text:
Submitted on 火曜, 04/14/2015 - 11:29am
Toronto IWW is pleased to announce opening of registration for the Spring 2015 edition of Organizing 101: Build the Committee Training, our crash course on grassroots, direct action-driven solidarity unionism.
Submitted on 木曜, 04/09/2015 - 10:31am
From WobblyCity.org
Several former workers at Beverage Plus and their supporters visited the company in Maspeth Queens this week to demand that the owner Yun Cho pay the more than $1.2 million owed to the workers from a wage theft lawsuit. Cho was present but did not come out to talk to the workers who then left a letter demanding payment. The company, which has also operated under the names YS Beverage and Grand Beverage, is a distributor in New York City, handling products such as Coca Cola and Poland Spring beverages. It was found liable in 2012 for multiple violations of the Fair Labor Standards Act for unpaid minimum wage and overtime for dozens of workers. The workers are still seeking the payments in court but have also called upon supporters to join their campaign to get the money owed to them. The workers have been long time members of the Brandworkers and NYC Industrial Workers of the World (IWW) campaign to organize and improve conditions in New York City's food production and distribution sector.
Stay tuned for more actions in support of the Beverage Plus workers!
Submitted on 水曜, 04/01/2015 - 9:21pm
Download a Free PDF of this issue. (Check out the April Fools' Day issue here.)
Submitted on 金曜, 03/27/2015 - 2:27pm
Click here to sign the petition!
By the London IWW
3 of us – hospitality workers employed on zero-hour contracts – have lost our jobs. This is at Friends House London, the head office of British Quakers where – despite the organisation's reputation as a 'good employer' – senior managers responded to objections to the use of zero-hour contracts by getting rid of the three remaining workers employed on them!
Two of us were elected Unite the Union workplace representatives, and a third is a Quaker. One worker has been subject to intimidatory disciplinary processes; we all feel we've been targeted for being principled and outspoken at work; and our health has suffered. With the support of the London IWW union, we are calling for:
Submitted on 金曜, 03/27/2015 - 1:45pm
By IWW Greece
At the present moment, the imprisoned anarchists, members of DAK (Network of imprisoned social fighters), A. Stampoulos, A. Theofilou, G. Karagianidis, D. Politis, F. Charisis, A. Dalios, D. Mpourzoukos, G. Sarafoudis, G. Michailidis, the members of the "Revolutionary Struggle" N. Maziotis and K. Gournas, the member of "17th of November" D. Koufontinas, a number of Turkish political prisoners and the prisoners G. Sofianidis and M. S. Eltsibach, are on a hunger strike, fighting against the repressive legal state of exception which has been established by the Greek state since the beginning of 2000.
Starting on March 2nd, along with the comrades outside the prison walls, we commenced a struggle for the abolition of type C high security prisons, the abolition of "antiterrorist" law, the abolition of the "hoodlaw", the radical change in the process of taking and identification of DNA samples, the release of the seriously sick member of 17N S. Ksiros.
Industrial Workers of the World
General Headquarters
PO Box 180195, Chicago, IL 60618, USA
tel: (773) 728-0996
Email - ghq [at] iww.org
Website - www [at] iww.org


text:
Integrated class A amplifier SE-100
We present a non compromise Class A integrated Amplifier
with the following characteristics:
- Class A.
- Parallel Mosfet output stage.
- Jfet amplifier input stage.
- Single Ended output characteristics.
- Unique JFet single ended preamplifier stage.
- Use of interstage transformers.
- Separate PSUs, for every stage of the amplifier and every stage of
preamplifier. Totally sixteen different psu are located inside the
chassis and twelve of them are regulated. Those who were not
regulated are left only with capacitors and chokes, because gave
better sonic results.
- Total capacitance bank ~600.000uF.
- Three separate transformers, and totally sixteen separate
windings. Two big toroidal transformers of 500 Watt each.
- Remote control.
- Big LCD Screen with backlight, Indicating input selection, and
volume intensity.
- Mute function.
- Push button control from Frontpanel.
- Luxurious and precisely machined chassis. Chassis made exclusively
of hi grade aluminium.
- With the use of internal jumbers the amplifier can work as a power
amplifier, separating completely the internal preamplifier. This is
very comfortable to those who would like to taste different preamplifier
- Four selectable inputs, one of them balanced.
- One set of speaker outputs.
- Separate inputs for those who will use the SE-100 as power
amplifier. Two sets, one Single Ended and one balanced.
- Top quality of input and output connectors.
- Top quality of materials used.
- Custom internal wiring.
- Through hole construction.
- Oversized thermal bank, using heatsinks.
- Three point feet support.
- Handcrafted.
- Inside detailed photo
Summary of the nominal specifications:
|Distortion @ 1 watt
|.01% @ 1 KHz
|Preamplifier input Impedance
|100 KOhm
|Power amplifier input Impedance
|100 KOhm
|Output Impedance
|0.51 Ohm
|Output power 8 ohms
|~50 watts @ 1% THD, 1KHz
|Pre-amplifier Voltage Gain
|10 dB
|Amplifier Voltage Gain
|25.6 dB
|Maximum unclipped output
|+/-29 Volts
|Frequency response
|-3 dB @ 2 Hz, -3 dB @ ~200 KHz
|~68uV unweighted, 20-20 Hz
|Power consumption
|~220 watts / per Channel
|slow blow type, 6 Amp for 240VAC
|Dimensions unpacked
|H x L x D / 22X45X52 cm
|Dimensions packed
|H x L x D / 30X55X65 cm
|Weight unpacked
|~ 41 Kgr
|Weight packed
|~ 46 Kgr

subset: commoncrawl


text:
Eastern Iowa schools dominated two national high school rankings released this week.
Eight Iowa schools, seven from the eastern part of the state, �were represented in Newsweek magazine's��America�s Best High Schools� list of the 1,000 top public high schools in America.
They are: Iowa City West (ranked 462nd overall), Cedar Rapids Washington (723), Cedar Rapids Kennedy (842), Iowa City High (880), Cedar Falls (886), Decorah (972), and Mid-Prairie in Wellman (999).
Ames High School came in at 791.
The rankings are based on graduation rate, college matriculation rate, test scores and Advanced Placement courses offered per student.
The Washington Post also released the results of its annual "High School Challenge," which included five Iowa schools, three from Eastern Iowa. Out of 1,900 top schools, Washington came in at 357, Kennedy at 643 and West at 1,210.
The other two Iowa schools on the list were Ames, at 831, and Des Moines Roosevelt, at 1,185,
The Post's methodology used many of the same statistics as Newsweek, but with a greater emphasis on Advanced Placement and International Baccalaureate courses.


text:
Volterra Semiconductor ReviewsVolterra Semiconductor 10
"Good Experience, & learn a lot of skills "
Neutral Outlook
- Work/Life Balance
- Culture & Values
- Career Opportunities
- Comp & Benefits
- Senior Management
I worked at Volterra Semiconductor full-time (More than 5 years)
There are many skills to learn
too many projects and alot of work
Volterra Semiconductor Awards & Accolades
Let us know if we're missing any workplace or industry recognition – Add an award
Additional InfoUnlock Profile
Volterra Semiconductor aims to usher in a new era in voltage control for microchips. The fabless semiconductor company designs and markets low-voltage power supply chips, primarily voltage regulators for the data networking, computer, consumer electronics, servers and storage, and telecomm industries. Its products are designed to replace several power management components with a single device. Volterra outsources fabrication of its products, primarily to GLOBALFOUNDRIES and Taiwan Semiconductor Manufacturing, among other contractors. IBM and Hon Hai are top customers of ... More


text:
Chris Tucker is facing foreclosure on his waterfront Orlando home. The actor owes the bank $4.4 million on the $6 million home, which has five bedrooms, three fireplaces, and a basement designed to look like a pirate ship. Wow. I'd heard Chris Tucker was crazy, but I didn't realize he was Nicolas Cage crazy.
Tucker should realize that as crappy as it seems right now, things could always be much worse. Like in the case of these seven celebrities who have lived in some of the dingiest or strangest homes. Keep your head up, Chris Tucker. And whatever you're thinking of doing, don't. The world doesn't need another Rush Hour movie.
Mike Tyson's abandoned Southington mansion is about the only thing that could get me to roadtrip to Ohio. Complete with a moss covered indoor swimming pool, a massive, trashed living room, holes punched through walls, and rusty tiger cages, it's like a dark-humored Disney World. Oh, I guess that would make it Disney Land.
The boxer lived here for a period in the 1990's and it has sat abandoned ever since. If I were a high schooler, I would so party in there.
Home Improvement's youngest son Taran Noah Smith has had a bit of a tough time. He was kicked out of his Sherman Oaks home shortly after he and his wife decided to open a vegan restaurant in it. They left the house looking like the site of an acid trip orgy. Cryptic messages and strange drawings were found drawn on the walls that were left standing. That's why he should have gone to college. That's were you go to get vandalism out of your system.
Consider posting a comment.


text:
Two Cute Girls Get Picked Up And Gets Fucked
The following tags exist in this scene. You can find similar scenes by combining tags in this scene together using the checkboxes, or just click a tag name to browse...
Starring Mischel Channson and Chicita, DVD: A Brotha's P.O.V.
Do you recognise pornstars in this scene? Begin typing a pornstar name, then click on the button to suggest them!
Tag team Mischel and Chicita take turns pleasuring a lucky guy in this hot sexual threesome.
Guy takes girls off the street home and gets them naked to suck and fuck his big dick in their bald twats
Featured in playlists: 3some (90 Videos), Fun55 (300 Videos), The Dark Truth Vol. 1 (42 Videos)
Video Suggestions
- 23:56Friends On Vacation Get Their First Taste Of Pussy And AnalOct 10, 2013
- 21:28Jasmine Shares A Dick With Her Friend And Takes A FacialOct 10, 2013
- 21:13Carrie Cruise And Jessica May Share Karl Kinkaid's CockApr 15, 2009
- 31:29Hottie Alissa Strips To Show Her Peachy AssetsOct 10, 2013
- 16:59Katalin Has Big White Ass Rammed And Cum On FaceJun 9, 2011
- 19:38Hotties Janavi And Tera Joy Share A Stiff CockDec 13, 2013
- 17:23Hot Blond Joelean Got Her Pussy Fucked By Black CockJun 9, 2011
- 25:39Enjoy Hot Blonde Lisa Rose in Sexy P.O.V Style!Jul 24, 2014
- 19:34Two Sexy Chicks Make Out And Fuck Their MenJan 24, 2014
- 15:56Naughty Katie Ditches Her Prim Duds To Take A Deep DickingJun 9, 2011


