately, the PEG-APGL-PEG-RGD polymerization by UV light failed and made it impossible to work with our cells (e.g. exchange media). (Possible reasons for this were incorrect UV wavelength and old, degraded PEG and/or activator.) Also unfortunately, the bioreactor chambers melted during autoclave sterilization and were rendered unusable. This left us with just the first two described cultures (the 2D and 3D alginate). The 2D culture was seeded with approximately 500,000 cells, and the 3D culture was seeded with 1.1M cells (per replicate).
After seven days, we harvested some of our cells. We recovered 155,000 cells per mL from our 3D culture and 500,000 from our 2D culture. Then we did a Live/Dead© (Invitrogen) cell count assay. Images of our assay can be found on the Day 4(?) talk page. Our results were these:
|500,000 cells/mL
|3D Alginate
Based on this data, both 2D and 3D cultures were quite viable, though as expected the 2D culture was somewhat more viable. We also looked at our cells under 40X magnification with visible light to observe morphology. The 2D culture cells were mostly fibroblasts, where as the 3D culture cells were mostly chondrocytes. [Because the other groups all decided to add big, fancy tables, and we just decided to have one small table, we are compensating with a big, fancy border.]
We then did RT-PCR on the RNA from the samples to determine relative production of type I and II collagen. We loaded 200 ng of total RNA in our RT-PCR reactions. (Our total RNA concentrations were 570.4 μg/mL for 2D and 24 μg/mL for 3D.) The gel (shown in Day 5 Talk page, with lanes labeled there) showed a higher percentage of chondrocytes in the 3D culture than 2D culture, based on the relative amounts of mRNA present in the cells. (This is based on the fact that chondrocytes express collagen II in higher amounts than fibroblasts do.)
We did indirect ELISAs for both types of collagen. We normalized the amount of cells from the two cultures, using 1 mL of 3D culture and 400 μL 2D culture. Unfortunately many of our samples had concentrations below the range of our standards. We can say, though, that there was more collagen I produced than collagen II, according to these results. (Note that these results apparently contradict out transcript analysis results. This is more likely due to a poor regression equation.) We can also say that there was more collagen II produced in our 3D culture than in our 2D culture.
Wednesday/Friday Blue
Background and Set-up:
From our research we found that the rate of attachment of cells increases with cross-link density. So we planned on increasing the cross-link density of one our 3D cultures(from now on 3D experimental) to test if this increased the dedifferentiation of chondrocytes into fibroblasts. We accomplished this by increasing the calcium concentration ten-fold (1002 mM instead of 102 mM). Our 3D control sample used a standard calcium concentration (102 mM). We used a 2% alginate for both samples with G/M ratio of 70/30, because the G segments are involved in the cross-linking. We actually noticed a difference in the size of our alginate beads: The higher calcium concentration beads were much smaller in size. Our 2D sample was set-up at normal conditions. We plated the 3D cultures with about 1.3 million cells per well and the 2D culture with 100,000 cells.
Morphology and Cell counts:
We saw differences between our samples, as expected. The 2D culture cells looked more like fibroblasts than chondrocytes. They were very flat and spread out. The 3D control cells looked more like chondrocytes since they were large and spherical. The 3D experimental cells still looked like chondrocytes but they were significantly smaller in size than the 3D control cells. Our 2D cells had about 720,000 cells in total. Our 3D control and experimental samples had 49,500 cells total.
Live/Dead Cell Assay:
Unfortunately, since we had a low cell count and then we also had low cell recovery, we were not able to get good data from this assay. We saw about 4-5 live cells and 1-2 dead cells in the 2D sample. For our 3D control, we could more live than dead cells, but this was for a small sample size. The live cells were in a clump, so we were not able to count them.
When we prepared our cells for analysis, we had a low cell count for our 3D control sample and no cells in the 3D experimental.
Culture Type : Cell Count : Total RNA Concentrations
2D: 705,555 cells/ml: 144 ug/ml
3D control: 44,444 cells/ml: 20.4 ug/ml
3D experimental: 0 cells/ml (50,000 cells/ml dead): 5.2 ug/ml
Sample - CNII:CNI
3D control - 2.354
3D experimental - 0.120
The gel results are mostly consistent with our predictions. The 3D control and 2D samples show a high CN II:I ratio, indicating that a significant portion of the cells remained as chondrocytes. However, our 3D experimental sample shows a low CN II:I ratio. There are many reasons that account for the low ratio. It is possible that the chondrocytes de-differentiated into fibroblasts; but the low cell count in the 3D experimental sample does not allow us to make that conclusion.
