text:
Flag City RV Resort provides recreational vehicle travelers with a premier five star RV Resort destination or overnight stop that is located in the heart of the San Joaquin Valley, near Lodi and convenient to Sacramento and Stockton.
21 amazing specialties - 1 Fantastic Camp! One of the best California summer camps for kids and teens! Located in Southern California, Pali Adventures is the adventure sleep-away camp of choice where kids get to follow their dreams!
We offer scenic helicopter tours over Los Angeles, Hollywood, Santa Monica, and Malibu coast. Open 7 days a week. Four different tours are available ranging from 30 minutes to 90 minutes. Tours depart from Van Nuys Airport, 15 mins from Hollywood
We offer the nicest, newest and usually the biggest travel trailers for rent in the southern California area. We put a lot of work in our trailers to make sure they are the cleanest and best maintained trailers.
Beautiful property isolated from traffic with parking spaces in front of rooms. Pool and Jacuzzi. Free abundant Continental Breakfast. Free Wi-Fi and Business Center. HD TV's in most rooms. VHS & DVD Players. Recreation Center & Fitness Room.
Located just minutes away from Disneyland Resort®, pristine beaches, museums, aquariums, and sporting events.With its warm weather, family friendly environment and premier entertainment, you don't have to look for it because fun finds you!
Happy Bear Vacation Rentals specializes in accommodations in beautiful Big Bear Lake, Southern California's premier four-season resort. Our rentals range from one to seven bedrooms and will accommodate 1 to 20 persons.
Los Angeles Horseback Riding Adventure at Westwind Ranch offers you affordable Dinner Rides, Romantic Rides, located 30 miles north of the San Fernando Valley. Creat a memorie you will never forget!.
The Knickerbocker Mansion (c1920) is a magnificently restored unique log home and carriage house with rustic elegance, now a well know Bed and Breakfast with quite atmosphere and personal attention. So close, yet so far away.
Select your destination in Victorville California. Browse thousands of listings for accommodations, adventures, tours and travel services.


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In Photos: Vladimir Putin Assumes The Presidency In Russia
As we've reported, Vladimir Putin's return to Russia's presidency was fraught with drama. But a disputed parliamentary election and many unprecedented protests later, Putin took the oath of office for a third time today.
Putin took the oath amid protests. The New York Times reports 300 were detained, following the round up of 400 detained after a surprisingly large anti-Putin demonstration popped up on Sunday.
We'll leave you with some pictures that tell the story of today.
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text:
updated 11/14/2006 AT 3:00 PM ET
•originally published 11/14/2006 AT 3:15 PM ET
It's down to the final dances for Mario Lopez and Emmitt Smith. The two men go toe-to-toe Tuesday night on ABC's Dancing with the Stars to see who will walk away with the season 3 disco-ball trophy.
For the first time on the show, each celebrity and his professional dance partner will be required to perform three numbers. Both couples will dance the samba and a freestyle, then for their third dance, Smith and partner Cheryl Burke will do the mambo while Lopez and partner Karina Smirnoff will perform the paso doble.
"I'm very nervous," says Burke, 22, who won the season 2 title with celebrity partner Drew Lachey. "Emmitt, on the other hand, is very cool. We balance each other out."
Smith says his years with the Dallas Cowboys have prepared him well for the kind of pressure and scrutiny Tuesday night will bring.
"We prepare during rehearsals and (on Tuesday nights) I don't think about the judges. I don't have a care in the world. I just enjoy the moment," Smith says.
Lopez, who has been teased throughout the season for having a short attention span and a fiery on- and off-screen relationship with Smirnoff, is a bit giddier. "I'm going up against Emmitt Smith! He was like a hero of mine!" Lopez tells PEOPLE, noting that he thrives on stress. "I'm a hyper, energetic person and I like to keep busy and do a bunch of other stuff. I think I work best under pressure."
He praises his wife, Pat, for being his faithful teammate. "She has made our home very happy and the kids feel love in the house. When Daddy is away, they know Daddy is coming back."
Lopez's mother, Elvia, has been in the audience each week to support him, as have a slew of celebrity pals including Eva Longoria, Jose Canseco, George Lopez, Oscar de la Hoya and Elizabeth Berkeley.
Lopez jokes that he is "withering away" – down three belt notches thanks to daily eight-hour dance rehearsals. What he's looking forward to most when it's all over is "just chilling." And eating. "My mom will be cooking and I'm visiting my sister over (Thanksgiving) and she'll be cooking. They'll take care of me."
Burke, meantime, confesses that while the end is near and "I really want that trophy," she's already stressing about what's next. "I'm going to miss Emmitt," she says. "I'm already missing him."
Burke will re-team with Lachey for the 38-city DWTS tour, which kicks off Dec. 19 in San Diego. Smirnoff, meanwhile, will team up with season-3 contestant Harry Hamlin for the tour.
But first, there's a trophy to win. And bragging rights. "It's always been my philosophy: It's not where you start, it's where you finish," Smith says. "I want to finish strong."


text:
MANHATTAN, Kan. (AP) _ More freshmen in the ROTC program at Kansas State University will be on scholarship this fall, thanks to increased funding.
Lt. Col. George Belin, head of Kansas State's military science department, said 41 students will be on full four-year scholarships, up from the original allotment of 18.
Nationally, the Army Cadet Command is seeking to graduate 25 percent more second lieutenants, increasing the number of scholarships accordingly.
Belin said adding to the program is its location near Fort Riley, where students can train and learn from soldiers and officers in the Army.
This year, 24 second lieutenants graduated and got their commissions at Kansas State.
On the Net:
Kansas State University ROTC: http://armyrotc.ksu.edu/index.php
(Copyright 2008 by The Associated Press. All Rights Reserved.)


text:
Women's Basketball
Feb. 19, 2014
LOS ANGELES, Calif. - Behind a career-high 31 points from senior guard Amy Kame, the University of San Diego women's basketball team picked up a 87-74 West Coast Conference win over Loyola Marymount University Thursday evening at the Gersten Pavilion.
With the win, the Toreros improve to 21-6 overall, move to 10-6 in the WCC and have completed a season sweep over the Lions. With the loss, LMU falls to 8-18 overall and 5-10 in the WCC.
Kame scored her career-high 31 points, 29 of which came in the second half, on 10-of-14 shooting from the field, while also pulling down six rebounds and dishing out six assists. Sophia Ederaine posted a double-double with 16 points and 11 rebounds, while Cori Woodward provided a spark off the bench and scored a career-high 12 points, all from beyond the arc. Brooke Jelniker was the final Torero in double figures, as she had 10. Kameron Knutson also had a great game, as she pulled down a team-high 12 rebounds. As a team, USD shot 48.5% from the field, and out rebounded the Lions 41-30.
In the first half, USD came out firing on all cylinders, as they opened the game on an 8-2 run over the first three minutes of action. The Lions would then respond with a 5-0 run of their own to pull back to within one at 8-7 with 16:13 to go.
However, an Ederaine lay-up then sparked a 6-0 Torero run to give USD its biggest lead of the half at 14-7 with 14:24 to go. LMU then responded with an 8-0 run to take a one-point lead at 15-14. The two teams then began to trade baskets, and LMU continued to dare USD to shoot the 3-pointer. So insert Woodward, and she produced nine first half points, all form beyond the arc, allowing USD to take back the lead and go into the break up four at 40-36.
In the second frame, USD and LMU were just about even in the early going, trading basket-for-back over the first eight minutes of action. Then a Malina Hood lay-up with 11:32 to go, gave USD its first double-digit lead of the game at 57-47. USD would see the Lions get back to within six a few times, but the Toreros continued to respond and quickly would retake a double-digit margin. USD never looked back, as they hit key free throws down the stretch to claim the win.
The Toreros will return to action on Saturday, as they travel to Malibu to take on the Pepperdine Waves in a 2 p.m. WCC game inside of the Firestone Fieldhouse.
Meet the Toreros: Jose Martinez
6'10" freshman forward from Cayey, Puerto Rico.
Meet the Toreros: Frank Ryder
6'10" freshman forward from Boulder, Colo.
2015-16 Highlight Video
All the top plays from 2015-16
Jordan Angus - Male Torero of the Year
Angus reflects on his time at USD
Malina Hood - Female Torero of the Year
Malina Hood looks back at her USD career
NCAA Tournament Preview
San Diego faces Vanderbilt in first round
2016 Celebration of Excellence
Behind the Scenes at the COE
Toreros Participate in Linda Vista Parade
Toreros Participate in Linda Vista Parade
Men's Tennis senior Day
The Toreros host LMU and Pepperdine Saturday and Sunday
Softball WCC Preview
The Toreros host Saint Mary's Saturday and Sunday
2016 San Diego Crew Classic
The Toreros show speed against the nation's top crews
The Torero Baseball Experience
Kris Bryant & Brian Matusz reflect on their time at USD
MBB Senior Night
Jito Kok and Duda Sanadze prepare for the final home game
USD Baseball Season Preview
Season starts at No. 3 Vanderbilt February 19
USD Softball Season Preview
Season starts February 12
UCLA Preview
USD travels to No. 9 UCLA Friday
Toreros Host Kentucky Saturday
USD looks to defend home court against No. 22 Kentucky
Toreros Host Pepperdine & LMU
USD returns home after four-game road trip
Toreros Team Up with PYBL
Men's Basketball hosts annual clinic for Challenged Division
Men's Tennis 2016
USD opens at home vs UC Irvine and UCSD Saturday
BYU Preview
Preview of USD women's basketball WCC opener vs. BYU.
City Championship Highlights
Relive the Toreros' Upset over SDSU at Petco Park
Toreros to Host Hope International
The 9-1 Toreros will host Hope International in their final game before WCC play
Toreros Stun Aztecs at Petco
Postgame reaction and press conferences
Toreros Return Home
USD will host Bethesda before Sunday's City Championship at Petco Park
Men's Basketball Season Tickets
Starting as Low as $99
Butler Preview
Pono Keni and Coach Lindsey talk about Saturday's Senior Day game against Butler
CSUN Preview
Coach Fisher and forward Maya Hood talk about Tuesday's home-opener against CSUN
USC Preview
Coach Smith and Brett Bailey preview the season opener at USC
Davidson Preview
Jonah Hodges and Dale Lindsey talk about Jonah's return and preview Davidson game
San Diego Fall Classic Preview
Coach Dagman previews the San Diego Fall Classic
Nevada Preview
USD's Women's Basketball team will start their season at Nevada this weekend
USD Men's Basketball - The Origins (1955-1973)
A Glance at the Beginnings of Torero Hoops
Campbell Preview
Coach Lindsey and Jeff Starr preview Campbell
Stetson Preview
Coach Lindsey and Jereke Armstrong talk about the season so far
WCC Media Day
Behind the scenes with Men's and Women's Basketball at WCC media day
Warner Recap
Dale Lindsey and Anthony Lawrence on USD's 45-14 win
Warner Preview
Anthony Lawrence and Coach Lindsey preview game vs. Warner
Torero Community Service
USD Athletics helps out at Father Joe's Village in downtown San Diego
Men's Basketball Camp Update
Kok and Carter talk about the upcoming season
Drake Preview
Toreros host Bulldogs for Homecoming game
Dayton Preview
Toreros look for 4th straight win
LMU Preview
Pack the Stadium Night
ITA All-American's Preview
Petronijevic, Angus, & Vittek Prepare
Marist Preview
USD Looks to Extend Home Win Streak
Valparaiso Preview
Toreros First True Road Game of 2015
CSUN and UCSB Preview
Toreros Look to Extend Win Streak
Aztec Invite Recap
Both Squads Finish Second
SDSU Preview
Toreros Take on Cross-town Rival
WNMU Recap
Highlights from 45-21 win
2015 Home Opener
Previewing Western New Mexico
Volleyball Tournament Preview
A preview of the USD Tournament
Men's Soccer Season Opener
Previewing No. 16 Cal
Training Camp Update
Coach Lindsey Recaps the First Two Weeks
2015 WSOC Preview
A brief preview of the 2015 Toreros.