text:
Sometimes it's the journey, sometimes it's the destination—and sometimes, it's both. National Geographic Traveler has scoured the globe for the world's most beautiful, interesting, and off-beat road trips. Dive in to get directions, quizzes, photos, and more.
Road Trips
Hill Country, Texas
Head down south for a visit to Texas's famed hill country, quirky cowboy towns and all.
Hudson Valley, New York
Follow the trails of Indians, adventurers, George Washington, and Gilded Age millionaires.
The James River, Virginia
Wind your way deep into American history and see the forces that shaped our nation.
Lake Michigan
Soak up the scenery and stop along the way for wine and cherries along the shores of Lake Michigan.
Los Angeles
Explore the back roads and ocean drives of Los Angeles and beyond.
Low Country, South Carolina and Georgia
Few regions in the United States pack in as much history, culture, and natural beauty as the Low Country.
Manitoba, Canada
The region's richest farm country provides the backdrop for rolling prairie hills and river valleys.
Maryland's Eastern Shore
Maryland's Eastern Shore offers up-close encounters with skipjacks, blue crabs, and wild ponies.
Maui's Hana Coast
Maui's Hana coast delivers black-sand beaches, plunging waterfalls—and a doozy of a drive.
Montreal, Canada
Brush up on your French as you cruise through Montreal's picturesque wine country.
Mount Hood, Oregon
The views here are just as spectacular today as they were when the first settlers arrived.
Navajo and Hopi Lands, Arizona
Watch the serene culture of Native Americans come alive in a drive through Hopi and Navajo lands.
Nebraska's Pioneer Trail
Follow a trail dotted with pioneers' legacy: historical villages, preserved log cabins, and lots of memorabilia.
The New Old West, Arizona
Saddle up at this bastion of true Americana where the Old West meets the new.
New York's Finger Lakes
The wines of this region stand alongside California's best, and the scenery is likewise first rate.
New Zealand's North Island
Get into the adventure with some of the most varied and rugged landscapes on Earth.
New Book
Photo Galleries
- Banff and Jasper National Parks
- Big Island, Hawaii
- The Borderlands of Texas
- Brandywine Valley
- California's Pacific Coast
- Canada
- Cape Breton, Nova Scotia
- Cheese Trail, Vermont
- Cornwall, England
- Florida Keys
- The Flower Route
- Italy's Amalfi Coast
- Jamaica
- Mississippi River
- Mount Hood, Oregon
- New York's Finger Lakes
- New Zealand's North Island
- Provence, France
- Rocky Mountains
- San Luis Valley, Colorado
- U.S. Atlantic Coast
- U.S. Midwest
- U.S. Mountains and Plains
- U.S. South
- U.S. West
National Park Road Trips
Top Picks for Travel
- Florida's Pristine Parks
- Road Trip: Ireland's Wild Atlantic Way
- Best Fall Trips 2015
- NEW National Geographic Journeys
- How to Make an Epic Photo
- Best Travel Photos from September
- 2015 Traveler Photo Contest Winners
- 101 Reasons to Travel Now
- NEW! National Geographic Unique Lodges of the World
- Request a Nat Geo Trips Catalog
2015 Traveler Photo Contest
See the Winners
Explore the top photos, share your favorites, and browse all entries.
Shop National Geographic
Travel Wallet
"My Town" Custom Map Puzzle


text:
AIFF Official Selection of Shorts and Musical Entertainment
Presented by Arlington International Film Festival
October 6, 2012
The second annual Arlington International Film Festival begins Wednesday, October 17, but you are invited to preview an OFFICIAL SELECTION of SHORTS – Saturday, October 6, Screenings with appearance by local artists & entertainers.
Jam'n Java Cafe, 594 Mass Ave, Arlington, 6:30pm - 9:00pm
Free to the public
More information about the selections and entertainment can be found at http://www.aiffest.org/, where one can also sign up for the virtual mailing list.
View the 2012 Festival program at http://www.aiffest.org/2012-festival-program/
Join the Festival's Facebook page -
AIFF Mission Statement: To foster appreciation for different cultures by exploring the lives of people around the globe through independent film — to nurture the next generation of filmmakers within our
Jam'n Java
594 Massachusetts Ave.
Arlington, MA 02474
Admission Info:
Phone: 857.209.1122
Accessibility Information:


text:
|MLB Career Stats
Red Sox catcher Christian Vazquez will take at-bats in the Instructional League, starting later this week, and should be fully healthy for the start of Spring Training.
Thursday marked the first time Red Sox catcher Christian Vazquez had thrown a ball since undergoing Tommy John surgery on his elbow in April.
|March 30, 2015
|Boston Red Sox placed C Christian Vazquez on the 60-day disabled list. Right elbow sprain
|July 9, 2014
|Boston Red Sox recalled C Christian Vazquez from Pawtucket Red Sox.
|March 17, 2014
|Boston Red Sox optioned C Christian Vazquez to Pawtucket Red Sox.
|November 1, 2013
|Christian Vazquez roster status changed by Boston Red Sox.
|September 1, 2013
|C Christian Vazquez assigned to Pawtucket Red Sox from Portland Sea Dogs.
|March 15, 2013
|Boston Red Sox optioned Christian Vazquez to Portland Sea Dogs.
|November 20, 2012
|Boston Red Sox selected the contract of Christian Vazquez from Portland Sea Dogs.
|August 3, 2012
|Christian Vazquez assigned to Portland Sea Dogs from Salem Red Sox.
|April 4, 2012
|Christian Vazquez assigned to Salem Red Sox from Greenville Drive.
|March 7, 2012
|C Christian Vazquez assigned to Boston Red Sox.
|March 20, 2011
|C Christian Vazquez assigned to Boston Red Sox.
|April 5, 2010
|Christian Vazquez assigned to Greenville Drive from Lowell Spinners.
|July 8, 2009
|Christian Vazquez assigned to Lowell Spinners from GCL Red Sox.
Graduated from Puerto Rico Baseball Academy High School.2014
Played 55 games for the Red Sox, his 1st career ML action, including 54 games at catcher (50 starts)...Took over as the club's regular catcher after July recall...Reached base safely in 40 of his 50 starts for BOS, hitting .240 (42-for-175) with 9 2B, HR, 20 RBI, and 19 BB...Threw out 51.7% of attempted base stealers (15 of 29), best in the majors among catchers with at least 50 games behind the plate...Was the best such mark in Red Sox history (since CS began being tracked in the AL in 1920)...Did not benefit from a pitcher caught stealing, making his 51.7% catcher caught stealing rate the best among major leaguers with at least 50 games behind the plate since STL's Yadier Molina in 2005 (54.8%; 17 of 31)...Was the best such mark by a rookie catcher since CCS began being tracked in 1974...Posted a team-best 4.04 catcher's ERA...His 54 games, 50 starts, and 458.1 innings at catcher all ranked 2nd on the team to A.J. Pierzynski (64 games, 60 starts, 540.0 inn.)...Had 4 pickoffs, the most by a Red Sox catcher in a season since Rich Gedman had 4 in 1985...Hit .286 (20-for-70) at Fenway Park compared to .210 (22-for-105) on the road...Went 7-for-24 (.292) with 5 walks with 2 out and runners in scoring position...Began the year with Triple-A Pawtucket, hitting .273 (68-for-244) with 17 2B, 3 HR, 20 RBI, 35 R, and 21 BB in 66 games...Threw out 39.6% of attempted base stealers (19 of 48)...Named a Mid-Season International League All-Star and was also selected to the 2014 SiriusXM All-Star Futures Game...Did not participate in either contest...Recalled 7/9 and made his ML debut that night, starting behind the plate in a 5-4 win vs. CWS...Started 50 of Boston's 72 remaining games following his recall...The Red Sox won his first 5 career starts from 7/9-21, over which Red Sox pitchers allowed only 9 runs...Was the 1st Red Sox ever to start as a catcher and win each of his first 5 major league games, and the 1st AL player to do it since MIL's Bill Schroeder in 1983 (Source: Elias)...Went 3-for-4 with 2 R, 2 2B, and 3 RBI in his 2nd ML game on 7/11 at HOU...Was the fastest Red Sox to a 3+ hit game since Nomar Garciaparra's on 9/1/96 at OAK (2nd game, 3 H)...Appeared in 13 of the club's last 14 games beginning on 9/12 (11 starts)...Hit safely in 8 of his last 9 starts (.400, 12-for-30, 2 2B, HR, 5 RBI, 4 BB)...Went 4-for-4 with a double and his 1st ML home run on 9/25 vs. TB...The homer was a 2-run, go-ahead shot off Jeremy Hellickson in the 2nd inning...Was the 1st Red Sox rookie catcher with 4 or more hits in a game since Scott Hatteberg had 4 hits on 8/8/97 vs. KC...Following the season, played 33 games for San Juan of the Puerto Rican Winter League...Ranked 9th among qualifiers with a .288 AVG (32-for-111) and had 6 2B, HR, 12 RBI, 10 BB, and a .365 OBP...Tied for the league lead among catchers with 10 CS (21 attempts, 47.6%)...Was named by Baseball America as the best defensive catcher in the International League in 2014.2013
Led Double-A Portland with a .289 AVG (99-for-342) and was named team MVP. Including 1 game with Triple-A Pawtucket, ranked 5th among full-season Red Sox minor league qualifiers with a career-best .287 AVG (99-for-345)…Also posted a career-high .375 OBP. Ranked 9th in AVG among Eastern League qualifiers…Had the 4th-highest ratio of plate appearances per strikeout (9.1). Topped Eastern League backstops throwing out 46.5 percent (47 of 101) attempted base stealers…Also led EL catchers in total chances (871), putouts (781), and assists (tied, 80). Named Red Sox Minor League Defensive Player of the Month for April (11 CS in 16 G). Reached in all 5 PA on 5/22 vs. New Hampshire (4-for-4, 2 2B, 3B, HR, 2 RBI, BB). Started behind the plate for the Eastern Division in the Eastern League All-Star Game on 7/10 in New Britain. Posted a season-long 12-game hitting streak from 7/31-8/16 (.429 AVG, 21-for-49, 3 2B, 7 RBI)…Matched the longest hit streak by a Sea Dog all year. Hit .347 (50-for-144) after the All-Star break…Promoted to Triple-A Pawtucket on 9/1, where he made his Triple-A debut the next day in the PawSox' regular season finale. Hit .273 (3-for-11) with 2 RBI and 2 walks in 3 playoff games for Pawtucket. After the season, played for Santurce of the Puerto Rico Winter League and led the league with 32 games behind the plate…Threw out a league-best 63.0% (17 of 27) of attempted base stealers. Following the season, ranked by Baseball America as the best defensive catcher in the Red Sox farm system for a 3rd straight year…Also named the best defensive catcher in the Eastern League.2012 Played the majority of the year with High-A Salem, where he was a Carolina League Post-Season All-Star...Also made his Double-A debut with 20 games for Portland. Combined to throw out 49 of 122 base stealers (40.2%), including a Carolina League-best 41.9 percentage (44 of 105)...Also topped the circuit with 110 assists at the position. Led the Red Sox system with 96 games caught. Named Red Sox Minor League Defensive Player of the Month for June after throwing out 10 of 23 attempted base stealers (43.5%). Combined to set a single-season career-high with 48 walks, including 8 walks compared to 9 strikeouts in Double-A. Recalled for Double-A debut on 8/3...At time of transfer, ranked 13th in the Carolina League with a .360 OBP. Over his last 28 games with Salem from 6/23-8/2, led the Carolina League in AVG (.353) and OBP (.455), and ranked among CL leaders in SLG (5th, .598) and OPS (3rd, 1.053). Notched 4 straight multi-hit games from 6/23-28 and again from 7/4-9. Batted .291 (51-for-175) over his final 48 games of the season beginning 6/23. Following the season, hit .257 (9-for-35) with 8 walks in 10 games for Surprise in the Arizona Fall League... Then played 9 contests for Manati of the Puerto Rico Winter League, going 7-for-24 (.292) with 2 doubles, a triple, and a homer. 2011 Spent the year with Single-A Greenville and was named Red Sox Minor League Defensive Player of the Year. Threw out 37 of 111 (33.3%) of opposing base stealers and posted a .991 fielding percentage (9 E/904 TC)... Led South Atlantic League catchers in chances (904), putouts (788), and assists (108). Reached single-season career-highs in homers (18) and RBI (84)...His 84 RBI led the Drive and ranked 4th among Red Sox minor leaguers. Also paced the Drive with 18 homers and ranked among club leaders in at-bats (3rd, 392), hits (2nd, 111), doubles (T-2nd, 27), and walks (T-2nd, 43). Named SAL Player of the Week from 6/20-26 (.467, 7-for-15, 4 HR) and from 8/29-9/4 (.381, 8-for-21, 3 HR). Selected as the Red Sox Minor League Defensive Player for July. Following the season, was rated byBaseball America as best defensive catcher in the SAL and in Red Sox system. After the season, played in 3 games with Indios de Mayaguez of the Puerto Rico Winter League. 2010 Played his 1st full season with Single-A Greenville and led the club with 60 games behind the plate...Threw out 31 of 76 (40.8%) attempted base stealers. Made his 1st pro appearances at 1st base (3 games) and 2nd base (1). Hit 1st career grand slam on 4/17 at Lexington (KC). Hit .370 (20-for-54) during a 14-game hit streak from 5/31-7/3. Went 5-for-21 (.238) with 3 doubles and 2 RBI in 6 playoff games for the Drive. 2009 Split the year with 31 games between the Rookie-level Gulf Coast Red Sox and ShortA Lowell. Combined for 21 games at catcher, 9 at 3rd base, and 1 at DH. Threw out 6 of 26 (23.1%) attempted base stealers. Began the year in the GCL before transferring to Lowell on 7/8. His 1st hit for the Spinners was a game-tying pinch-hit homer in the 9th inning on 7/9 vs. Tri-City (HOU). 2008 Led the Gulf Coast League Red Sox with 21 games caught in his pro debut. Threw out 5 of 32 (15.6%) attempted base stealers.