Our ELISA results are inconsistent with our transcript level results. This could be due a difference in the specificities of the two antibodies (cross-reactivity). Only the 3D Control pepsin digested had a measurable amount of collagen II, while all of the samples had collagen I. The 3D control pepsin digested Collagen II:I = 0.1685.
Wednesday/Friday Purple
Background/Experimental Setup
For our experiment, we decided to add a calcium chelating agent (mix of Sodium Citrate, EDTA, NaCl) to one of our 3D samples. We hypothesized that the addition of a calcium chelating agent would reduce the mechanical strength of our scaffold by inhibiting the G block interactions between our alginate beads. We predicted that this reduced mechanical support would be detrimental to chondrocyte phenotype preservation and would cause more chondrocyte de-differentiation to fibroblasts. The other 3D sample was used as a control (no calcium chelator), in order to have a basis of comparison for the level of de-differentiation. Our 2D sample was used to compare the effects of chondrocyte development in an environment lacking a scaffold. Due to low cell recovery, we initially seeded only about 100,000 cells in the 2D sample and about 1.5 million cells in each of our 3D samples. We added chelating agent to 3D-sample 2 at a concentration of 1%. We determined this concentration after some discussion of the fact that too high a concentration might completely obliterate our alginate matrix, while too low would yield no interesting results.
Cell Recovery & Live/Dead Fluorescence Assay
Upon trypan exclusion on day 3, we found cell counts to be extremely low. We determined that our 2D sample had around 500,000 cells while each of our 3D samples had only around 11,000 cells. Preliminary microscopy showed no difference in the morphology of our 3D samples. However, we noticed that most of the cells in our 2D sample had an oblong shape consistent with fibroblast morphology. Because of our low cell count, we took the maximum amount of each sample (1.5mL) for our Live/Dead fluorescence assay. Despite this, cell counts were too low for us to observe many live or dead cells.
RT-PCR & Agarose Gel
We measured the collagen II to collagen I ratio of our samples by examining the RNA content as well as collagen expression of our samples. With the first half of our samples, we extracted all RNA for RT-PCR. Using collagen specific primers, we reverse transcribed the collagen RNA to cDNA and then amplified this cDNA through PCR. Upon running our RT-PCR products on an agarose gel we found that our Col II:Col I ratio was high in all of our samples. Using ImageJ, we analyzed the intensity of the agarose gel bands and found the following ratios:
|CN-II : CN-I Mean Intensity
|3D Sample 1
|3D Sample 2
Note: 2D Sample Results are inconsistent with our microscopy observations - we will continue to address this after the results of our ELISA.
Wednesday/Friday Pink
Our Experiment
For our experiments, we chose to test the effects of mechanical compressive stress on chondrocyte phenotype. Our 2D sample was cultured normally. Both of our 3D samples were cultured in Petri dishes: the control had no weight added and was cultured in 1% alginate, our test sample was compressed using the bottom of the inverted Petri dish, covered with filter paper to allow diffusion of nutrients and oxygen into the beads. We hypothesized that in compressive situations, similar to those seen by the cartilage in the knee, would result in maintained chondrocye phenotype.
Cell Observations
Observation of the cells after one week of growth confirmed our prediction, at least initially. Our 2D sample was mostly de-differentiated, but not completely. It was hard to tell what happened with our 3D samples, but the cells within the beads looked circular. We recovered 356000 cells/mL of our 2D sample, and less of both of our 3D samples; 33300 cells/mL for the weighted sample and 116600 cells/mL for the control sample.
Live/Dead assay
Our Live/Dead assay revealed very few dead cells, but also very few lives cells. This is most likely the result of relatively low recovery and incomplete staining. We think, after looking at the cells under normal lighting that it was a staining problem, and that most of our cells remained alive regardless of conditions.