text:
Say for example I post a blog entry. Later notice I have misspelled something in the title, and then go back edit the post and fix the misspelling. The text is fixed on the site, but the actual URL to the permalink is still misspelled. Is there anyway I can get the URL adjusted too?
I tried OPTIONS - PERMALINKS - UPDATE PERMALINK STRUCTURE, but that doesn't seem to take.
Any help appreciated!
The post I'm talking about is here in case you're curious.
I can't spell restaurant...
- Harvey


text:
Stock has taken it on the chin but there are clear signs of a rebound
oh I made 12% up from this AMLN. :D
$15 after a 1:15 split at $1 LOL!!!
um... if you're buying in 50K blocks, I wouldn't look for investing advice on this msg board. I doubled down close to ten, hoping that averaging down can salvage my position.
I pick up some.How many shares did u buy? Should I buy another 50,000 shares from here?
Buy 100,000 shares. So every $1 it goes up you can buy a new car! :D
lol, you're buying $500K blocks of stock and you are posting to this message board? We'll file that post in the "yeah right" category.
anguejin, put 10,000 shares into AMLN with a sell order at 17.25 and 40,000 shares into ASTI (ASTI will be 12-16 by end of of the year).


text:
Jacksonville Jaguars target a big-bodied, physical wideout
So far, free agency has gone quite well for the Jacksonville Jaguars. GM David Caldwell seems to have a vision that he is putting to work and steering the franchise in the right direction. Caldwell has clearly made the defensive line and getting after quarterback a priority as evidenced by the signings of Red Bryant, Chris Clemons, Ziggy Hood, and the re-signing of Jason Babin....
Via cover32 - Jaguars
March 18, 2014 | Discuss
Jaguars Not Interested In Being Featured On Hard Knocks
A few years ago, the Jaguars came out and said that they were interested in being featured on HBO's "Hard Knocks," but their feelings towards the show have apparently changed since GM David Caldwell was hired. "Our reaction would be that we would try to defer," Caldwell said in an interview with Pro Football Talk on NBC. "But it wouldn't be an ideal situation for...
Via NFL Trade Rumors
February 25, 2014 | Discuss
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Comments about 'Balancing act: OECD notes U.S. work-life balance problems'
- Oldest first
- Newest first
- Most recommended
One possibility is that we're still coming out of a deep recession. If the
boss wants you to put in extra hours as a salaried person and take on more than
is reasonable, it's still harder to get out of that position while still
maintaining an income for your family.
Another possibility is cultural. We do work hard here, but so do those in countries that have a population that lives on a dollar's worth a day. We've got multinational companies importing a cultural norm that lowers the value of humanity from south and southeast Asia. That makes the idea of vacations and 'work-life' balance laughable to them. You work for nearly nothing until you die, then they replace you with another dollar-a-day worker.
- 6:57 p.m. June 26, 2013
- Like
- Report abuse
Just the other day, there was a news report about a study on how about 1/3 of
Americans are "disengaged" from work due to overwork and they're
not feeling appreciated. I see a lot of that at my own work, and it is
frustrating to see how management is so blinded to the fact of worker
dissatisfaction -- even with our high turnover and people avoiding coming to the
office (working at home or in the field).
An important concept in management that has emerged over the last two decades is the idea that enriching employee satisfaction is the major driver of profitability in service-based firms. In short, happy employees are more productive, serve customers better, which in turn drives up sales and profits. If you short-change employees, it drives that customer satisfaction and profitability down.
Sadly, I don't see many Utah companies recognizing this fact.
- 6:59 a.m. June 27, 2013
- Like
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DeseretNews.com encourages a civil dialogue among its readers. We welcome your thoughtful comments.
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text:
A new developer went crazy on a large git repository, really messed it up and pushed. All his changes can be thrown away.
My problem: Our git hoster does not allow push --force, so I have to pull first.
If I do so, I have A LOT of merges and conflicts, and there is no point in resolving them. What is the best way to just revert all the changes done by the other guy so I can push my current version?


text:
Few experiences can match the beauty and tranquility of floating above the treetops and riding the peaceful Washington breeze. A hot air balloon ride is one of the most fascinating adventures you will ever embark upon. At first, the burners roar as you rise into the sky but soon you are overcome by the silence of floating as you drift with the winds and look down upon the breathtaking Washington landscape. Washington Balloon Rides cannot be matched in beauty or magic by any other experience in the Evergreen State!
Washington Hot Air Balloon Rides are something special in comparison to other forms of flight. You don't feel the wind... You are a part of it! You may not even realize that you are moving. Hot air balloons drift gracefully above Washington at heights of up to 10,000 feet. However, most hot air balloon flights are 500 to 3,000 feet above ground where peace and tranquility reign in the gentle low-altitude breeze. As the balloon rises, your bird's-eye view of Washington's beautiful landscape, and all who inhabit it, is put into a perspective that you won't soon forget.
For your convenience, Washington Balloon Rides provides it's services throughout the great state of Washington. Whether you live or are vacationing in Washington, we have certified hot air balloon pilots near you! We can provide you with a panoramic view of many of Washington's favorite vistas.
Washington Balloon Rides has professional and knowledgeable customer service representatives available to answer any of your questions and help you make your reservation seven days a week! We pride ourselves on our customer service and are looking forward to serving you!
Contact Washington Balloon Rides at 1-800-611-1298 today and prepare to embark on a uniquely Washington adventure!
Popular Search Terms for Washington Balloon Rides:
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text:
Who Else Can Slob On The Nob Like Riley Evans? She Can't…
Views: 175 | Added: 4 years agoMoreWatch Video
Riley Evans blows the horn like no one else in the biz. She could put the shine on any prick. She also has one of the best around when it comes to working her dressing sexy for her man scrumptious. Don't forget to check Madthumbs everyday for updates on the hottest models around.
Submitted by: fuckyou


text:
The University of Arkansas at Pine Bluff Adult Education Department is offering morning, afternoon and evening classes for adult students interested in earning their Arkansas High School Diploma.
Interested participants should come into the office at 414 Walnut St. between 8 a.m. and 5 p.m. Monday through Friday and sign up for assessment.
Also, the UAPB Adult Education Department will be testing students who are in the process of taking the official GED Test and have not completed one or more areas. All scores will expire Jan. 1, 2014.
Details: Call 534-0749.