text:
Should the County Be Able to Take Your Property?
October 29, 2010 2:03 PM
Should the laws be changed in Clark County regarding eminent domain? That's what voters will be deciding when they vote on Question 4.
Alan talked with former Clark County Commissioner Bruce Woodbury and current Attorney General candidate, Joel Hansen, regarding repealing PISTOL (People's Initiative to Stop the Taking of Our Land).


text:
Flip Doll - What a cute idea, especially for toddlers!
Ruby Doll - So sweet.
A collection of felt dolls - including a modern looking boy and girl (pictured at the right).
Prairie Flowers Doll - For all those Little House fans.
Rosie the Rag Doll - Sweet!
Realistic baby doll - Really impressive.
Belinda and Lucinda - Old fashioned dollies.
Wide Eyed Wendy - A good, basic doll.
Mimi Kirchner's Felt Doll - Cute and designed to hand sew.
Flat girl doll - Simple and sweet.
Martha Stewart's Black Apple Doll - Modern and cute.
Sleepy Time Baby - Uber simple.
Dutch Doll - or Irish, Scottish, Balkin, Russian, and Swiss dolls.
Tiny Girl - an ity bitty doll.
Infant Doll - an even tinier dollie.
Babyland Rag Doll - a classic.
Martha's Gingham Dolls - Simple dolls made from gingham fabric.
Rag Hair Dolls - Fun!


text:
Limited Edition Fisheye2 Camera with Flash & FIlm
Qty: 1
- 1
- 2
- 3
- 4
- 5
- 6
Please select an available size.
Please select a size.
Description: Limited Edition Fisheye2 Camera with Flash and FIlm:
- Point-and-shoot fisheye camera with built-in flash
- Produces 170 degree shots
- Uses all kinds of film (color negative, black and white, slides, redscale)
- Multiple exposure switch and long exposure function
- Premium quality Japanese fisheye lens
- Requires 35 mm film (included)
Third image depicts the quality of photographs produced by this camera.
Material: Plastic and glass
Care: Dust with a dry cloth
Brand: Lomography
Origin: Imported


text:
Receiving the Day
Christian Practices for Opening the Gift of Time
(If any tax is payable it will be calculated and shown at checkout.)
Preview (read now) Add to my own site Give this ebook to a friend Add to my wishlist Author's page Publisher's page
moreIn this spirituality of time, Dorothy Bass invites readers into a way of living in time that is alert to both contemporary pressures and rooted ancient wisdom. The celebrated editor of Practicing Our Faith asks hard questions about how our injurious attitude toward time has distorted our relationships with our innermost selves, with other people, with the natural world, and with God.
As an alternative to the rhetoric of management and mastery, Receiving the Day offers a language of attention, poetry, and celebration. Bass encourages us to reevaluate our understanding of the temporal and thereby to participate fully in the Christian practice of knowing time as God's gift. Embraced in this way, time need not be wrestled with each day. Instead, time becomes the habitation of blessing.less
Wiley; March 2002
161 pages; ISBN 9780787959272
Read online, or download in secure PDF format
Title: Receiving the Day
Author: Dorothy C. Bass
161 pages; ISBN 9780787959272
Read online, or download in secure PDF format
Title: Receiving the Day
Author: Dorothy C. Bass
Buy, download and read Receiving the Day (eBook) by Dorothy C. Bass today!
More Body Mind
The Alchemist 2015 US$ 9.99 208 pages
E-Squared 2013 US$ 6.99 194 pages
Man's Search For Meaning 2015 US$ 14.00
The Untethered Soul 2007 US$ 16.95 200 pages
Subject categories


text:
Prospective Resident Information
Thank you for your interest in post-graduate surgical education at the University of North Carolina at Chapel Hill. All Accreditation Council on Graduate Medical Education (ACGME) residencies and fellowships are available through the Department of Surgery at the University of North Carolina Hospitals.
For information on salaries, benefits, and more, see UNC Health Care's Office of Graduate Medical Education.
Residency Programs:
Cardiothoracic Surgery
Program Director: Michael Mill, MD
Program Coordinator: Shelby Long, 919-966-3381
General Surgery
Program Director: Mark J. Koruda, MD
Program Coordinator: Kathie Patterson, 919-966-4653
Program Director: Matthew G. Ewend, MD
Program Coordinator: Smyrna Robinson, 919-843-4608
Plastic Surgery
Program Director: C. Scott Hultman, MD
Program Coordinator: Sharon Sullivan, 919-843-1287
Surgical Critical Care
Program Director: Renae E. Stafford, MD
Program Coordinator: Jesse Deshane, 919-966-4389
Program Director: Culley C. Carson III, MD
Program Coordinator: Lynn West, 919-966-2574
Vascular Surgery
Program Director: Mark A. Farber, MD
Program Coordinator: Debbie Alberti, 919-966-3391
The Department of Surgery also offers serveral advanced residency programs, fellowships and post-graduate training programs.


text:
The Pacifica Sea Lions swim team is a competitive swimming program for youth 5-18 years of age. Sign-ups are held on a daily basis with the head swim coach. The competition team practices Monday through Friday between the hours of 4:00 pm-6:15 pm. Specific practice time varies depending on individual's ability. The pre-competition team practices Monday through Thursday from 6:15-7:00 pm.The cost for joining the competition swim team is $72 per month plus a $5 sign-up fee for the first month. The cost for joining the pre-competition team is $62 per month plus a $5 sign-up fee for the first month. All swimmers must join USA Swimming. Membership for USA Swimming is $65 per year. The head coach has paper work at the Jean E. Brink Pool for sign-up with both the Pacifica Sea Lions and USA Swimming. The team is affiliated with Pacific Swimming.
The Pacifica Sea Lion Aquatics Club Parent Board requires families to pay yearly dues to support the competitive aspect of the team. Please refer to parent handbook for more information.
See the Pacifica Sea Lions website for more information: http://www.teamunify.com/Home.jsp?team=psl.