Cell Concentrations
Concentration analysis of our samples showed high protein amount in two out of three cell conditions. Our 2D sample had a concentration of 120.4 ug/mL, 3D control was 32.4 ug/mL, and 3D weighted was very low, at 6.8 ug/mL. We decided to run the full 200 ng of RNA in each RT-PCR reaction, but ran out of our 3D weighted sample. We ended up using only 75% as much for the 3D weighted as for the other two.
RT-PCR Results
We performed RT-PCR on all three of our samples, but got very little yield. Our 2D samples yielded a strong band of collagen II (around 400bp), and a very faint but visible on the gel (not on the picture) around 100bp that was collagen I. None of our 3D samples showed visible bands, despite the high protein concentration evidenced in spectrophotometer. Regardless, based off the striking results of our 2D sample, we can conclude that even the cells displaying the more fibroblast phenotype were still producing markedly more collagen II than collagen I. Using the 2D results on the agarose gel, our collagen II to collagen I ratio was 23.25 (100/4.3).
|11 uL
|2D Collagen I 5uL
|18 uL
|very weak
|3D Control Collagen I 5uL
|18 uL
|not visible
|3D Weighted Collagen I 5uL
|18 uL
|not visible
|2D Collagen II 5uL
|18 uL
|strong band
|3D Control Collagen II 5uL
|18 uL
|not visible
|3D Weighted Collagen II 5uL
|18 uL
|not visible
|Collagen I Mock
|18 uL
|no band
|Collagen II Mock
|18 uL
|no band
ELISA Result
Our ELISA results were inconclusive, because our Collagen II data was not within the bounds of the assay. The Collagen I assay worked, however, only when it was oversaturated. Interestingly, our pepsin cell samples had less protein yield than our non-pepsin cell samples. We also had very high background.
(sup = supernatant, c = control, w = weighted) Collagen I (y = 0.0456x + 0.0065) Collagen II (y = 0.0519x + 0.2045)
Our Collagen Ratio Table:
|Collagen I (in ug/mL)
|Collagen II (in ug/mL)
|CI : CII ratio
|2D Sup
|2D cell
|3D C Sup
|3D C Cells
|3D W Sup
|3D W Cells
Within Collagen I our results make sense: Our weighted sample (under compressive stress) shows the least amount of Collagen I, thus we can assume it had higher levels of Collagen II and kept its chondrocyte phenotype.
Our data was normalized to 3D control, using 187000 cells (for both 3D control and 2D). The weighted 3D sample did not have enough, and thus we used half of what was left (from setting aside a certain amount for RT-PCR), for a total of 16700 cells. As such, our weighed sample was not normalized to the other two samples used.


text:
Santini Zest Cotton Summer Standard Profile Sock Black
Save 73% on the Zest Cotton Summer Standard Profile Socks by Santini.!
Santini Zest Standard Profile Sock
Light and breathable 69% cotton 29% nylon 2% lycra Keep feet feeling fresh in the summer months Elasticated Standard profile Temperature Control Flexi Properties
Cycling Socks Deals
Santini Socks
Santini have been producing performance road cycling gear for many decades, and like their rivals Castelli have been responsible for several world's firsts, such as creating bib shorts out of lycra rather than wool and the antibacterial fabric chamois in 1976. In 1988 they introduced the world to breathable yet windproof Windstopper garments, and to this day are constantly improving their product ranges with their carbon fabric, acquazero and interactive cycling kit. Whilst not quite as pricey as other premium brands, the Santini name is very much associated with quality. It's aimed at those professional and recreational road riders requiring performance cycling gear which won't let them down. This is further underlined by Santini's strong association with the Giro d'Italia and UCI World Championships. As a popular brand amongst UK riders, my Santini section is regularly updated with fresh deals, to help save you money on your premium cycling kit.
For more information on Santini, you can visit their UK website or their wikipedia page for a more in-depth guide to their rich history in cycling.
Rutland Cycling
This particular deal was found at Rutland Cycling - you can see their P&P charges on their Delivery page, as well as learn more about their bike shop.


text:
PPIx::Regexp::Token::GroupType::Switch - Represent the introducing characters for a switch
use PPIx::Regexp::Dumper;
PPIx::Regexp::Dumper->new( 'qr{(?(1)foo|bar)}smx' )
PPIx::Regexp::Token::GroupType::Switch is a PPIx::Regexp::Token::GroupType.
PPIx::Regexp::Token::GroupType::Switch has no descendants.