text:
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Description: Six circular images on a glass plate negative from the Stirn Concealed Vest Camera. One of the images is of a black dog standing on its hind legs; another...
Date: ????-02-07
Description: Accident scene between truck driven by Clyde Holmes and Donald Marking, age eight, who was struck and killed while sledding. View shows the alley between 1...
Date: 1950-04-11
Description: Twenty-one members of the Marquette school Girl Scout troop pose by a bus for a trip to Milwaukee.
Description: Children riding the carousel (merry-go-round) at Henry Vilas Zoo (Vilas Park Zoo), donated by the citizens of the Madison area. A stone bridge is in the ba...
Date: 1983-11-01
Description: Virginia Dillman and Richard Lund, the last customer for Dillman's Service Station at 421 South Park Street.
Date: 1949-11-27
Description: Car wreckage and a Milwaukee Road switch engine are shown at the University Avenue railroad crossing. The four persons in the car were hurt. They were Mr...
Date: 1898
Description: Armory (Red Gym or Old Red) on Langdon Street on the University of Wisconsin-Madison campus. A boathouse is behind the Armory on Lake Mendota.
Date: 1920
Description: A page from the Webb family album, compiled and captioned by Andrew Webb Jr., son of Caroline Webb, with two images of friends, an image of the corner groc...
Date: 1935 ca.
Description: View of Regent Street from the intersection of Regent and Monroe Streets.
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Description: A woman (identified as Elizabeth Was) crouches inside two triangular walls open on one side. In front of her two painted signs lay on the grass and read 'S...
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Description: Elevated view of ornate carriage and animal cage with tents and people looking on at the annual Wisconsin State Fair held at Camp Randall.
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Description: Four Dane County Highway Police Ford squad cars with one officer standing next to the cars.
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Description: Man in a wheelchair on the sidelines of the Wisconsin-Illinois football game at Camp Randall Stadium. He is possibly Robert Butts, the founder of the annua...
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Description: Elevated view looking down State Street taken from the Wisconsin State Capitol. On the right on Mifflin Street men are working on scaffolding on the facade...
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Description: A pick-up truck from Hult's Capital Garage parks in front of 115 East Gorham Street with several derby car racers in the truck bed.
Date: 1953-06-15 before
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Description: White and dark European fallow, and a Sika Nippon deer, at the Vilas Park Zoo (Henry Vilas Zoo) in Madison.
Date: 1926 ca.
Description: Exterior view of the South Madison Chicago and North Western Railroad Station located next to the railroad tracks that cross Lakeside Street. In the foregr...
Date: 1954 ca.
Description: View of the Sears-Roebuck store on East Washington Avenue. The portion of East Washington in front of the store is full of parked automobiles.
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Description: Howard Morey delivering the film "Hell's Holiday" to R.E. Mutchler of American Legion Post No. 57. (The man in the top coat is unidentified.) Morey flew th...
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text:
March 9, 2015
Rising acid levels in oceans imperil region's shellfish Changes from surge in carbon dioxide take toll
By David Abel GLOBE STAFF MARCH 07, 2015
WALPOLE, Maine — Something was wrong with the larvae.
Bill Mook noticed the newly born oysters of his coastal hatchery often failed to thrive after heavy rainfalls. The storms left the brackish seawater he was pumping into his tanks from the nearby Damariscotta River estuary too acidic for the pinhead-sized mollusks.
To stay in...
February 24, 2015
University of Maine marine scientist Bob Steneck is part of an international team that has unlocked an underwater time capsule in the North Pacific that has been monitoring the climate for centuries.
The time capsule is the long-living, slow-growing alga Clathromorphum nereostratum that creates massive reefs in shallow coastal regions of Alaska's Aleutian archipelago. These solid calcium carbonate structures have fine growth rings — similar to tree growth rings — which Steneck...
February 6, 2015
2015 National Ocean Sciences Bowl
Northern New England Regional Competition
February 7, 2015
Hosted by
University of Maine School of Marine Sciences
Regional Co-Sponsors
Maine Sea Grant College Program
University of New England
New Hampshire Sea Grant
Welcome to the University of Maine!
The 2015 Nor'easter Bowl is a regional competition of the National Ocean Sciences Bowl (NOSB). This event is co-sponsored by the...
December 15, 2014
Extreme weather
Researchers look at the effects of a changing environment on Maine's marine waterways, croplands and municipalities
by Beth Staples
Study, strategize, prepare and adapt
https://www.youtube.com/watch?v=kCYjrPenfAw#t=15Video transcript
Paul Mayewski:
Climate change has always happened. There are natural climate changes and then today, we of course have the dramatically added influence of human activity. Weather in general make, those are the building blocks of climate,...
November 18, 2014
Marine Ecosystem Health
Understanding the biodiversity of bacteria associated with marine algae that contribute to marine ecosystem health is the focus of a study led by three University of Maine researchers.
Susan Brawley, a professor of plant biology in the School of Marine Sciences and a cooperating professor in the School of Biology and Ecology, is leading the three-year project. At UMaine, Brawley is working with John Singer, a professor of microbiology, and Benildo de los Reyes, a...
November 10, 2014
Academic partnership allows for students to work in Gulf of Maine
By Chase Brunton
Posted on Nov. 09, 2014, 11:42 p.m. at
The College of Natural Sciences, Forestry and Agriculture (NSFA) has established a new work-study/internship program with the National Oceanic and Atmospheric Administration's (NOAA) Northeast Fisheries Science Center. The newly announced internships will allow undergraduate students in the college of NSFA the opportunity to become involved in different...
October 21, 2014
Studying the movement of carbon dioxide into the deep ocean to improve climate projections and understanding of deep-sea ecosystems will be the focus of a two-year research project by a University of Maine marine scientist.
Feb. 1, Nathan Briggs begins a two-year postdoctoral fellowship research project in France that's funded, in part, by a $194,000 grant from the National Science Foundation (NSF). He will collaborate with Hervé Claustre, a senior scientist...
October 16, 2014
Current Biology is widely valued among life scientists for its unique blend of important research papers and informed, lively commentary. 5-Year Impact Factor: 10.227.
Bob Steneck
Bob Steneck is professor of marine biology, oceanography and marine policy in University of Maine's, School of Marine Sciences. He is a marine ecologist whose 'laboratories' includecoral reefs in the Caribbean and Indopacific oceans and kelp forests of the North Atlantic and North Pacific
October 14, 2014
Going Deep
When planning a trip, travelers often consider their destinations' peak tourist seasons and weather.
Rhian Waller schedules her voyages around whale migration, glacial melting and ocean clarity.
This spring, the associate research professor in the University of Maine School of Marine Sciences will be chief scientist on an expedition to explore, map and survey underwater habitats and ecosystems of Glacier Bay National Park and Preserve (GBNP) in Alaska.
Waller has been...
September 11, 2014
Researchers discover 'spectacular' coral formations in the Gulf of Maine
Gulf of Maine Deep Coral Science Team 2014/NURTEC-UCONN, NOAA Fisheries, UMAINE
Pollock and herring swim past newly discovered Primnoa coral in the Schoodic Ridges area of the Gulf of Maine earlier this summer.
Gulf of Maine Deep Coral Science Team 2014/NURTEC-UCONN, NOAA Fisheries, UMAINE
Herring swim past Primnoa coral in the Schoodic Ridges area of the Gulf of Maine earlier this summer. Red dots in the photo are...


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The W. Cary Edwards School of Nursing at Thomas Edison State College in Trenton, N.J., held a pinning ceremony Sept. 21 for the graduates of its inaugural accelerated, second-degree BSN program. According to a news release, the program is designed for adult learners who already possess a non-nursing bachelors degree and who are interested in becoming RNs.
Of the three program components that the cohort completed — hands-on clinical nursing experience, online courses and classroom-based instruction — the latter is a departure for the W. Cary Edwards School of Nursing, at which RN-to-BSN students customarily complete their course work online, according to the release.
"The students put forth an extraordinary effort and had to be fully committed to putting their lives on hold for the program," Filomela "Phyllis" Marshall, RN, EdD, CNE, dean of the W. Cary Edwards School of Nursing, said in the release. "The programs pace and rigorousness demanded that they exclude most other activities from their lives, including work, while they fulfilled their BSN course requirements. This first accelerated, second-degree BSN class is a remarkable group. They will bring a wealth of knowledge, compassion, adaptability and professionalism to the field of nursing."