text:
Tumor-associated macrophages (TAMs) are alternatively activated cells induced by interleukin-4 (IL-4)-releasing CD4+ T cells. TAMs promote breast cancer invasion and metastasis; however, the mechanisms underlying these interactions between macrophages and tumor cells that lead to cancer metastasis remain elusive. Previous studies have found microRNAs (miRNAs) circulating in the peripheral blood and have identified microvesicles, or exosomes, as mediators of cell-cell communication. Therefore, one alternative mechanism for the promotion of breast cancer cell invasion by TAMs may be through macrophage-secreted exosomes, which would deliver invasion-potentiating miRNAs to breast cancer cells.
We utilized a co-culture system with IL-4-activated macrophages and breast cancer cells to verify that miRNAs are transported from macrophages to breast cancer cells. The shuttling of fluorescently-labeled exogenous miRNAs from IL-4-activated macrophages to co-cultivated breast cancer cells without direct cell-cell contact was observed. miR-223, a miRNA specific for IL-4-activated macrophages, was detected within the exosomes released by macrophages and was significantly elevated in the co-cultivated SKBR3 and MDA-MB-231 cells. The invasiveness of the co-cultivated breast cancer cells decreased when the IL-4-activated macrophages were treated with a miR-223 antisense oligonucleotide (ASO) that would inhibit miR-223 expression. Furthermore, results from a functional assay revealed that miR-223 promoted the invasion of breast cancer cells via the Mef2c-β-catenin pathway.
We conclude that macrophages regulate the invasiveness of breast cancer cells through exosome-mediated delivery of oncogenic miRNAs. Our data provide insight into the mechanisms underlying the metastasis-promoting interactions between macrophages and breast cancer cells.
Breast cancer is the most common malignant tumor in women, and distal metastasis of highly invasive breast cancer cells is the major cause of death in these women. Tumor-associated macrophages that infiltrate the breast cancer stroma are the most notable migratory hematopoietic cell-type in the tumor microenvironment and function to promote the invasiveness of breast cancer cells [1-4]. Macrophages are heterogeneous cells that respond differently to various stimulating signals and display numerous phenotypes [5]. Fully polarized M1, or classically activated, macrophages are stimulated by microbial agents or pro-inflammatory factors, such as lipopolysaccharides (LPS); whereas M2, or alternatively-activated, macrophages are responding to anti-inflammatory molecules, such as interleukin-4 (IL-4). The M1 and M2 macrophage phenotypes represent the two extremes of a broad range of macrophage functional states. Among these functional states, M2 macrophages activated by IL-4 have been associated with breast cancer invasion, metastasis and poor patient prognosis [2,6-10].
Previous studies have shown that TAMs promote breast cancer progression and metastasis by releasing a variety of cytokines that regulate the survival and invasiveness of tumor cells and stimulate tumor angiogenesis [11,12]. More recent data have demonstrated that macrophages are able to produce microvesicles, also known as exosomes, which shuttle proteins or microRNAs (miRNAs) into adjacent cells within the microenvironment [13,14]. Exosomes are derived from multivesicular endosomes that fuse with the plasma membrane and are shed into the extracellular space. These particles range in size from 50 to 100 nm. A wide variety of cells may release exosomes, but their contents vary depending on the cell-type of origin and its activation status [15]. One topic of considerable interest is that exosomes contain miRNAs that mediate intercellular communication [16-19]. miRNAs are short, non-coding RNAs that regulate the expression of complementary mRNAs [20]. The shuttling of these molecules between cells aids in regulating the biology of target cells. miR-223 is specific for alternatively-activated M2 macrophages induced by IL-4 and is associated with the regulation of human granulopoiesis [21].
In the present study, we demonstrate that exogenous miRNAs transfected into IL-4-activated M2 macrophages can be shuttled into co-cultivated breast cancer cells in the absence of direct cell-cell contact with the macrophages. Exosomes containing miR-223 were released by M2 cells and were then internalized by co-cultivated breast cancer cells that did not express this miRNA. The exosome-shuttled miR-223 promoted the invasiveness of breast cancer cells in vitro. This process of invasion could be inhibited by transfecting miR-223 antisense oligonucleotides (ASO) into the tumor cells. Our study provides evidence for the delivery of invasion-potentiating miR-223 by IL-4-activated macrophages to breast cancer cells via exosomes and may highlight a novel communication mechanism between TAMs and cancer cells.
Isolation and activation of human monocyte-derived macrophages
Institutional approval from the local research ethical committees (Internal Review and the Ethics Boards of the Sun-Yat-Sen Memorial Hospital, Sun-Yat-Sen University) was obtained prior to conducting the study. Human mononuclear cells were isolated from the peripheral blood of healthy donors by Ficoll density gradient centrifugation at 450 × g for 25 min at room temperature. The mononuclear cells were washed three times with PBS and plated at a density of 5 × 106 per well in 24-well plates and incubated for 1.5 h in DMEM alone. Subsequently, non-adherent cells were washed away with warm Hank's solution, and the adherent monocytes were cultured in DMEM containing 10% fetal bovine serum (FBS). Media was changed every 3 days, and the resulting monocyte-derived macrophages (MDMs) were activated by adding IL-4 (45 ng/ml) to the culture medium for 3 days.
Cell cultures and co-cultivation
HEK-293T cells and the breast cancer cell lines, SKBR3 and MDA-MB-231, were cultured in DMEM containing 10% FBS. Co-cultivation of macrophages and breast cancer cells was performed in 24-well Boyden chambers (Corning, cat. no. 3413). Macrophages were seeded on the 0.4 μM inserts, which are permeable to supernatants but not to cellular components. Breast cancer cells were seeded in the lower chambers and grown for the indicated periods of time.
MicroRNA expression profiles were generated as described previously [22], and hybridization using DiscovArray miRNA arrays was performed according to the manufacturer's instructions.
Quantitative reverse transcription-PCR
Quantitative real-time reverse transcription-PCR (qRT-PCR) for miR-223 was conducted using Real-Time PCR Universal Reagent (GenePharma Co., Ltd.) and the MX-3000P Real-Time PCR machine (Stratagene). All reactions were performed in a 20 μL reaction volume in triplicate. The primers used for miR-223 and U6 snRNA are as follows: miR-223 forward, 5'CATTGTCAGTTTGTCAAATACC3'; miR-223 reverse, 5'CCATGAGAGATCCCTAGCG3' and U6 snRNA forward, 5'ATTGGAACGATACAGAGAAGAT3'; U6 snRNA reverse, 5'GGAACGCTTCACGAATTT3'. PCR amplification consisted of an initial denaturation step at 95°C for 3 min, followed by 40 cycles of 95°C for 30 s, 62°C for 40 s, and 72°C for 30 s. Standard curves were generated, and the relative amount of miR-223 was normalized to the amount of U6 snRNA (2- ΔCt).
Luciferase reporter plasmid construction
The pMIR-REPORT miRNA expression reporter (firefly luciferase reporter plasmid; Ambion) was used for plasmid construction. The 3'-UTR of Mef2c, containing two miR-223 target sequences, miR-223 complementary sequence (TGGGGTATTTGACAAACTGACA) and lin-4 complementary sequence (CTAGTCACACTTGAGGTCTCAGGGA), were separately cloned into the 3'-UTR of the pMIR-REPORT plasmid according to the manufacturer's instructions (Ambion).
Invasion assay
Invasion was measured by assessing the cell migration rate through an artificial basement membrane in a modified Boyden chamber (Corning, cat. no. 3422). The membrane consisted of polycarbonate (8 μm pore diameter) and was coated on ice with Matrigel (BD Biosciences) diluted in serum-free DMEM. Cells resuspended in DMEM were seeded into the upper well of the chamber (100 μl), while the lower well was filled to the top (approximately 600 μl) with complete medium (DMEM containing 10% FBS). Cells were incubated for 4-8 h. The cells in the upper well that did not migrate were scraped off, and the cells that migrated onto the outer side of the upper well membrane were stained with crystal violet. Invading cells were observed under a microscope and counted for statistical analysis.
Transfection of miRNA mimics and miRNA ASO
MicroRNA mimics and inhibitors (miRNA antisense oligonucleotides (ASO)) were purchased from GenePharma Co., Ltd. The sequences are provided in Table 1. In vitro transfection of miRNAs (miR-223, lin-4 and miR-NC) and miRNA ASO (miR-223 ASO and miR-NC ASO) were performed using X-tremeGENE siRNA Transfection Reagent according to the manufacturer's instructions (Roche, cat. no. 04 476 093 001).
Table 1. The sequences of microRNA mimics and inhibitors
Luciferase assays
To determine whether miRNAs were shuttled from macrophages to breast cancer cells, the levels of miRNAs in the target cancer cells were assessed using luciferase assays. Briefly, the pMIR-REPORT vector with either the miR-223 or the lin-4 complementary sequence in its 3'-UTR (firefly luciferase reporter vector, Ambion) were transfected into SKBR3 cells using Lipofectamine2000 (Invitrogen) according to the manufacturer's instructions. A Renilla luciferase vector (Promega) was co-transfected as an internal control for normalization. After transfection, cells were washed to remove untransfected plasmids or miRNAs. SKBR3 cells were then co-cultivated in Boyden chambers with macrophages activated with IL-4 or transfected with lin-4 mimics, as described above, for 24-72 h. pMIR-REPORT and Renilla luciferase activities were assayed using the Dual-Luciferase assay kit (Promega). pMIR-REPORT luciferase activity was normalized to the Renilla luciferase activity. To determine whether Mef2c mRNA is a target of miR-223, the pMIR-REPORT vector containing the 3'-UTR of Mef2c was co-transfected with miR-223 into HEK-293T cells. After 24-48 h of incubation, the cells were lysed, and luciferase activity was detected as described above.
Shuttling assays for fluorescently-labeled miRNA
To further visualize the shuttling of miRNAs, Cy3-labeled miRNAs (GenePharma) were transfected into macrophages, as described above. Macrophages were washed to remove the residual transfection reagent 24-h after transfection. Macrophages carrying Cy3-miRNA were then placed onto transwell inserts, and SKBR3 cells were seeded in the lower wells of Boyden chambers. After incubation for 24-48 h, SKBR3 cells were collected for fluorescence microscopy and flow cytometric analyses.
Western blots
Cells were lysed with RIPA lysis buffer and protease inhibitors (Sigma-Aldrich). Nuclear protein was collected according to previously described protocols [23]. A total of 20 μg of protein per sample was separated on SDS-PAGE gels and transferred onto nitrocellulose membranes. Membranes were blocked and incubated with antibodies against β-catenin (1:1000, Cell Signaling Technology, #9587) or Mef2c (1:1000, Cell Signaling Technology, #9792) overnight at 4°C. Primary antibody incubation was followed by incubation with HRP-conjugated secondary antibodies (Amersham Pharmacia). HRP signals were then visualized by enhanced chemiluminescence (Pierce).