This class represents the characters right after a left parenthesis that introduce a switch. Strictly speaking these characters are '?(', but this class only takes the '?'.
This class provides no public methods beyond those provided by its superclass.
Support is by the author. Please file bug reports at http://rt.cpan.org, or in electronic mail to the author.
Thomas R. Wyant, III wyant at cpan dot org
Copyright (C) 2009-2016 by Thomas R. Wyant, III
This program is free software; you can redistribute it and/or modify it under the same terms as Perl 5.10.0. For more details, see the full text of the licenses in the directory LICENSES.
This program is distributed in the hope that it will be useful, but without any warranty; without even the implied warranty of merchantability or fitness for a particular purpose.


text:
Red variety of Friuli with harsh tannins that inspired its unique name, translated as "cutting the tongue."
Red variety of Trentino with deep color, ripe berrry fruit and good acidity.
High acid white variety of Piemonte. Often used in the production of grappa, though a few producers – most notably Massa – make an excellent dry version.
White variety planted throughout much of Italy; generally a blender with modest aromatics and high acidity. Trebbiano di Soave is one of the most highly regarded subvarieties.
Uva di Troia
Also known as Nero di Troia. Excellent red variety found in northern Puglia. Moderate tannins, good acidity and cherry, berry fruit. The principal red variety of Castel del Monte DOC.
White variety of Marche. Generally aged in stainless steel, though a few producers age in oak. Many versions are light with pleasant pear and apple fruit; there are some excellent bottlings that offer more fruit intensity and spice.
Verduzzo Friulano
White variety of Friuli, primarily in the Colli Orientali DOC. Used to produce a dry white, but also a famous sweet white named Ramondolo. Apricot and pear flavors with lively acidity.
White variety grown in Sardegna, Liguria and the coast of Tuscany (especially in Bolgheri). Very high acidity with flavors of pear, lime and pine and often notes of sea salt with a distinct minerality.
Two distinct examples: Vernaccia di San Gimignano is a refreshing, dry white wiht moderate acidity made as a light, dry white. Vernaccia di Oristano is a sweeter white from Sardegna that is sherry-like.
White from Veneto made as a lush, apricot and honeyed dessert wine called Torcolato.
Name for Moscato in Sicilia; the word is Arabic for "raisin." Used in the production of Passito di Pantelleria. Honey, apricot and marzipan flavors.


text:
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CISCO Catalyst 2960-XR, L2, Managed, Gigabit Ethernet (10/100/1000), RJ-45, black, UL 60950-1, CAN/CSA-C22.2 No. 60950-1, EN 60950-1, IEC 60950-1, AS/NZS 60950-1
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Brand: CISCO
Product code: WS-C2960XR-24PD-I
- 882658598098
Warranty: 12 months
Cisco Catalyst 2960-XR. Switch layer: L2, Switch type: Managed. Basic switching RJ-45 Ethernet ports type: Gigabit Ethernet (10/100/1000), Console port: RJ-45. Colour of product: black, Safety: UL 60950-1, CAN/CSA-C22.2 No. 60950-1, EN 60950-1, IEC 60950-1, AS/NZS 60950-1. Built-in processor: APM86392, Memory type: DRAM. AC input voltage: 100-240, AC input frequency: 50/60
Cisco® Catalyst® 2960-X Series Switches are fixed-configuration, stackable Gigabit Ethernet switches that provide enterprise-class access for campus and branch applications (Figure 1). Designed for operational simplicity to lower total cost of ownership, they enable scalable, secure and energy-efficient business operations with intelligent services and a range of advanced Cisco IOS® Software features. - 24 or 48 Gigabit Ethernet ports with line-rate forwarding performance. - Gigabit Small Form-Factor Pluggable (SFP) or 10G SFP+ uplinks. - FlexStack Plus for stacking of up to 8 switches with 80 Gbps of stack throughput (optional). - Power over Ethernet Plus (PoE+) support with up to 740W of PoE budget. - Reduced power consumption and advanced energy management features. - USB and Ethernet management interfaces for simplified operations. - Application visibility and capacity planning with integrated NetFlow-Lite. - LAN Base or LAN Lite Cisco IOS® software features. - Enhanced limited lifetime warranty (E-LLW) offering next-business-day hardware replacement.
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