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|Publication number
|Publication type
|Application number
|Publication date
|Aug 5, 2003
|Filing date
|Mar 5, 2001
|Priority date
|Mar 5, 2001
|Fee status
|Also published as
|Publication number
|Vladimir B. Serikov
|Original Assignee
|Vladimir B. Serikov
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (14), Referenced by (12), Classifications (10), Legal Events (3)
|External Links: USPTO, USPTO Assignment, Espacenet
The present invention relates generally to devices and methods for performing separation of biological macromolecules (electrophoresis or gel chromatography) and transfer (electro-blot, capillary blot or other means of blotting). The present invention particularly relates to a device, which can perform separation and blotting of protein and nucleic acid samples within one capillary element.
Separation of macromolecules such as proteins and nucleic acids is a necessary step in numerous applications of protein and DNA-RNA analyses in multiple biological, scientific, medical, and forensic applications. The most prominent and widely used techniques for separating macromolecules are chromatography and gel electrophoresis. Following separation by said principles, proteins and nucleic acids are then generally collected in separate volumes or fixed (blotted) to special chemical compounds, mostly nitrocellulose or nylon (F. Ausubel et al (Ed.), Current Protocols in Molecular Biology, Ed. Current Protocols, Wiley, N.Y., 1994), for further processing and analyses. Separation and blotting must be performed independently in time and space, otherwise highly chemically reactive blotting membrane may be contaminated with separated molecules. For this reason after separation is performed, the gel with separated molecules is placed in contact with blotting membrane by mechanical transfer.
Electrophoresis, used for separation, utilizes a physical phenomenon of charged particles' ability to migrate toward the pole possessing the charge opposite to that of a particle, when it is suspended between opposite poles in an electric field. Conventional gel electrophoresis utilizes a gel slab assembly. Pore matrix of a gel slab is usually made of agarose or polyacrialamide. Pores, which retain macromolecules depending on their physical properties (size, hydrodynamic radius, weight, composition, electrical charge, etc.), form an interacting or sieving matrix. Wells are formed in the upper part of the gel slab during casting procedure. A gel slab assembly is placed into a device for electrophoresis. Samples are then introduced in a band within wells; and an electric field is applied across the slab. The upper and the lower portions of the gel are submerged into separate buffer solution reservoirs. The electric field forces macromolecules of the samples to migrate through the gel. During migration, the macromolecules are "sieved" by their properties, most often their molecular weight. Specific species of macromolecules will be in bands arranged from top to bottom of the gel (Andrews, A. T. Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications. Clarendon Press-Oxford (1986), 2nd Ed).
In the process of DNA separation, the DNA fragments (DNA ladders) can be labeled before or after separation with either radioactive or florescence labels (F. Ausubel et al (Ed.), Current Protocols in Molecular Biology. Current Protocols, Wiley, N.Y., 1994, Chapter 2). In DNA sequencing procedures, each of the four types of a nucleotide can be labeled with specific probe, which will appear at termination of DNA fragment. A mixture of different reactions can be electrophorized and separated according to the label from one reservoir in the gel.
After separation is complete, deposition membrane with blotting material is placed in contact with gel. A common transfer process is called "electro-blot" transfer. In the "electro-blot" transfer process, the macromolecules in the gel slab move under an electric field to a blotting membrane. In designing an electro-blot transfer system it is essential that the blotting membrane be in close contact with the gel slab. Presence of gas bubbles between the gel slab and blotting membrane will prevent the band images from being transferred properly. It is also important to maintain a uniform electric field across the electro-blot sandwich. Transfer of the gel slab onto the nitrocellulose membrane must be carefully performed so that the macromolecules on the gel membrane are not removed or contaminated. After transfer, a labeling procedure must be employed, and a detection technique must be utilized so that the samples can be analyzed. A commonly used detection method involves staining and de-staining of the gel slab. This technique imposes staining of the entire gel with a dye that only adheres to the macromolecules. Then a de-staining process is performed, wherein dye not adhered to the macromolecules is washed away; bands of macromolecules thus become detectable. Another common detection method is the use of antibodies. Bands of proteins or samples are blotted or transferred to a binding membrane, which binds macromolecules. Then, a known antibody is introduced. The antibody combines with a specific protein if it is present in a sample. In order to detect the antibody-protein combination, the antibodies are labeled with fluorescent or radioactive tags or have enzyme activity, which is further detected by separate methods (F. Ausubel et al (Ed.), Current Protocols in Molecular Biology, Ed. Current Protocols, Wiley, N.Y., 1994, Chapter 10).
Capillary electrophoresis offers some advantages not available with other separation methods, such as slab gel separation, HPLC, or column chromatography (Krylov, S. N., and Dovichi, N. J. Capillary electrophoresis for the analyses of biopolymers. Analytical Chemistry, 2000, Vol. 72, No. 12:111R-128R). The major advantage of the capillary electrophoresis is the speed of the analysis (few minutes, compared to few hours by other methods). Highly efficient dissipation of electro-resistive generated heat in the capillary is provided by a large surface to volume ratio. Decoupling of gel temperature from electro-resistive generated energy provides greater effective field strength. Migration rate varies directly with the field strength over a linear range, which can be extended using gel-filled capillaries. Thus, separations at higher fields can be performed with reduced running time. The advantage of a single capillary can be further extended by use of an array of coupled capillaries with equal characteristics. A possibility to process them together under identical conditions allows technology for protein separation and DNA sequencing (Dolnik V. DNA sequencing by capillary electrophoresis. J Biochem. and Biophys. Methods. 1999, V.41, No. 2-3:103-119).
Collection of samples in capillary electrophoresis poses, however, substantial technical problems (Altria, K. D. Overview of capillary electrophoresis and capillary chromatography. J. Chromatography, 1999, Vol. 286, No 1-2:443-63; Swinney, K., Bornhop, D. J. Detection in capillary electrophoresis. Electrophoresis, 2000, Vol. 21, no. 7:1239-50). Samples are eluted from the capillary at a certain point by either pressurized flow or electroelution. Method for sample collection by electroelution employs standard capillary electrophoretic equipment. Several parameters of the system must be precisely controlled (velocity of migration of a sample, distance between the detection point and the end of the capillary, etc). To collect fractions in appropriate vials, capillaries, etc, one must know the exact time, when a zone appears at the exit end of the capillary, as the variation of migration rates in capillary electrophoresis can be more than 2%. The time necessary for the zone of interest to move through the distance between the detection point and the capillary exit is calculated after detection of the zone of interest is done. Electric current is then turned off, and the capillary is removed from the apparatus and placed into a collection vial. Current is applied for a predetermined time so that the zone migrates into the collection reservoir with buffer from the capillary. Pressure can also be applied to remove the sample from the capillary. After collection of the zone is accomplished, the capillary is placed back into the electrophoretic device, and separation continues. A set of collection reservoirs, or capillaries, containing a collection buffer and electrode, can be positioned close to the exit end of the capillary; collection of samples can be performed, while the electric current is temporarily turned off.
Another approach for collecting samples from a capillary is positioning samples on a membrane. According to the technique, the exit end of the capillary is in contact with a wet surface of a moving blotting membrane, which also serves as an electrode. Separated proteins, or DNA fragments, are transferred to deposition membranes either by electrophoresing the bands onto a moving deposition membrane (direct electroblotting) or by various elution techniques implying gradient of hydrostatic pressure, capillary forces, and others. Standard direct blotting devices use a deposition membrane attached to a conveyor belt in the lower buffer chamber that remains totally submerged during electrophoresis. These devices require physical removal of the nylon or other membrane from the conveyor belt. Submersion of the deposition membrane makes detection and processing a difficult task.
Prior art:
U.S. Pat. No. 4,622,124 discloses a device for horizontal electroblotting of electrophoretically separated material. A liquid tight container is provided having a support assembly, horizontally disposed in a chamber defined by the container. Electrodes are located below and above the support assembly, and a barrier is provided to prevent bubble attachment and uneven blotting. U.S. Pat. No. 4,589,965 presents a method for electroblotting, whereby an electrophoretically resolved material in a gelatin sheet is transferred to a membrane. The gelatin sheet is in contact with an immobilizing material, sandwiched between two plate electrodes. U.S. Pat. No. 4,994,166 to Fernwood et al describes a single apparatus for slab gel electrophoresis and blotting, both of which are performed in a single tank cell, which contains separation electrodes along opposing vertical walls and blotting electrodes arranged horizontally above and below the level of gel placement. The cell is operated in separatory and blotting modes, in which separatory and blotting electrodes are separately energized. No means for separation of gel and blotting membrane are provided.
U.S. Pat. No. 4,812,216 to Hurd explain a method and apparatus for supporting and handling blot membranes during the course of blotting, analysis, and storage. U.S. Pat. No. 5,039,493 to Oprandy describes a positive pressure blotting apparatus having a bottom section, a middle section, and a top section. The first volume of space is encompassed between the top section and the middle section. Positive pressure is maintained in the first volume of the apparatus. A hydrophobic filter means for binding biological materials is positioned on, or in, the middle section, and a means is provided to secure the top, middle and bottom sections together to form a pressure blotting apparatus. U.S. Pat. No. 5,149,408 to Perlman shows a capillary blotting sandwich for transferring soluble macromolecules in a liquid medium from a liquid-permeable matrix to a semi-permeable receiving membrane. The blotting sandwich includes: (i) a liquid-permeable matrix layer comprising soluble macromolecules, said matrix having at least one flat surface; (ii) a semi-permeable membrane sheet layer disposed on said flat surface; (iii) an interlocked hydrophilic absorbent fiber-containing material in the form of a non-woven absorbent felt sheet, which facilitates capillary transport of a solution through said membrane.
U.S. Pat. No. 5,013,420 to Schuette presents electrophoresis-blot transfer apparatus, in which a buffer tank is common to both the electrophoresis and electro-blot transfer. Combination of two procedures (separation and transfer) requires a two-step operation with reassembly of an electro-blot sandwich. U.S. Pat. No. 4,889,606 to Dyson et al describes a method and apparatus for use in electrophoresis and blotting, in which electrophoresis is carried out on a gel, supported by a rigid porous plate. The gel is cast onto the porous plate with a transfer membrane between the porous plate and the gel. The method consists of the first stage, using electric field produced by electrodes to draw the samples through the gel; and the second stage, drawing the distributed macromolecules onto the transfer membrane. Since separating gel and transfer membrane are not separated by a barrier, contamination of the blotting membrane occurs while separation. U.S. Pat. No. 4,589,965 to Kreisher describes a method for electroblotting, whereby an electrophoretically resolved material in a gelatin sheet is quickly and efficiently transferred to a membrane with high pattern definition and resolution.
U.S. Pat. No. 5,593,561 to Cognard and Hache shows a multiple electrophoresis method for controlled migration of macromolecules and transfer thereof to the membrane in a vessel, containing a plurality of parallel elongate electrodes. The first electric field, established between electrodes, provides means for macromolecular separation in a gel, and the second electric field, perpendicular to the first, provides means for transferring the macromolecules onto the membranes. In the described method, at first, electrodes and transfer membranes are assembled in the vessel, which is then filled with gel. After the separation of macromolecules in a gel and transfer to membranes, gel is liquefied, dissolved, or decomposed, allowing the removal of membranes. Closely related U.S. Pat. No. 5,102,524 to Dutertre describes a multiple electrophoresis method, where different sets of electrodes are used in a two-step process to first separate macromolecules and then to transfer them to a deposition membrane. No means to separate the deposition membrane from the gel during the first step of electrophoresis are provided. U.S. Pat. Nos. 4,849,078 and 4,911,816, both to Love et al, present apparatus for carrying out horizontal electrophoresis and subsequent vacuum-assisted transportation of macromolecules to a support membrane. Process is performed in two steps: first, electrophoresis is performed; and second, deposit membrane is put in contact with gel, and vacuum transfer is conducted.
U.S. Pat. No. 5,217,592 to Jones describes apparatus, which includes the combination of submarine gel tank for electrophoresis separation with a vacuum applying means, which transfer the separated molecules from the gel into the deposition membrane, to a filter membrane by means of controlled vacuum. No means to separate the deposition membrane from the gel during the first step of electrophoresis are provided. U.S. Pat. No. 5,155,049 to Kauvar teaches a technique for passage of liquid through a membrane putatively containing in its interstices at least one substance, for which detection is desired. Further, it comprises positioning donor and acceptor bibulous matrices onto either surface of the membrane, squeezing the resulting sandwich. This technique permits the application of small volumes of reagents or wash to the membranes and the facile recovery of the waste.
U.S. Pat. No. 5,445,723 to Camacho describes blotting apparatus for transferring electrophoretically separated molecules from the gel into a transfer stack. Apparatus includes a resilient anode surface for transfer of molecules, which is mounted on a mechanical carrier arm, which moves the anode surface over the membrane during transfer—mechanically moved device.
U.S. Pat. No. 5,234,559 to Collier et al explains an apparatus for direct blotting and automated electrophoresis, transfer, and detection of biomolecules. Separation and transfer module consists of a separating gel and transfer membrane, stabilized by a frame, which is moved mechanically in orthogonal direction to the gel. Moving frame with the membrane is in contact with the edge of the gel, towards which separated fragments are driven electrophoretically.
U.S. Pat. No. 5,279,721 to Schmid describes an automated electrophoresis and transfer apparatus for separating macromolecules and blotting them to a transfer membrane, which includes a housing having a perforated intermediate horizontal partition defining an upper reservoir and a lower chamber. A transfer membrane is positioned over the partition; an impermeable sheet is interposed between the membrane and the gel member and withdrawn prior to transfer operation. Cooperation with the impermeable sheet substantially prevents chemical solutions from permeating the transfer membrane prior to transfer operation. In this embodiment, subdivision of separation and transfer processes is provided by means of mechanically moving an impermeable membrane.
U.S. Pat. Nos. 4,631,120 and 4,631,122, depict apparatus and method for a direct blotting process, which provides one or more collecting surfaces mounted on a conveyer belt or tape and advances the belt so that it slides over the end of the gel to collect separated particles and transport them with the belt away from the gel to a location, where further processing may be performed. Both vertical and horizontal embodiments are disclosed. Contact of this nature is damaging to the surface and may remove separated material. Also apparatus does not allow handling and identification of a multiplicity of samples in sequence. U.S. Pat. No. 5,514,256 to Douthart et al describes a direct blotting electrophoresis unit for DNA separation. The DNA sequence is deposited on a membrane attached to a rotating drum. Separation of DNA is performed in a capillary-like mini-gel system. A rotating drum with a deposition membrane is adjacent to the edge of the gel, and transfer of molecules to the gel occurs by means of electrophoretic transfer.