Confocal microscopy
Cells prepared on coverslips were fixed in 4% PFA, treated with 0.3% Triton X-100, blocked with 5% BSA and incubated with an anti-β-catenin antibody (1:200, Cell Signaling Technology, #9587) overnight at 4°C. After being washed with PBS, cells were incubated with a FITC-conjugated secondary antibody for 1 h and counterstained with PI prior to inspection under a confocal microscope.
SKBR3 breast cancer cells were co-cultured with Cy3-preloaded macrophages for 24-48 h. After co-culture, both macrophages and SKBR3 were fixed in 4% PFA, treated with 0.1% Triton X-100, blocked in 3% BSA and incubated with an anti-CD68 antibody (1:200, Dako) for 2 h at room temperature. After being washed, cells were incubated with an Alexa Fluor 488-conjugated secondary antibody, and then counterstained with DAPI before inspection under fluorescence microscope.
All tumor samples of invasive breast cancer were obtained from female patients at the No. 2 Affiliated Hospital, Sun-Yat-Sen University. All patient samples were collected with informed consent according to the Internal Review and the Ethics Boards of the Sun-Yat-Sen Memorial Hospital, Sun-Yat-Sen University. Samples were fixed, paraffin embedded and sectioned into 5- μM slices. Macrophages were visualized by immunohistochemistry staining using an anti-CD68 (1:200, Dako) antibody. Bound primary antibody was detected by using a horseradish peroxidase conjugated secondary antibody, which was then developed in DAB solution (Dako). Pictures were taken under a light microscope.
Exosome purification and labeling
The same number of IL-4-activated or unactivated macrophages were cultured in exosome-free medium (DMEM containing 10% FBS that was depleted of exosomes by ultra-centrifuging at 100,000 × g for 18 h). Conditioned media were collected after 3-5 days of incubation. Exosomes were purified by differential centrifugation. Briefly, the conditioned media were centrifuged at 500 × g for 30 min and 16,500 × g for 20 min to eliminate cells and cellular debris, respectively. Supernatants were filtered through 0.22 μm filters. Exosomes were pelleted by ultracentrifugation at 120,000 × g for 180 min, washed in PBS, pelleted again and resuspended in PBS. Exosome preparations were stained with CM-DiI (CellTracker, C7000), a fluorescent dye that labels the plasma membrane, according to the manufacturer's instructions. Next, exosomes were diluted in complete medium (DMEM with 10% FBS) and were added into the cell cultures. At the indicated time points, cells were examined under a confocal microscope and analyzed using flow cytometry.
RNase treatment of Exosomes
The culture of unactivated and IL-4-activated macrophages and the process through which exosomes were collected are described above. Exosomes were treated with RNase according to previously described protocols [24]. Briefly, separated exosomes were incubated with RNase A at a final concentration of 100 U/ml, with or without 1% Triton X-100, at room temperature for 30 min. Exosomes were washed with PBS to remove residual RNase and Triton X-100. Exosomes were incubated with breast cancer cells prior to performing invasion assays.
Electronic microscopy
Exosome preparations were mixed with equal quantities of freshly prepared 4% paraformaldehyde for 20 min. Samples were washed in water, pelleted by ultracentrifugation and then fixed for 5 min in 1% glutaraldehyde. After this process, exosomes were re-suspended in water, and 5 μl of the samples were loaded onto carbon-coated formvar grids. Exosomes were stained for 10 min with saturated aqueous uranyl and examined using an electron microscope (Hitachi S-4800 FEG SEM).
Statistical analyses
All data are expressed as mean ± SD. Statistical analyses were performed using paired Student's t-tests.
Co-cultivation with IL-4-activated macrophages elevates miR-223 levels in breast cancer cells
Because TAMs located in the stroma of breast cancers are primarily M2 macrophages activated by IL-4-producing CD4+ T cells [25], we mimicked this TAM-populated microenvironment by co-cultivating SKBR3 breast cancer cells with IL-4-activated MDMs in a Boyden chamber, which prevents direct cell-cell contact. To optimize a physiologically-relevant cell number ratio for the co-culture experiments, we quantified the amount of macrophage infiltration present in patient samples that were pathologically diagnosed as invasive breast cancer. As shown in Additional file 1 Figure S1(A), a large number of macrophages (CD68-positive) infiltrated breast tumors, especially in the tumor-associated stromal border, where many invasive tumor cells were also located. Because previous studies suggested that macrophages produce exosomes, which shuttle proteins or microRNAs (miRNAs) into adjacent cells within the microenvironment, we focused on the neighboring tumor cells and macrophages. We calculated the cell ratio based upon the neighboring tumor cell and macrophage populations in a local context as indicated in Additional file 1 Figure S1(B). The ratio of tumor cells to their adjacent macrophages ranged from 1:1 to 1:7. Subsequently, we tested the effects of macrophage to breast cancer cell ratios, ranging from 1:1 to 5:1, in the co-culture system. The effects of macrophages on breast cancer cells were observed at ratios starting from 1:1. First, we screened for miRNAs that were differentially expressed between macrophages and breast cancer cells using a DiscovArray miRNA microarray. All the microarray data were listed in Additional file 2 Table S1. We found three microRNAs that were abundantly expressed in macrophages but not in SKBR3 or MDA-MB-231 breast cancer cells (Figure 1A). Using qRT-PCR, we confirmed that miR-223 was overexpressed in IL-4-activated MDMs but was not highly expressed in either SKBR3 or MDA-MB-231 breast cancer cells (Figure 1B). Additionally, miR-223 is involved in cancer progression [26,27]; therefore, we focused on miR-223 expression levels in breast cancer cells after co-cultivation with IL-4-activated MDMs. We seeded breast cancer cells and macrophages in co-culture Boyden chambers, as described in Figure 2A. Interestingly, breast cancer cells co-cultured with IL-4-activated macrophages exhibited a profound increase in cellular miR-223 levels (2.7 ± 0.5- and 3.3 ± 0.98-fold in SKBR3 and MDA-MB-231 cells, respectively) relative to cells that were not co-cultured (blank) or were co-cultured with unactivated macrophages (Figure 1C). To evaluate the function of elevated miR-223 in breast cancer cells, we used a luciferase reporter gene containing a sequence complementary to miR-223 in its 3'-UTR. Co-culturing with IL-4-activated macrophages reduced luciferase reporter activity in SKBR3 breast cancer cells (Figure 1D and Additional file 3 Figure S2), which suggests that the elevated miR-223 levels observed in breast cancer cells are capable of silencing target gene expression. Furthermore, direct transfection of miR-223 mimics, but not a scrambled negative control miRNA (miR-NC), also suppressed the reporter gene activity in SKBR3 cells (Figure 1D). These data suggest that co-culturing breast cancer cells with IL-4-activated macrophages increases the level of functional miR-223 in breast cancer cells.
Additional file 1. Figure S1. Macrophages infiltration in breast tumor. Immunohistochemical staining of macrophage marker: CD68 in invasive breast cancer samples. (A) Macrophage distribution in invasive breast cancer of patients #1 and #2. Representative regions in #1 and #2 (200X) images were marked by rectangle and shown in (B). (B) Typical examples of tumour cells with neighbouring macrophages. In the marked regions, macrophages were those brown cells and adjacent tumour cells were marked with *.
Format: PDF Size: 2.5MB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 2. Table S1. Microarray data. MicroRNA expression profiles of breast cancer cell SKBR3, unactivated and IL-4 activated macrophages were generated using DiscovArray miRNA arrays. All the microarray data were listed in the table.
Format: DOC Size: 1.2MB Download file
This file can be viewed with: Microsoft Word Viewer
Figure 1. miR-223 was up-regulated in breast cancer cell lines co-cultured with IL-4-activated macrophages. (A) miRNA profiling in macrophages and the breast cancer cell line SKBR3 using the DiscovArray miRNA array. Three of the miRNAs that were present at high levels in macrophages but absent in SKBR3 cells are shown. (Un-Mac, unactivated macrophages; IL4-Mac, IL-4-activated macrophages). (B) Quantitative real-time PCR (qRT-PCR) confirmed a high expression level of hsa-miR-223 in macrophages but not in SKBR3 or MDA-MB-231 cells. * p < 0.05. (C) Co-culture of breast cancer cells with macrophages increased miR-223 levels in breast cancer cells. Breast cancer cell lines (SKBR3 and MDA-MB-231) were cultured alone (blank) or co-cultured with unactivated or IL-4-activated macrophages without direct cell-cell contact. miR-223 expression in SKBR3 and MDA-MB-231 cells was detected using qRT-PCR. Relative levels of miR-223 expression normalised to U6 rRNA levels are presented. * p < 0.05. (D) Functional assay of miR-223 in breast cancer cells. miR-223-targeting luciferase reporter containing a miR-223 complementary sequence within the 3'-UTR of the luciferase reporter gene was constructed. SKBR3 cells were transfected with the reporter gene and cultured alone (blank) or co-cultured with IL-4-activated macrophages. As controls, SKBR3 cells were co-transfected with the reporter gene and miR-NC, miR-660 or miR-223. Relative luciferase activities (normalised to Renilla luciferase activity) are presented. * p < 0.05.
Figure 2. Functional microRNA is shuttled from macrophages to breast cancer cells. (A) Schematic illustration of a miRNA transfer model. Macrophages were preloaded with Cy3 fluorescently-labeled miR-223 or the non-mammalian miRNA lin-4. A transwell system with a 0.4- μm pore size film, which allows small size materials (e.g., miRNAs), but not cells, to pass through, was used to separate SKBR3 cells from macrophages. (B) and (C) SKBR3 cells were cultured alone (blank) or co-cultured with macrophages that were pre-transfected with reagent (mock), unlabeled miR-223 or Cy3-miR-223. The Cy3 fluorescence signal in SKBR3 cells was determined by fluorescence microscopy (B), and the percentage of Cy3-positive cells in the images was calculated in (C). Arrowheads indicate Cy3-positive SKBR3 cells, and images are shown at 200×. (D) SKBR3 cells were cultured as shown in (B), and Cy3-positive cells (%) were quantified by flow cytometry. (E) A lin-4-targeting luciferase reporter gene carrying a lin-4 complementary sequence in the 3'-UTR was transfected into SKBR3 cells. SKBR3 cells were then co-cultured in a transwell with macrophages pre-transfected with miR-NC or lin-4. SKBR3 cells that were directly transfected with lin-4 were used as a control. Luciferase activities in SKBR3 cells were measured, and relative luciferase activities (normalised to Renilla luciferase activity) are presented. * p < 0.05. (Un-Mac, unactivated macrophages; IL4-Mac, IL-4-activated macrophages).