Claimed apparatuses and methods possess substantial disadvantages: after separation is completed, direct blotting in combination with slab gel electrophoresis requires mechanical means for changing the configuration of the system in order to put the blotting element in contact with separating gel. In capillary electrophoresis system, direct blotting appears to be impossible because a very thin layer of interactive matrix is in a very close proximity to the walls.
Use of semipermeable membranes for assisting the capillary separation has been claimed previously, though for different purposes. For example, U.S. Pat. No. 5,985,121 to Wu et al describes apparatus for capillary electrophoresis carrying out on-line sample preparation by means of a semipermeable membranes connected to the capillary separation column for selective introduction of ampholytes into the capillary separation column. Transfer procedures are not described in this invention. U.S. Pat. No. 5,338,427 to Shartle teaches a disposable cartridge for a capillary electrophoresis instrument, in which short capillary tube segments are used for simultaneous multiple lane separations. The cartridge contains all separation components of the instrument, which come in contact with the sample and is capable of automatically loading a quantitative portion of a bulk sample into the capillary tube segments. Electrophoresis occurs without bulk flow through the capillaries, which are scanned in situ by the instrument. U.S. Pat. No. 5,169,511 depicts capillary tube for electrophoresis with a sample cup, consisting of two wells, bottoms of which are covered with semipermeable membrane. Membrane allows the flow of buffer, but not the migration of separated sample. U.S. Pat. No. 5,482,613 describes a method for making a gel plate with a microporous membrane by means of rectangular frame cassette and means of tensioning a membrane deposited on a frame. U.S. Pat. No. 4,512,896 to Gershoni teaches a method of transfer of macromolecules to a mobilizing matrix, which is a charge modified microporous membrane. U.S. Pat. No. 5,897,817 shows a process for making medical device from a cellulose acetate hollow fiber semipermeable membrane. In this process, a molten liquid, comprising cellulose acetate, is extruded to produce a membrane. The solvent and the non-solvent are removed from the membrane to produce a semipermeable membrane, having water permeability. The semipermeable membrane can be incorporated in casing in order to produce a product. U.S. Pat. No. 5,131,994 to Shmidt and Cheh explains a method and apparatus for affecting an electrophoretic separation of charged particles. Fractionation chamber comprising a semipermeable membrane is used, wherein a trans-membrane force holds the particles to be separated against the membrane surface, while a voltage gradient is applied to separate particles. U.S. Pat. No. 4,964,961 to Brautigam and Gorman describes apparatus for electroelution of components, separated by preparative electrophoresis on a gel comprising a means for separation of molecules and elution of a selected fraction through a side of separation vessels using a dialysis membrane.
U.S. Pat. No. 5,840,169 to Andersen presents an apparatus and process for electroelution of a gel, comprising of a device with parallel adjacent chambers. Gel with separated macromolecules is placed on top of the apparatus; and electroelution occurs through a semipermeable membrane, placed on the other open sides of the chambers. Macromolecules in the gel migrate into the elution buffers in the chambers. Apparatus and method do not provide means for blotting.
U.S. Pat. No. 5,284,559 to Lim and Hixton describes device for electrophoresis comprising of a pair of spaced apart plates with a separating gel in between and a semipermeable membrane connected to the end of the plates. Membrane forms a collection channel for receiving molecules, which have traveled through the gel, and allows performance of fraction collection. Means for blotting are not disclosed. U.S. Pat. No. 5,427,664 to Stoev demonstrate a free solution electrophones-membrane-filter trapping assay apparatus, which includes a container and at least one porous membrane. The porous membrane and container define a chamber capable of holding run solution. A specimen containing particles to be classified is juxtaposed with the run solution. Particles, which are smaller in size than the pores of the membrane, pass through the membrane, while particles having a larger size do not. A method of classifying particles includes steps providing a sample of particles and a porous membrane, positioning a run solution between the sample and the membrane, and applying an initial voltage across the sample, run solution and membrane.
U.S. Pat. Nos. 4,992,172 and 5,160,626, both to Pemawansa et al, are examples of various membrane compositions, including the ones, which use microporous composites for direct blotting. U.S. Pat. No. 4,992,172 describes a blotting composition, comprising a substrate applied to an activated microporous membrane comprising a membrane polymer and having (1) an internal surface comprising interstices or pore surfaces and (2) an external surface; wherein substantially all said surfaces of the microporous membrane are coated without significant chemical reaction to substantially all said available surface areas of said microporous membrane and thereby activated by a polyaldehyde-containing mixture in an amount sufficient to effectively activate the microporous membrane; and wherein the mixture is soluble enough to penetrate substantially all said interstices of the microporous membrane without substantial damage to the pore structure. Means of separation and/or modification of membranes in the process of transfer are not claimed. U.S. Pat. No. 5,160,626 describes a method of transferring a biological sample to an immobilizing matrix, comprising applying the sample to an activated microporous membrane having: 1) an internal surface comprising interstices or pore surfaces; and 2) an external surface, which membrane comprises a membrane polymer that is non-covalently activated by having all said surfaces of the membrane physically coated with a polyaldehyde mixture in an amount sufficient to provide effective free aldehyde functionality on said surfaces for covalent bonding between the thus coated membrane and aldehyde reactive material of said sample and to thereby non-covalently activate the microporous membrane for said bonding without substantial damage to the pore structure. Means of combined separation and blotting are not disclosed.
The major disadvantage of the prior art is that the methods and apparatuses described allow performing only one of the two processes at a time and require a mechanical rearrangement of the geometry of the system to combine blotting and separating elements. Two separate units increase cost, labor and time of the procedure, which is a significant disadvantage with conventional devices. Mechanically based devices are complex and expensive; they cannot be used as disposables. In a standard setup for capillary separation molecules of interest, if direct blotting to the walls of the capillary is attempted, the molecules of interest will be attached to the walls along the whole length of the capillary, making distinction impossible. For that reason, the entire wall of the capillary element must be inert and should not allow any interaction between walls and separated molecules. Thus, detection of capillary electrophoresis or chromatography was previously possible only at the exit end of the capillary. The method of the instant invention allows modification of chemical properties of the walls of separating element, so that walls are transformed from impermeable to permeable state after separation by means of chemical or physical modification and thus allow a free passage of separated molecules towards the blotting element through the walls of the capillary. Previously described capillary-based separation techniques do not allow to perform separation and direct blotting, as they require detection or blotting only after separated materials leave the capillary. In formerly described capillary separation devices, these processes cannot be combined together in one capillary element. The capillary electrophoresis system and method of the instant invention have solved these problems of prior art systems.
It is an object of the present invention to provide an apparatus that positions a transfer membrane (blotting membrane) relative to a separating element (sieving or interacting matrix such as gel) by means of permeable membrane, whose properties can be chemically or physically modified, and when the gradient of driving forces is changed from the direction of separation to direction favorable to transfer, the transfer to the membrane is conducted in a time-controlled fashion. It is a further object of the present invention to combine separating element and blotting element in one composite to eliminate the necessity to perform two separate processes—separation and blotting. A feature of the present invention is the means to provide a combined apparatus for separation and blotting in one capillary-based unit with means of direct blotting to the walls of a disposable capillary. It is yet another advantage of the present invention, that it allows to use very long capillary elements, where length to diameter ratio of capillary can be more than a 100, thus providing a high resolution of separation. It is a further advantage of the present invention that the apparatus and method disclosed are amenable to automated process and allow to provide uniform, repeatable and well-controlled conditions for performing separation and blotting even in non-specialized environment (field environment). These and other objects, features and advantages of the invention will become apparent upon having reference to the following description of the preferred embodiments. The present invention meets the above objectives and provides advantages theretofore unavailable in conventional devices.
The invention is directed to a system for separation of macromolecules according to their dimensions and/or charge (capillary or blot electrophoresis or gel chromatography) and method for precise post-separation blotting of said molecules to the accepting element. A disposable separation element (capillary) contains inside a sieving or interaction matrix; an external layer of blotting material, positioned close to the boundary of said sieving or interaction matrix; and/or a membrane with changeable permeability for separated material, which separates blotting layer from the sieving or interaction matrix. After separation of macromolecules in capillary with initially non-permeable walls, physical of chemical modification of the intermediate membrane is performed, which is followed by change in the vector of driving forces for transfer, so that separated molecules are moved through the walls of the capillary and blotted to the outer layer of blotting material. The system of the invention includes a disposable separation element (capillary), which can be stretched or coiled inside reservoir; an electrophoresis buffer reservoir to supply electrophoresis buffer to the entrance and exit of the capillary; reservoirs to supply buffer; and modifying solution to a blotting material positioned as an outer layer at the body of capillary element. Means of modification of separation membrane include, but not limited to, chemical or physical modification to change permeability of the membrane for separated material after accomplishment of the separation in said sieving or interaction matrix. Change in driving force for the separation material may include, but is not limited to electrical charge application, bulk flow of fluid, hydrostatic pressure, or gravity.
Post-blotting processing of the sample components in the said system can include any type of analysis, reaction, modification, collection, or purification. For example, the blotting element may be used to transfer the separated and blotted sample components to analytical devices and/or further processed in antigen-antibody reaction or hybridized with DNA or RNA probes. Also blotted samples may be purified or processed by radioactive analysis or electrochemical analyses.
FIG. 1. Arrangement of separating and deposition membranes in a separating element.
FIG. 2. Unit for the capillary electrophoresis—direct electro-blotting.
FIG. 3. Positioning of the blotting element by rewinding the deposition material around the separating element.
In FIG. 1, a diagram of a separating element is shown. The separating system comprises of a capillary tube 1 with an entrance end 2 and an exit end 3. Capillary is filled with separating media or interactive matrix 4. Sample is introduced into the entrance end and enters the capillary tube by a pipette, hydrostatic flow or by electromigration and/or electroosmosis. Walls of the capillary tube 5 are made of the first layer of semipermeable separating membrane with changeable permeability 6, and the layer of deposition membrane 7. The membrane contains electrolyte in order to maintain electrical contact between electrode and deposition membrane. The boundary between layers 6 and 7 may be comprised of an additional layer of porous material 8. A key component of the apparatus of an instant invention is a unique unit with chemically or physically modifiable membrane 6, which allows direct blotting of the separated proteins or sequence ladders onto a nylon or another suitable deposition membrane affixed to external surface of the separating membrane.
The basis of the method of a present invention is membrane 6, with changeable permeability characteristics (Kesting, R. E. Synthetic polymeric membranes. John Wiley and Sons, Inc., 1985, p. 224-329). Such membrane separates the interactive matrix 4 in the capillary and deposition membrane 7 and thus forms a structure of the capillary wall. This membrane can be made of a variety of materials and utilize a variety of methods to modify its permeability. One of the examples of separation membranes is a phase inversion process of membrane formation, in which polymer solution (in which the solvent system is in continuous phase) inverts into swollen three-dimensional macromolecular network or gel (polymer in the gel is a continuous phase). Gel constitutes a phase-inversion membrane. The latent solvent is removed (process named leaching) from the membrane gel after the gel attains sufficient strength. Typical leaching agents include isopropanol, ethanol, heptane, methyl alkyl ketone, and others. The most appropriate leaching processes for the described invention are those, in which preformers are low-molecular-weight surfactants (preferably ionic), which form high molecular weight, evenly dispersed micelles in the fluid state and maintain this structure in the solid polymer matrix. After leaching the solid matrix, the volumes previously occupied by surfactant micelles become pores. Polymer-assisted phase inversion process uses a solution consisting of one solvent and two physically compatible polymers. After solvent evaporation, film is immersed in liquid such as water, which serves as a solvent for polymer, but not the other. The insoluble network remains after leaching, forming microporous polymer-assisted phase-inversion membrane. Polymer, which is leached, serves as a non-solvent pore former ("assistant").
Another example is liquid and dynamically formed membranes. Liquid membranes are thin semipermeable barriers, which are in liquid state. Immobilized liquid membranes are confined within a microporous solid. The most widely used and simplest process of preparing porous membranes is the Gelgard� process, in which the semicristalline films or fibers are extruded from the melt. Porosity induced by stretching solid-state polymer, like polypropylene. Gelgard� is available in both film and hollow fiber form with pore sizes up to 0.04 μm. Gelgard� hollow fibers are available with 100-1500 μm ID and 25 μm wall thickness and are used for example, in blood oxygenators. Gelgard� is compatible with ethyl alcohol, ethylene glycol, and isopropyl alcohol. Another example is Gore-Tex�, microporous poly (tetrafluoroethylene), which is a widely used porous membrane, also manufactured by stretching process. Gore-Tex� is one of the most chemically inert and hydrophobic synthetic polymeric membranes.
Dynamically formed membranes initially exist in the liquid state and are formed into semipermeable barrier layer from suspensions of finely divided particles, when the later are allowed to impinge upon a porous layer. Support liquid membranes are formed by placing liquid membrane on a dense membrane or by internal containment of the liquid membrane within a microporous support. Liquid surfactant membranes are formed by concentration on the interface between a liquid solution and a dense membrane. The bilayer of dense membrane and surfactant membrane provides a composite structure, whose components act in series to provide permeability characteristics of the particular membrane. Liquid surfactant membranes can form spontaneously on every impermeable interface, which is in contact with surfactant solution. Surface films of soluble surfactants influence material transport across phase interfaces in the same manner as insoluble films. Internally supported membranes can be used for facilitating selective transport across porous membranes. Variation in permeability and permselectivity of various liquid-surfactant membranes is due to two main factors: hydrophilic/hydrophobic balance and packing arrangement within and between individual micelles.
Separating membrane 6 with changeable permeability characteristics can be made by process of leaching of the preformed membrane; by changing permeability of liquid-deposited, liquid surfactant membrane by changing chemical composition, pH or the temperature of the surrounding buffering solution 11. Leaching of the solid matrix of membrane makes pores in volumes previously occupied by surfactant micelles. For example, such porous membrane can be made from mixture of viscose solution and 200% sodium dodecyl benzene sulfonate (R. E. Kesting, Synthetic Polymeric Membranes. John Wiley and Sons, Inc. 1985, p. 224-329). The preferred leaching agents are water, lower alcohols or toluene. After solidification of the polymer, it can be swollen in liquid, which has the effect of breaking down the micelles of surfactant to individual molecules and their rapid extraction. Leaching of the surfactant micelles can be applied to a variety of solutions, including cellulose and methoxymethylated nylon, polyacrilic, poly (vinyl acetate), and polyethylene-paraffin. For example, lauryl pyridinium chloride in toluene can be utilized as the surfactant micelle to form a microporous polyethylene membrane.
Liquid-surfactant membranes can be precoated on a dense supporting membrane, for example by coating the membrane substrate with 0.1% solution of poly (vinyl methyl ether) in CCl4. Internal support to liquid membranes can be provided by several ways, for example by addition of a gelling agent such as Cabosil� or Cellosize�, confinement of liquid membrane with a microporous support (immobilized membrane), deposition of a film such as poly (vinyl methyl ether), which is potentially soluble in water, and controlling swelling by introduction of covalent cross-links.
For dynamically formed membranes support like any suitable filter or hollow fiber can be used. Phospholipid layer is an example of temperature-sensitive permeability membrane (Sada, S., Katoh, S., and Terashima, M. Transfer of solutes through composite membranes containing phospholipids. In: Membranes and Membrane Processes. E. Drioli. M. Nagasaki (Ed.), Plenum Press, N.Y. (1986), pp. 455-459), where permeability characteristics change by an order of magnitude around the melting temperature of phospholipids.
Other polymeric substances, which can be used for these purposes, are, but not limited to, starch sulfate, Sephadex (dextran) polyacrylic acid, sulfonated polysterene, poly (vinyl pyrrolidone), hydroxyethylcellulose, poly (ethyleneoxide) and its block copolymers, and poly (propylene oxide). Permeability and permselectivity of positively charged membranes is a function of pH and temperature. Membranes can be made wet by contacting the membrane with a water miscible organic solvent such as ethanol, methanol, acetone, or the like. Representative membranes may include polyolefins such as polyethylene, polypropylene, polymethylpentene, or the like; polystyrene or substituted polystyrenes; fluorinated polymers including poly (tetrafluoroethylene), polyvinyldiene fluoride, or the like; polysulfones, such as polysulfone, polyethersulfone, or the like; polyesters including polyethylene terephthalate, polybutylene terephthalate, or the like; polyacrylates or any polycarbonates; polyamide nitrocellulose, vinyl polymers such as polyvinyl chloride, copolymers of butadiene and styrene, fluorinated ethylene-propylene copolymer, ethylene-chlorotri-fluoroethylene copolymer, or the like. Deposition membrane element 7 comprises an electrically conductive layer electrically connected to a source of electrical energy through the buffering electrolyte solution made of a material suitable for deposition of protein or DNA molecules, like for example, nitrocellulose paper, nylon, and others.
The diagrammatic scheme of a separating apparatus, based on the above-described separating element 1, is shown in FIG. 2. Referring to FIG. 2, the capillary 1 is submerged into reservoir and support structure 10, filled with buffering solution 11. Reservoir 10 can be in the form of rectangular parallelepiped or in the form of cylinder. Reservoir 10 may contain multiple separating elements 1. A separation-blotting system of the invention includes, in general, an array of capillaries 1. Capillary 1 is in contact at the loading end 2 with a capillary electrophoresis buffer reservoir 16 and at the exit end 3 with a capillary electrophoresis buffer reservoir 12. Buffer, contained within reservoirs 10, 12, 16, can be any buffer commonly used in capillary electrophoretic separation: a Tris buffer or a phosphate buffer. Capillary electrophoresis reservoir 16 and collection buffer reservoir 12 are in electrical contact through electrodes 19 and 18, respectively, and are connected to a high voltage power supply 27 to provide electrical current. An additional electrode structure of this invention comprises an electrically conductive wire 21 connected to power supply 22. The electrically conductive wire 25 is connected to the electrical battery or electrical power supply 27 through switches 24 and 26, which are turned on during the separation phase and are turned off during the blotting phase.
Support structure 10 is a generally circular reservoir or rectangular block of electrically insulating material. Reservoir 10 has at least two separate electrolyte and buffer fillings, which are represented in the form of ducts 14, each including opening and closing means (for example mechanically operated stop-cocks or solenoid valves). One of the ducts is provided at the bottom of reservoir and the other one at the top. According to the invention, these ducts 14 are used for multiple purposes: to fill the reservoir with electrolyte or buffer, to provide circulation of the buffer between reservoir 10 and heat exchange unit to provide means for cooling the set of capillaries during separation phase. Reservoirs 12 and 16 have openings 20 and 15 respectively, which serve the same purposes. The reservoir 10 includes electrodes 17, which are constituted by electrically conductive metal wires and are connected by the electrically conductive wire 21 at their ends to power supply 22 for applying electrical potentials during the blotting phase. The electrode structure 17 can be flat or cylindrical structure. Capillary tube 1 can be supported by an additional supporting structure 13 for mechanical stability. Capillary tube 1 may have support structures made of any clear, low reflection, low absorbance material such as plastic, glass, or silica. Capillary tube 1 is usually from about 1 to about 10 cm in length, although longer length may be used.
Devices may be fabricated from a wide variety of materials, including glass, fused silica, acrylics, thermoplastics and the like. The components may be fabricated from the same or different materials, depending on the particular use of the device. For example, materials like polymethylmethacrylate, polycarbonate, polyethylene, polystyrene or styrene copolymers, and the like. The devices may be fabricated using any convenient means, including casting techniques, by etching or laser micromachining.
The apparatus of the invention is then used as follows:
1. Application of sample and separation of sample. Array of capillaries 1 are placed in chamber 10 and fixed. Chamber 10 and 12 are filled with electrolyte buffer. The lid 9 is opened and samples are deposited at the entrance end 2 of the capillary 1. Then lid 9 is closed and upper chamber 16 is filled with electrolyte buffer through openings 15. Different electrical potentials are applied to electrodes 19 and 18 to create an electrical field between the entrance end 2 and exit end 3 of the capillary, causing the macromolecules to move through the gel, until separation is completed. During this separation phase switches 23 and 28 are opened and switches 24 and 26 are closed. Other means of separating macromolecules (for example gradient of hydrostatic pressure) may be applied instead of electrical field at this stage.
2. Modification of the capillary wall-separating membrane. After separation is complete, electrical switches 24 and 26 are opened. Modification of the separating membrane is achieved by means of changing chemical composition of the buffer solution in chamber 10, or changing its temperature or pH. After a certain amount of time, required for the modification of the membrane to be completed, buffer or other solution is washed from chamber 10, and it is filled with electrolyte buffer.
3. Blotting by change in the gradient of driving force. Electrical switches 24 and 26 remain opened, electrical switches 23 and 28 are closed. Electrode 17 in chamber 10 thus becomes a cathode and electrodes 19 in chamber 16 and electrode 18 in chamber 12 with the capillary structure 1, form an anode. Established electrical driving force moves separated macromolecules in capillary structure 1 in horizontal direction through the membrane 6. Macromolecules are then deposited on the blotting membrane 7.
Other means of transferring macromolecules, for example gradient of hydrostatic pressures, which will cause the flow of fluid through the capillary from reservoirs 12 and 16 into reservoir 10, can be used instead of electrical force. Negative pressure in reservoir 10 can be also applied through ducts 14.
4. Processing and detection. After a certain period of time macromolecules become fixed on the blotting membrane 7, after which the current is disconnected. The respective fixing solutions or means of detecting macromolecules on the blotting membrane may be applied into chamber 10. The outer wall of the capillary element becomes a carrier of a blotted material, which can be further processed for specific detection. In other embodiments the deposition membrane can be removed without disturbing the separation membrane. The deposition membrane can then be moved intact to suitable downstream detection and processing units. FIG. 3 shows an example of such method, where a thread 29, made of a composition amenable for blotting, is winded around the capillary 1. After blotting is completed, element 29 can be easily unwind and stored for purposes of detection of separated molecules.
The capillary unit of the instant invention has both the data compaction and energy dissipation characteristics of gel-filled capillaries and the ease of preparation, reliability, and easy recasting characteristics of slab gels. The separation capillary can also be formed as a channel or a groove or micromachined on a planar surface or molded in a block of a suitable polymer (Plexiglas or polycarbonate), or on a microchip (Regnier, F. E., He, B., Lin, S., and Busse, J. Chromatography and electrophoresis on chips: critical elements of future integrated, microfluidic analytical systems for life science. Trends Biotech, 1999, Vol. 17, no. 3:101-116; U.S. Pat. No. 6,007,690 to Nelson et al). Electrophoretic separation can be carried out with or without the use of a sieving or interaction matrix and in combination with any other appropriate technique, such as isoelectric focusing. In addition, separation can take place in an open tube as capillary zone electrophoresis, with or without the use of electroosmotic flow. After deposition of the sample on the deposition membrane, the sample can be reacted with conventional reagents used in available assay or detection techniques (radio-labeled or fluorescent labeled antigens or antibodies, staining reagents or dyes).
The described apparatus and method allows combination of advantages of capillary electrophoresis and direct blotting in one single element. Multiple arrays of such elements allow performing numerous sample analyses in short periods of time automatically, saving labor and time. Optimization of geometrical variables allows using very small volumes of expensive reagents, like antibodies.
To demonstrate the potential applicability of the described process and apparatus to the commonly used procedures, Southern and Western blot procedures were performed with DNA samples and tissue protein samples. In Southern blotting procedure samples of prokaryotic DNA from cultures transfected with plasmids carrying sequence for I-II loop of α1 subunit of L-type calcium channel were separated using the described procedure, blotted and probed for the presence of sequence of interest. In Western blot procedure presence of β subunit of L-type calcium channel was determined in protein extracts from heart tissue. To perform capillary electrophoresis, Gelgard� hollow fibers were used. For Southern analyses 6 cm long fibers were filled with 1% agarose solution at 80� C.; and for Western analyses—with 10% polyacrilamide solution at 50� C., both of which become gels upon cooling to room temperature. Walls of capillary fibers were bathed in a mixture of organic triglycerides at 50� C., removed, cooled to room temperature, washed in buffer, and wrapped in nitrocellulose paper (Hybond, Amersham Pharmacia Biotech) in a manner, shown in FIG. 3. The resulting capillary element was placed in the electrophoresis bath, schematically depicted in FIG. 2. Element contacts with walls, which separate the reservoirs, were sealed with glue. Samples of procariotic DNA were digested with EcoRI and introduced into the entrance of the capillary with syringe for chromatographic sample injection. Positive controls were samples of DNA from transfected cultures of E. coli expressing I-II loop of L-VDCC; negative controls were wild type cultures. Separation was performed at 150V with potential difference of 10 min; temperature of bath was kept below 10� C. After electrophoresis separation element was washed several times in elution buffer with methanol concentration of 40% at 60� C. by changing solution in intermediate reservoir. Transfer to nitrocellulose membrane was performed in 10 min by changing the electrophoretic gradient, as described in the summary of the invention. After transfer the nitrocellulose paper was detached from the separating element, the DNA was probed with a 1350 bp EcoRI digested complementary fragment from the full-length α1C cDNA (bases 2098-3448). As a control, standard procedures of Southern blotting, utilizing slab blot electrophoresis and electrotransfer, were used. Both methods showed presence of DNA fragment of interest (I-II loop of L-VDCC) in positive controls and its absence in negative controls. Capillary transfer was accomplished in a small fraction (10%) of time required for standard procedure without mechanical transfer of elements.
Western blot analysis of the β-subunit was performed in the same manner. Protein extracts from mice hearts were prepared by standard techniques. Membrane and cytosolic fractions (0.5 μg) were introduced into separating capillary elements, separated and transferred as described above to nitrocellulose membranes (Hybond-ECL, Amersham Pharmacia Biotech). Nitrocellulose membranes were then detached from separating element, blocked with 5% nonfat milk for one hour at room temperature (22-25� C.). Blots were incubated with β1-subunit of L-type calcium channel primary antibody (Swant) for 1 h at 22� C. Horseradish peroxidase-conjugated secondary antibody was added for 1 hour at room temperature, and the signals were visualized by the enhanced chemiluminescence procedure (Amersham Pharmacia Biotech). Standard stained molecular weight markers (Amersham) were loaded to determine the molecular weight of the fraction. Standard electrophoresis and electroblotting procedure was performed with same samples as a control. Both methods demonstrated similar results of location of fraction of interest (β1-subunit) relatively to the known molecular weight markers.
This example demonstrated that the described apparatus and method allows to shorten time, cost and labor of the procedure, not to mention a possibility of usage of multiple separating elements for simultaneous analyses of a very large number of samples.
Although the invention has been shown and described with respect to illustrative embodiments thereof, it should be appreciated that the foregoing and various other changes, omissions and additions in the form and detail thereof may be made without departing from the spirit and scope of the invention as delineated in the following claims.
|Cited Patent
|Filing date
|Publication date
|Nov 14, 1984
|May 20, 1986
|International Biotechnologies, Inc.
|Method for electroblotting
|Aug 22, 1989
|Feb 19, 1991
|Bio-Rad Laboratories, Inc.
|Single apparatus for slab gel electrophoresis and blotting
|Apr 19, 1990
|May 7, 1991
|Life Technologies Inc.
|Gel electrophoresis/electro-blot apparatus
|May 4, 1990
|Aug 13, 1991
|The United States Of America As Represented By The Secretary Of The Navy
|Positive pressure blotting apparatus with hydropholic filter means
|Apr 29, 1991
|Sep 22, 1992
|Brandeis University
|Capillary blotting pad for molecular transfer to membranes
|Aug 22, 1989
|Oct 13, 1992
|Terrapin Technologies, Inc.
|Blotting technique for membrane assay
|Sep 7, 1990
|Nov 3, 1992
|Gelman Sciences Inc.
|Blotting methods using polyaldehyde activated membranes
|Apr 22, 1993
|Jan 18, 1994
|Peter Schmid
|Apparatus and method for an automated electrophoresis system
|Jun 16, 1992
|Feb 8, 1994
|Rhode Island Hospital
|Preparative electrophoresis device and method
|Mar 9, 1994
|Sep 12, 1995
|Verma; Sumeet
|Multi-purpose electrophoresis apparatus
|Dec 2, 1993
|Jan 14, 1997
|Bertin & Cie
|Multiple electrophoresis method and apparatus for migration and transfer of macromolecules
|Feb 25, 1992
|Nov 24, 1998
|Andersen; Peter
|Apparatus and process for electroelution of a gel containing charged macromolecules
|Jul 8, 1997
|Apr 27, 1999
|Althin Medical, Inc.
|Process for making a medical device from a cellulose acetate hollow fiber semipermeable membrane
|May 10, 1996
|Nov 16, 1999
|Wu; Jiaqi
|Fast sampling device and sampling method for capillary electrophoresis
|Citing Patent
|Filing date
|Publication date
|Sep 2, 2004
|Dec 2, 2008
|Shmuel Bukshpan
|Methods and apparatus for rapid crystallization of biomolecules
|Jul 8, 2004
|Feb 16, 2010
|Siemens Water Technologies Corp.
|Membrane post treatment
|Oct 18, 2011
|Shmuel Bukshpan
|Methods and apparatus for rapid crystallization of biomolecules
|Dec 28, 2009
|Nov 15, 2011
|Siemens Industry, Inc.
|Membrane post treatment
|Aug 10, 2011
|Sep 11, 2012
|Siemens Industry, Inc.
|Membrane post treatment
|Jul 4, 2005
|Sep 3, 2013
|Siemens Industry, Inc.
|Hydrophilic membranes
|Sep 24, 2003
|Jun 17, 2004
|Igor Touzov
|Components for nano-scale Reactor
|Dec 19, 2005
|Jun 29, 2006
|Shmuel Bukshpan
|Methods and apparatus for rapid crystallization of biomolecules
|Jul 8, 2004
|Jul 20, 2006
|Daniel Mullette
|Membrane post treatment
|Jul 4, 2005
|Mar 6, 2008
|Siemens Water Technologies Corp.
|Hydrophilic Membranes
|Nov 4, 2008
|Apr 23, 2009
|Shmuel Bukshpan
|Methods and apparatus for rapid crystallization of biomolecules
|Aug 26, 2010
|Daniel Mullette
|Membrane post treatment
|U.S. Classification
|204/464, 204/455, 204/451, 204/601, 204/605, 204/613
|International Classification
|Cooperative Classification
|European Classification
|Feb 21, 2007
|Maintenance fee reminder mailed
|Aug 5, 2007
|Lapse for failure to pay maintenance fees
|Sep 25, 2007
|Expired due to failure to pay maintenance fee
Effective date: 20070805