Additional file 3. Figure S2. Luciferase assay of miR-223 activity in SKBR3 breast cancer cells. miR-223-targeting luciferase reporter containing a miR-223 complementary sequence within the 3'-UTR of the luciferase reporter gene was constructed. SKBR3 cells were transfected with the reporter gene and cultured alone (blank) or co-cultured with IL-4-activated or unactivated macrophages. As controls, SKBR3 cells were co-transfected with the reporter gene or miR-NC. Relative luciferase activities (normalised to Renilla luciferase activity) are presented. * p < 0.05.
Format: PDF Size: 197KB Download file
This file can be viewed with: Adobe Acrobat Reader
miRNAs released by macrophages are shuttled into breast cancer cells
To determine whether miRNAs released by IL-4-activated macrophages are shuttled into co-cultured breast cancer cells, we transfected macrophages with either Cy3-labeled miR-223 or non-mammalian lin-4 miRNA prior to co-culture with SKBR3 cells. Co-culture was performed in a 24-well Boyden chamber with a 0.4- μm insert (Figure 2A). Fluorescence microscopy analysis indicated the presence of Cy3-miRNA in SKBR3 cells, with approximately 15 positive cells per field of view, when co-cultured with macrophages transfected with Cy3-labeled miR-223. Fluorescence was not detected in cells that were not co-cultured or that were co-cultured with macrophages transfected with unlabeled miR-223 (Figure 2B & 2C). Theoretically, macrophages cannot penetrate through the 0.4- μm pore size membrane. However, to verify that the co-cultivated fluorescent tumor cells were not contaminated with macrophages, we stained these cells for CD68, a macrophage marker. As shown in Additional file 4 Figure S3, after being co-cultured with Cy3-preloaded macrophages, no CD68 staining was detected among the Cy3-positive cells. Furthermore, flow cytometric analysis confirmed that 13.8% of SKBR3 cells co-cultured with IL-4-activated macrophages that were preloaded with Cy3-labeled miR-223 were positive for Cy3-miRNA (Figure 2D). These data suggest that miRNAs within macrophages can be physically transported into adjacent cancer cells. To determine whether the miRNAs shuttled from macrophages retained their gene-silencing function in the recipient cells, we used a non-mammalian miRNA, lin-4, and its target reporter gene (luciferase reporter containing a lin-4 complementary sequence in its 3'-UTR). Prior to co-cultivation, IL-4-activated macrophages were transfected with either control (NC) or lin-4 miRNA, and SKBR3 breast cancer cells were transfected with a luciferase reporter gene with a lin-4 target sequence at its 3'-UTR. Luciferase activity was suppressed in SKBR3 cells co-cultured with macrophages transfected with lin-4, while this suppression was not observed in cells co-cultured with the control NC miRNA macrophages. Transfection of SKBR3 cells with lin-4 was used as a control to demonstrate a significant reduction in luciferase activity of the lin-4 reporter gene (Figure 2E).
Additional file 4. Figure S3. The fluorescent Cells co-cultured with Cy3-proloaded macrophages were CD68 negative. SKBR3 were cultured alone or co-cultured with IL-4 activated or unactivated macrophages that were pre-transfected with Cy3-miR-223. Both macrophages and SKBR3 were then stained for macrophage marker: CD68. DAPI was used to visualize nucleus. Fluorescence signals were determined by fluorescence microscopy. Arrows indicate Cy3 signal in SKBR3 cells and images are shown at 1000×.
Format: PDF Size: 685KB Download file
This file can be viewed with: Adobe Acrobat Reader
Exosomes released from IL-4-activated macrophages mediate miRNA shuttling
Previous studies have demonstrated that microvesicles, or exosomes, secreted from macrophages may serve as vesicles that mediate cell-to-cell exchange of small RNAs. To further confirm that exosomes released from macrophages mediate miR-223 transfer, exosomes released from macrophages were purified by gradient centrifugation. Consistent with previous findings, electron microscopy revealed that exosomes released from the unactivated and IL-4-activated macrophages were nanometer-sized particles with bilayer membranes (Figure 3A) [16]. The exosomes derived from unactivated macrophages were much larger in size, with a diameter of 222 ± 52 nm, while those derived from IL-4-activated macrophages were smaller, with a diameter of 57 ± 21 nm (Figure 3B). Additionally, qRT-PCR data demonstrated the presence of miR-223 in the exosomes of both subtypes of macrophages, while those released from IL-4-activated macrophages contained higher levels of miR-223 than those from the unactivated cells (p < 0.01) (Figure 3C). To visualize exosome uptake in breast cancer cells, SKBR3 cells were incubated with CM-DiI-labeled exosomes that were isolated from macrophages. Internalization of CM-DiI-labeled exosomes was detected in SKBR3 cells by confocal microscopy (Figure 3E). The number of cells with internalized exosomes increased in a time-dependent manner and reached a plateau at 24 h (Figure 3D). Interestingly, exosomes released from IL-4-activated macrophages were internalized more efficiently than those released from unactivated macrophages (Figure 3D). In parallel, Cy3-labeled miR-223 from IL-4-activated macrophages was more efficiently transported into SKBR3 breast cancer cells (13.36 ± 3.52%) than the labeled miR-223 from unactivated macrophages (7.85 ± 2.84%) (Additional file 5 Figure S4). Taken together, these data suggest that macrophage-secreted exosomes mediate miR-223 shuttling.
Figure 3. Exosomes secreted from macrophages shuttle miR-223 to breast cancer cells. (A) Exosomes secreted from unactivated and IL-4-activated macrophages were collected and observed with an electron microscope. Exosomes derived from unactivated macrophages (un-Mac-origin) were larger in size (100-250 nm) than those derived from IL-4-activated macrophages (IL-4-Mac-origin) (50-80 nm). Scale bars are as indicated. (B) The diameter of exosomes derived from unactivated and IL-4-activated macrophages were quantified. Data are averages of more than 10 exosomes from each group. (C) Levels of miR-223 expression in exosomes were quantified by qRT-PCR. ** p < 0.01. (D) and (E) Exosomes derived from IL-4-activated macrophages were more readily internalised by breast cancer cells. Exosomes derived from unactivated and IL-4-activated macrophages were labeled with CM-Dil and incubated with SKBR3 cells for the indicated times (0-40 h). Uptake of exosomes by cancer cells was visualized by confocal microscopy (representative images are shown in (E) and quantified by flow cytometry in (D)). (Un-Mac, unactivated macrophages; IL4-Mac, IL-4-activated macrophages).
Additional file 5. Figure S4. Cy3-miR-223 was more efficiently shuttled from IL-4 activated macrophages than that from unactivated macrophages to breast cancer cells. SKBR3 cells were cultured alone (blank) or co-cultured respectively with IL-4 activated or unactivated macrophages that were pre-transfected with reagent (mock), unlabeled miR-223 or Cy3-miR-223. Cy3-positive cells (%) were quantified by flow cytometry. ** p < 0.01. (Un-Mac, unactivated macrophages; IL4-Mac, IL-4-activated macrophages).
Format: PDF Size: 251KB Download file
This file can be viewed with: Adobe Acrobat Reader
miR-223 promotes breast cancer cell invasion
miR-223 has been implicated in the progression of renal [26] and hepatocellular cancers [27]. Consistent with previous studies, co-culture with IL-4-activated macrophages enhanced breast cancer cell invasion relative to control cells cultured alone or to co-culture with unactivated macrophages (Figure 4A). To determine the biological function of miR-223 uptake by breast cancer cells, we first examined the effects of miR-223 on breast cancer cell invasion by directly transfecting miR-223 mimics into SKBR3 or MDA-MB-231 cells. Cell invasiveness was determined using a transwell invasion assay. Indeed, significantly more miR-223-transfected breast cancer cells invaded across the Matrigel-coated inserts as compared to miR-NC-transfected cells (Figure 4B). We also tested the invasion-promoting potential of exosomes from both unactivated and IL-4-activated macrophages. SKBR3 cells were incubated with exosomes purified from equal numbers of IL-4-activated and unactivated macrophages. Exosomes isolated from IL-4-activated macrophages promoted SKBR3 invasion, whereas the same effect was not observed with those isolated from unactivated macrophages (Figure 4C). Additionally, the invasion-promoting activity by exosomes derived from IL-4-activated macrophages was alleviated by miR-223-ASO (Figure 4C). Next, we treated exosomes derived from macrophages with RNase plus triton X-100 and incubated those exosomes with SKBR3 cells. Exosomes derived from IL-4-activiated macrophages treated with RNase plus triton X-100 had decreased invasion potential compared to the blank and RNase alone groups. In contrast, no significant differences were observed in the invasion potential of RNase plus triton X-100 treated exosomes derived from unactivated macrophages (Additional file 6 Figure S5). Moreover, the invasiveness of the co-cultivated breast cancer cells decreased when we treated IL-4-activated macrophages with miR-223-ASO (Additional file 7 Figure S6). Similarly, breast cancer cells preloaded with miR-223-ASO had decreased invasiveness when co-cultured with IL-4-activated macrophages (Additional file 8 Figure S7). These data suggest that miR-223 uptake by breast cancer cells is associated with the promotion of invasiveness.
Figure 4. Exosomal shuttling of miR-223 from macrophages to breast cancer cells promotes breast cancer cell invasion. (A) IL-4-activated macrophages promote breast cancer cell invasion. The breast cancer cell lines SKBR3 and MDA-MB-231 were cultured alone (blank) or co-cultured with unactivated or IL-4-activated macrophages. Approximately 24 to 48 h after co-culture, SKBR3 and MDA-MB-231 cells were subjected to an invasion assay. Data are averages of triplicates from more than three independent experiments and are presented as the number of invading cells per field. (B) miR-223 mimics enhanced the invasion of SKBR3 and MDA-MB-231 breast cancer cells. miR-223 was transfected into SKBR3 and MDA-MB-231 cells. Cell invasion was then determined by a transwell invasion assay. Relative invasion activities are presented as fold increases in the miR-223 group. The miR-NC group was normalized to 1.0. (C) Exposure to exosomes derived from macrophages resulted in similar effects on breast cancer invasion as the actual macrophages. SKBR3 cells were incubated with exosomes derived from unactivated or IL-4-activated macrophages and then treated with miR-NC ASO or miR-223 ASO. Cell invasion assays were performed as shown in (A). Data are averages of triplicates from three independent experiments and are presented as the number of invading cells per field. * p < 0.05; ** p < 0.01. (Un-Mac-exosome, exosomes originated from unactivated macrophages; IL-4-Mac-exosome, exosomes from IL-4-activated macrophages).
Additional file 6. Figure S5. Exosomes derived from IL-4 activated macrophages treated by RNase plus triton X-100 had decreased invasion potentiate. Exosomes secreted from IL-4 activated macrophages or unactivated macrophages were treated by RNase with or without triton X-100. SKBR3 breast cancer cells were then incubated with those exosomes. Invasion assays were performed. Data are presented as the number of invading cells per field (A) and representative images of invading cells were showed in (B). ** p < 0.01. (Un-Mac, unactivated macrophages; IL4-Mac, IL-4-activated macrophages).
Format: PDF Size: 1.5MB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 7. Figure S6. Inhibition of miR-223 expression in IL-4 activated macrophages decreased co-cultivated breast cancer cell invasion. IL-4 activated macrophages were treated by miR-223-ASO, miR-NC-ASO or transfection reagent only (mock) and then co-cultured with MDA-MB-231 breast cancer cells. Breast cancer cell invasion assays were then performed. Data are presented as the number of invading cells per field (A) and representative images of invading cells were showed in (B). * p < 0.05.
Format: PDF Size: 802KB Download file
This file can be viewed with: Adobe Acrobat Reader
Additional file 8. Figure S7. SKBR3 pre-treated by miR-223-ASO showed decreased cell invasion when co-cultured with IL-4 activated macrophages. Unactivated or IL-4 activated macrophages respectively co-cultured with SKBR3 that pre-treated with miR-223-ASO (miR-223-ASO treated SKBR3) or miR-NC-ASO (miR-NC-ASO treated SKBR3). SKBR3 cell invasion was then determined by transwell invasion assay. Relative invasion activities are presented as fold changes in the miR-223-ASO group. The miR-NC-ASO group was normalized to 1.0. * p < 0.05.
Format: PDF Size: 216KB Download file
This file can be viewed with: Adobe Acrobat Reader
miR-223 targets the Mef2c-β-catenin pathway
Previous studies have suggested that miR-223 targets the myocyte enhancer factor, Mef2c, in myeloid progenitor cells to inhibit their proliferation and granulocyte function [28]. Using TargetScan, we identified two miR-223 target sites in the Mef2c 3'-UTR. To understand the mechanism by which miR-223 promotes breast cancer cell invasion, we assessed whether miR-223 targets Mef2c in breast cancer cells. HEK-293T cells were transfected with a luciferase reporter vector, pMIR-REPORTER-Mef2c, that contained the cloned Mef2c 3'-UTR. After approximately 24-48 h, cells were lysed and luciferase activity was determined, as described above. As depicted in Figure 5A, transfection with miR-223 inhibited the luciferase activity of the Mef2c reporter gene but did not reduce the activity of the control luciferase vector that did not have the Mef2c 3'-UTR. This inhibition was not observed in the miR-NC-transfected cells. Moreover, western blot analysis demonstrated that transfection with miR-223 reduced the levels of endogenous Mef2c in SKBR3 cells (Figure 5B). As Mef2c reduction has been linked to nuclear accumulation of β-catenin and the promotion of cell migration [29,30], we further examined whether miR-223 regulates nuclear translocation of β-catenin. Western blotting of cellular fractions demonstrated that transfection with miR-223 dramatically increased β-catenin expression in the nuclei of breast cancer cells (Figure 5B). Additionally, indirect fluorescence microscopy revealed localization of β-catenin in the nuclei of breast cancer cells transfected with miR-223 (Figure 5C). Based on these results, we conclude that miR-223 may target the Mef2c-β-catenin pathway to mediate breast cancer cell invasion.
Figure 5. miR-223 targets the Mef2c-β-catenin pathway during breast cancer cell invasion. (A) miR-223 transfection inhibited Mef2c gene expression. HEK-293T cells were co-transfected with miR-223 and the Mef2c 3'-UTR-directed luciferase reporter (luciferase-Mef2c; Mef2c 3'-UTR cloned into the pMIR-REPORTER). Luciferase activities were measured as described in Figure 2. Cells transfected with miR-223 and luciferase vector, miR-NC and luciferase-Mef2c, or miR-223 and luciferase-miR-223C (miR-223 complementary sequence cloned in the 3'-UTR of luciferase reporter) were used as controls. Data are averages of triplicates from three independent experiments. * p < 0.05. (B) and (C) miR-223 reduced Mef2c expression and resulted in nuclear accumulation of β-catenin. SKBR3 cells were transfected with miR-223 or miR-NC (control). The expression of Mef2c and localisation of β-catenin in breast cancer cells were then determined by cellular fractionation followed by western blot analysis with anti-Mef2c, β-catenin and β-actin (B). The localisation of β-catenin in SKBR3 was visualized by indirect fluorescence microscopy. Briefly, cells were incubated with an anti-β-catenin antibody followed by an incubation with a FITC-conjugated anti-mouse secondary antibody. PI was used to visualise the nuclei (C). (scale bar: 20 μm).
Interactions between macrophages and breast cancer cells result in more invasive cancer cells, metastasis and poor patient prognosis. Preventing malignant breast epithelium-macrophage communication may inhibit the metastatic cascade during cancer progression [2,9], and thereby, provide important treatment targets for breast cancer therapy. Consequently, the mechanisms underlying breast cancer cell-macrophage interactions require further investigation. Most current studies focus on the cytokines, molecules or enzymes that are secreted by macrophages; however, greater attention is needed on miRNAs due to their regulatory effects on cancer cell progression [31].
Previously, miRNA profiling has been limited to tissues or cells. Evidence for circulating miRNAs is increasing, and recent studies have detected miRNAs in the peripheral blood of patients [32]. These peripheral blood-isolated miRNAs could potentially be used as biomarkers for certain diseases [33,34]. In these studies, microvesicles/exosomes were found to serve as mediators of miRNA circulation. Additional studies have suggested that exosomes can be secreted by many cells, including T cells [35], B cells [36], mast cells [16], dendritic cells [37], cancer cells [38] and macrophages [39]. Using ultracentrifugation, we isolated exosomes that were released from macrophages. We demonstrated that these macrophage-derived exosomes have classic structures consistent with previous reports [16]. Exosomes have been shown to contain protein, RNA and DNA, and much work has focused on understanding the mechanisms of exosome-mediated cell-cell interactions [40]. Some of these interactions involve communication between different cell types and convey regulatory effectors [41,42]; whereas, others occur among cells of the same type [15]. Our knowledge regarding communication between macrophages and tumor cells, however, is limited to physical contact and cytokine or chemokine secretion [43]. More recently, human macrophages were found to release exosomes containing migration-promoting enzymes [44].
Therefore, exosomes may mediate the interactions between macrophages and target cells by shuttling functional miRNAs between them. Within the breast cancer microenvironment, it is possible for macrophages to communicate with cancer cells by transferring miRNAs via exosomes. For this study, we utilized a transwell system to mimic the tumor microenvironment; this system allows macrophages to communicate with breast cancer cells without physical contact. We detected a macrophage-dominant miRNA, miR-223, that showed an increase in breast cancer cells following co-culture with macrophages. To verify miRNA transport between macrophages and breast cancer cells, we transfected Cy3-labeled miR-223 into macrophages and tracked its movement within the co-culture system. Once fluorescently-labeled miRNAs were secreted from the macrophages and captured by breast cancer cells, the fluorescent Cy3 signal could be detected in the breast cancer cells. As demonstrated by confocal microscopy and flow cytometric analyses, Cy3-positive breast cancer cells were observed, which indicates miRNA transfer. Additionally, functional miRNA delivery was confirmed using lin-4, a miRNA that is not expressed in mammalian cells.
Next, we incubated breast cancer cells with exosomes treated with RNase and triton X-100 to degrade the RNA. The depletion of RNAs in exosomes significantly decreased the invasive potential of exosomes derived from IL-4-activated macrophages. In order to determine the specific function of miR-223, we transfected miR-223-ASO into SKBR3 cells and then incubated these cells with exosomes. The invasion-promoting activity of exosomes derived from IL-4-activated macrophages on miR-223-ASO-treated SKBR3 cells was dramatically reduced. Moreover, when miR-223 expression in IL-4-activated macrophages was reduced, the invasiveness of co-cultivated breast cancer cells also decreased. Thus, it is likely that the miR-223 delivered from macrophages, and not the endogenous miR-223, promotes breast cancer cell invasion.
MDMs (monocyte-derived macrophages), prior to activation by IL-4, were not polarized and were relatively quiescent. The invasion potential of breast cancer cells co-cultured with either unactivated macrophages or exosomes released from unactivated macrophages was not different from that observed for the untreated cells. When incubated with IL-4, macrophages became activated and polarized. IL-4, the signature cytokine of a TH-2 type immune response, is a major activator of the TAM phenotype in the tumor microenvironment, and the effects of IL-4 collectively prime TAM with the ability to promote tumor growth, invasion and metastasis [45]. When activated by IL-4, macrophages change their cellular morphology, gene expression profile and secretory pattern [46]. TAMs selectively secrete regulators or effectors that promote tumor proliferation, invasion and metastasis [47]. miR-223 is a miRNA associated with tumor progress. Therefore, when activated by IL-4, macrophages would selectively secrete more invasion-potentiating cytokines, enzymes and miRNAs, including miR-223, that are able to regulate the characteristics of target cells. Our data demonstrate that there are differences between exosomes from unactivated and from IL-4-activated macrophages in terms of size, miR-223 content, and invasion-potential.
There is a profound impact of macrophages on breast cancer cells relative to biological function. Therefore, the identification and verification of these cellular interactions may provide additional strategies to negate the tumor promoting functions of TAMs by targeting those regulators and/or effectors which could have significant potential in anticancer therapies. In our studies, IL-4-activated macrophages efficiently secrete and deliver miR-223 to breast cancer cells, and miR-223 is responsible for macrophage-promoting breast cancer cell invasion. Clinically, based on this data, we could identify macrophage infiltration using CD68 staining, and then separate tumor cells for analysis of miR-223 expression. If greater levels of macrophage infiltration were observed in combination with up-regulation of miR-223 expression in tumor cells, which would indicate a more aggressive breast cancer phenotype according to our data, then the patient may have a poor prognosis and alternative treatments might be necessary. Inhibition of miR-223 in tumor cells and/or TAMs might be helpful. Furthermore, quantification and/or detection of miR-223 in the serum may indicate tumor aggressiveness. Therefore, it may be of great significance to designate miRNAs as diagnostic or prognostic markers for breast cancer. This will require extensive additional work to detect miR-223 in patient serum and calculate the relationship of miR-223 with prognosis, survival rate, etc.