text: Does anyone know if you can set the default playback resolution on the YouTube app? I can do this on my Motorola RAZR V phone, but can't do it on my Samsung Galaxy Tab 2. Can I set it on the Nexus 10? Does anyone know of any other Android tablet that allows the selection of the default video resolution in the YouTube app?


text:
i have a simple question for us, it's possibile insert a code string to automatic grab the first 10 word of post
in this section of theme code header.php?
<meta name=\"description\" content=\" \" />
<meta name="description" content=" " />
have suggestion?
thanks for support and sorry for my english :)
kiss lory


text:
+ Post New Thread
Hi all,
I've read all the tutorials and examples but didn't find a way to add a grid dynamicly :(
I have taken the complex borderlayout example...
hey guys!
this is a very general question: i have a complexlayout, and i want to handle the panels. that means i want to open, hide, show etc the...
Hi guys,
after a bit of developing in Ext, my one file for everything approach started to get out of hand. I've tried to separate my code into...
Is there a way to get all defined comboxes on a page? (Or more specifically in a ContentPanel)?
Hi... please help me...
i was modified complex.html, and i added code :
loadContent : function(elid,url,param){
Hello all - I'm just getting started with Ext and I am very excited with some of the functionality! This may be a stupid question but I can't...
Hi there.
I would like to show a Dialog out of a menu.
I removed the Button were the animation usually starts from. But now FireBug tells me...
I was wondering why Ext loads certain images used by the interface remotely from the Ext site?
For example the dropdown image used by the...
Hello !
I'm trying to understand the tutorial but I have a problem with this function :
I scanned the source files looking for a mechanism to format a NumberField's display text. Is there anything like that? Specifically, I want a...
Hi all ~ could you tell how to set the node's order ?
the server side will return JSON like following
so , if I add a root node and two child...
Anyone know if there's a good example of how to connect to the grid's onRowsUpdated event?
I was assuming that I could do something like the...
how can i use the datagrid if i have a local variable that is basically a DOM object ? i have:
var parser=new DOMParser();...
Is there a way to call a UpdateManager update() to a <DIV/> which has been "converted"/rendered into a NestedBorderLayout, and have the inner divs...
I'd like to load a HTML website via ContentPanel.setUrl(asdfasfasdf)
At the top of the page I have:
--page: sayHi.html --
I got this error while running a filter on a grid:
" nsresult: "0x8057001e (NS_ERROR_XPC_JS_THREW_STRING)" location: "<unknown>" data: no]...
Where I work, all the data is in Microsoft Access databases. I know, that's lame, but that's what I have to work with. I do a lot of ASP and ASP.net....
Hi everyone
In the Datefield object, there is a property called menuListners:
menuListeners : {
select: function(m, d){
showBtn = Ext.get('lnk1');
showBtn.on('click', this.showDialog, this);
showBtn = Ext.get('lnk2');
The first element in my data array is an id number that is used only for grid.getSelectedRowId(). I don't want it to be displayed on the grid though....


text: Posted August 1, 2014 In the context of the show, I think it's worth discussing that they should change from talking on social issues. Yeah, we are all in agreement that Stephen A made a mistake, but the show censored him and they didn't debate this issue like others. Here is a youtube clip talking on the situation from a black perspective. (There's some cursing in it but it's not overly so. Probably curses 4 or 5 times in the video for those of you who are like me who wince at 'em) Overall I have to agree with this video. As I said I can't look at First Take in the same light anymore after they ran away from this topic. If you are going to let your anchors on all these sports shows discuss social issues, don't punish them when they say something that sets off a firestorm, and don't censor them. If you're going to do sports, do it. Don't have a sports version of "The View". (Notice how Whoopi Golberg wasn't suspended for her defense of Stephen A. That is because that show is about discussing social issues) Now when Stephen A. comes back, I'm going to question if something is his opinion or is he simply towing the company line? If I ever thought he was fake before, which many believe he is at times, I'm definitely going to believe he's fake now. I laugh at some of the things that are mentioned in that youtube clip because they are true. Smith offended black people for supporting Cuban's usage of walking on the other side of the street when he sees a black guy with a hoodie. For him to walk on the other side of the street when a white guy is coming at him, he has to be a skin head who's body is covered in tatoos. Can't you see the unfairness in those statements? All the black guy has to do is have on a "Bill Belichick" hoodie to appear menacing enough for Cuban, yet the white guy has to look like an all around thug. (Of course we know the "hoodie" thing brought back allusions to the Trayvon Martin case) People were offended by those statements, and Stephen A was unapologetic in his defense of Cuban. What did First Take do? They continued to discuss it. They brought on Michael Eric Dyson to debate with Smith. They didn't censor that one. Why censor the debate now? Why suspend Stephen A. now? So I agree with the youtube clip. If you are going to censor a guy for making comments on a social issue, no longer talk on social issues. Just talk sports, that's where these guys are experts, or suppose to be experts heehee! Stephen A. can offend as many people as he wants on sports issues, and that is fair game. (And that really goes to all the other ESPN shows as well.) Of course from time to time you sometimes will have to dive into social issues because athletes do live lives, but keep it in the realm as it relates to sports. Say Ray Rice should have been suspended for more games, don't let your anchors dive into the issue of domestic violence. 1 person likes this Share this post Link to post Share on other sites


text:
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Washi, or handmade Japanese paper, is still produced in every part of Japan. In 2014, UNESCO inscribed three of the most important washi traditions in its list of the intangible cultural heritage of humanity.
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- Ōmura Satoshi's Art Museum: A Nobel Winner's GenerosityNippon.com Staff
Ōmura Satoshi, emeritus professor at Kitasato University, has cemented his reputation as a world-class researcher by becoming a joint winner of this year's Nobel Prize in Physiology or Medicine. He is also known as a lover of fine art, though. In 2007 he donated a considerable collection of artworks, along with a new museum to house them, to the city of Nirasaki, Yamanashi Prefecture, where he …
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Today art from Japan has worldwide cultural impact and is a familiar part of the global artistic scene. Up through the early modern era, though, Japanese art was a bolt from the blue—something entirely new to viewers overseas, who saw it as something fresh and surprisingly sophisticated. This was the dawn of Japonism.
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Despite Japan's lucrative domestic music scene (recent tribulations notwithstanding), few mainstream Japanese artists have achieved significant success outside of Asia. There may have been recent sold-out tours in the West for the likes of Kyary Pamyu Pamyu and Babymetal, but their songs are not the ones sought by discerning sound-seekers overseas. For many of these aficionados, the buzzword tends…
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The woodblock print, or hanga, has played a centuries-long role in the history of Japanese art. The Edo-period masters, like Hokusai and Hiroshige, produced images that are familiar to people all around the world today, but this is a deep genre with many lesser-known creators of similar skill. Below we present five images by five artists worth looking into in more depth. Utamaro, Kushi (Comb)…
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Ōta Kayoko was appointed the commissioner of the Japanese Pavilion at the 2014 Venice Architecture Biennale, becoming the first woman to hold that position. In this interview, Ōta discusses how working overseas fostered her own global outlook and offers recommendations for Japan to transform its social outlook.
- Mountains, Mummies, and Modern Art: Ascetic Practice in Yamagata PrefectureDavid McMahon
For over a thousand years, Yamagata Prefecture, on the Sea of Japan side of the northern Tōhoku region, has drawn pilgrims and mystics to its mountains. As the native Shintō faith intertwined with imported Buddhism, Yamagata became the site for scores of shrines and temples, some of which remain to the present day. The holiest of all the sites in the region are the three sacred mountains of Dew…
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At first glance, they look much like people, and the village seems to be full of residents going about their daily business. These particular locals are not people, though, but scarecrows. The mannequins outnumber the village's 40 or so human residents by more than two to one, and on the surface—with their own distinctive personalities—do create a rather cheerful atmosphere. But in the context of …
- Kyotographie: An International Photo Festival in Japan's Ancient Capital
French photographer Lucille Reyboz launched the Kyotographie International Photography Festival in 2013 with Nakanishi Yūsuke as a way of drawing attention to the world of photography. Now in its second year, the festival is helping to raise the profile of photography in Japan and encourage dialogue among photographers around the world.


text:
5 (1) 4 (0) 3 (0) 2 (0) 1 (0)
5 stars 5/5 Stars Average score of 1 user reviews spread across 22 releases and 0 DLC
The sequel to the all-time classic, a great game in its own right 0
The original Donkey Kong was a hallmark in the platforming genre when Nintendo released it in 1981. It doesn't hurt that it also made some mad money in the arcades. Thus designer Shigeru Miyamoto got to (perhaps was put to) work on the sequel, Donkey Kong Jr. Premiering in 1982, Donkey Kong Jr. stars the game's namesake as he goes through four levels of platforming to rescue his father, the original Donkey Kong, who was captured by Mario at the end of the previous game. This time Mario is your e...0 out of 0 found this review helpful.
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