Additionally, we explored the targets of miR-223, and a possible mechanism through which miR-223 may promote breast cancer cell invasion. Our data do not clearly demonstrate the invasion pathway utilized; however, our observations indicate that the involvement of the miR-223/Mef2c/β-catenin pathway is plausible. Additionally, a miRNA has many targets and may function through multiple different pathways. Currently, we are trying to find additional targets and verify their effects using gain- and loss-of-function assays.
The results of this study provide the first evidence suggesting that macrophages can transfer miRNA via exosomes to breast cancer cells. We found that the vesicular miRNA is responsible for macrophage-promoting breast cancer cell invasion, and we have provided a rationale for therapeutically targeting miR-223 in M2 macrophages or exosomal miR-223 from M2 macrophages.
In conclusion, this study demonstrates that functional miRNAs can be transported from macrophages to breast cancer cells. Exosomes secreted from IL-4-activated macrophages shuttle miR-223 into breast cancer cells, and miR-223 promotes breast cancer cell invasion. Thus, our study provides insights into the mechanisms of cell-cell interactions through which macrophages regulate the invasiveness of breast cancer cells via the exosomal-mediated delivery of oncogenic miRNAs.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MY designed and conducted the majority of the experiments, interpreted and analyzed the data, and wrote the manuscript. JQC, LL and YJL performed a subset of the breast cancer cell-invasion assays. FS contributed to the macrophage cell culture and quantitative RT-PCR assays. BY contributed to the luciferase reporter construction. FXS provided the clinical breast tumor samples and helped revise the manuscript. JDH contributed to the project design, supervised the experiments and revised the manuscript. EWS designed the project, supervised the experiments and helped draft and revise the manuscript. All authors read and approved the final manuscript.
This work was supported by grants from the National Natural Science Foundation of China (NSFC30972785, 30921140312, 30831160515, and 30830110), the Research Grants Council of Hong Kong (N_HKU 719/08 and HKU1/CRF/10) to ES and JDH, "973" Program Projects (2010CB912800, 2011CB504203, and 2009CB521706) from the Ministry of Science and Technology of China to ES, the Key Laboratory of Malignant Tumor Gene Regulation and Target Therapy of Guangdong Higher Education Institutes, Sun Yat-Sen University (KLB09001) to ES, the Clinical Key Project of Public Health Administration of China, and the Grant for Development of Important New Drugs from Ministry of Health of China (2011ZX09102-010-02).
Lin EY, Pollard JW: Tumor-associated macrophages press the angiogenic switch in breast cancer.
Cancer Res 2007, 67:5064-5066. PubMed Abstract | Publisher Full Text
Leek RD, Lewis CE, Whitehouse R, Greenall M, Clarke J, Harris AL: Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma.
Cancer Res 1996, 56:4625-4629. PubMed Abstract | Publisher Full Text
Allavena P, Sica A, Solinas G, Porta C, Mantovani A: The inflammatory micro-environment in tumor progression: the role of tumor-associated macrophages.
Crit Rev Oncol Hematol 2008, 66:1-9. PubMed Abstract | Publisher Full Text
O'Brien J, Schedin P: Macrophages in breast cancer: do involution macrophages account for the poor prognosis of pregnancy-associated breast cancer?
J Mammary Gland Biol Neoplasia 2009, 14:145-157. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Martinez FO, Sica A, Mantovani A, Locati M: Macrophage activation and polarization.
Front Biosci 2008, 13:453-461. PubMed Abstract | Publisher Full Text
Luo Y, Zhou H, Krueger J, Kaplan C, Lee SH, Dolman C, Markowitz D, Wu W, Liu C, Reisfeld RA, Xiang R: Targeting tumor-associated macrophages as a novel strategy against breast cancer.
J Clin Invest 2006, 116:2132-2141. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Bingle L, Brown NJ, Lewis CE: The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies.
J Pathol 2002, 196:254-265. PubMed Abstract | Publisher Full Text
Lin EY, Gouon-Evans V, Nguyen AV, Pollard JW: The macrophage growth factor CSF-1 in mammary gland development and tumor progression.
J Mammary Gland Biol Neoplasia 2002, 7:147-162. PubMed Abstract | Publisher Full Text
Pollard JW: Tumour-educated macrophages promote tumour progression and metastasis.
Nat Rev Cancer 2004, 4:71-78. PubMed Abstract | Publisher Full Text
Joyce JA, Pollard JW: Microenvironmental regulation of metastasis.
Nat Rev Cancer 2009, 9:239-252. PubMed Abstract | Publisher Full Text
Joimel U, Gest C, Soria J, Pritchard LL, Alexandre J, Laurent M, Blot E, Cazin L, Vannier JP, Varin R, et al.: Stimulation of angiogenesis resulting from cooperation between macrophages and MDA-MB-231 breast cancer cells: proposed molecular mechanism and effect of tetrathiomolybdate.
Luo YP, Zhou H, Krueger J, Kaplan C, Liao D, Markowitz D, Liu C, Chen T, Chuang TH, Xiang R, Reisfeld RA: The role of proto-oncogene Fra-1 in remodeling the tumor microenvironment in support of breast tumor cell invasion and progression.
Ratajczak J, Wysoczynski M, Hayek F, Janowska-Wieczorek A, Ratajczak MZ: Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication.
Leukemia 2006, 20:1487-1495. PubMed Abstract | Publisher Full Text
Camussi G, Deregibus MC, Bruno S, Cantaluppi V, Biancone L: Exosomes/microvesicles as a mechanism of cell-to-cell communication.
Vallhov H, Gutzeit C, Johansson SM, Nagy N, Paul M, Li Q, Friend S, George TC, Klein E, Scheynius A, Gabrielsson S: Exosomes Containing Glycoprotein 350 Released by EBV-Transformed B Cells Selectively Target B Cells through CD21 and Block EBV Infection In Vitro.
J Immunol 2011, 186(1):73-82. PubMed Abstract | Publisher Full Text
Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO: Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.
Nat Cell Biol 2007, 9:654-659. PubMed Abstract | Publisher Full Text
Ohshima K, Inoue K, Fujiwara A, Hatakeyama K, Kanto K, Watanabe Y, Muramatsu K, Fukuda Y, Ogura S, Yamaguchi K, Mochizuki T: Let-7 microRNA family is selectively secreted into the extracellular environment via exosomes in a metastatic gastric cancer cell line.
Gourzones C, Gelin A, Bombik I, Klibi J, Verillaud B, Guigay J, Lang P, Temam S, Schneider V, Amiel C, et al.: Extra-cellular release and blood diffusion of BART viral micro-RNAs produced by EBV-infected nasopharyngeal carcinoma cells.
Luo SS, Ishibashi O, Ishikawa G, Ishikawa T, Katayama A, Mishima T, Takizawa T, Shigihara T, Goto T, Izumi A, et al.: Human villous trophoblasts express and secrete placenta-specific microRNAs into maternal circulation via exosomes.
Biol Reprod 2009, 81:717-729. PubMed Abstract | Publisher Full Text
Ambros V: The functions of animal microRNAs.
Nature 2004, 431:350-355. PubMed Abstract | Publisher Full Text
Fazi F, Rosa A, Fatica A, Gelmetti V, De Marchis ML, Nervi C, Bozzoni I: A minicircuitry comprised of microRNA-223 and transcription factors NFI-A and C/EBPalpha regulates human granulopoiesis.
Cell 2005, 123:819-831. PubMed Abstract | Publisher Full Text
Shingara J, Keiger K, Shelton J, Laosinchai-Wolf W, Powers P, Conrad R, Brown D, Labourier E: An optimized isolation and labeling platform for accurate microRNA expression profiling.
Rna 2005, 11:1461-1470. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Liu B, Wang J, Chan KM, Tjia WM, Deng W, Guan X, Huang JD, Li KM, Chau PY, Chen DJ, et al.: Genomic instability in laminopathy-based premature aging.
Nature medicine 2005, 11:780-785. PubMed Abstract | Publisher Full Text
Palanisamy V, Sharma S, Deshpande A, Zhou H, Gimzewski J, Wong DT: Nanostructural and transcriptomic analyses of human saliva derived exosomes.
PLoS One 2010, 5:e8577. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
DeNardo DG, Barreto JB, Andreu P, Vasquez L, Tawfik D, Kolhatkar N, Coussens LM: CD4(+) T cells regulate pulmonary metastasis of mammary carcinomas by enhancing protumor properties of macrophages.
Cancer cell 2009, 16:91-102. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Gottardo F, Liu CG, Ferracin M, Calin GA, Fassan M, Bassi P, Sevignani C, Byrne D, Negrini M, Pagano F, et al.: Micro-RNA profiling in kidney and bladder cancers.
Urol Oncol 2007, 25:387-392. PubMed Abstract | Publisher Full Text
Xu J, Wu C, Che X, Wang L, Yu D, Zhang T, Huang L, Li H, Tan W, Wang C, Lin D: Circulating MicroRNAs, miR-21, miR-122, and miR-223, in patients with hepatocellular carcinoma or chronic hepatitis.
Johnnidis JB, Harris MH, Wheeler RT, Stehling-Sun S, Lam MH, Kirak O, Brummelkamp TR, Fleming MD, Camargo FD: Regulation of progenitor cell proliferation and granulocyte function by microRNA-223.
Nature 2008, 451:1125-1129. PubMed Abstract | Publisher Full Text
Vanpoucke G, Goossens S, De Craene B, Gilbert B, van Roy F, Berx G: GATA-4 and MEF2C transcription factors control the tissue-specific expression of the alphaT-catenin gene CTNNA3.
Nucleic Acids Res 2004, 32:4155-4165. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Janssens B, Goossens S, Staes K, Gilbert B, van Hengel J, Colpaert C, Bruyneel E, Mareel M, van Roy F: alphaT-catenin: a novel tissue-specific beta-catenin-binding protein mediating strong cell-cell adhesion.
J Cell Sci 2001, 114:3177-3188. PubMed Abstract | Publisher Full Text
Yan LX, Huang XF, Shao Q, Huang MY, Deng L, Wu QL, Zeng YX, Shao JY: MicroRNA miR-21 overexpression in human breast cancer is associated with advanced clinical stage, lymph node metastasis and patient poor prognosis.
Rna 2008, 14:2348-2360. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Taylor DD, Gercel-Taylor C: MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer.
Gynecol Oncol 2008, 110:13-21. PubMed Abstract | Publisher Full Text
Rosell R, Wei J, Taron M: Circulating MicroRNA Signatures of Tumor-Derived Exosomes for Early Diagnosis of Non-Small-Cell Lung Cancer.
Clin Lung Cancer 2009, 10:8-9. PubMed Abstract | Publisher Full Text
Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O'Briant KC, Allen A, et al.: Circulating microRNAs as stable blood-based markers for cancer detection.
Proc Natl Acad Sci USA 2008, 105:10513-10518. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Blanchard N, Lankar D, Faure F, Regnault A, Dumont C, Raposo G, Hivroz C: TCR activation of human T cells induces the production of exosomes bearing the TCR/CD3/zeta complex.
J Immunol 2002, 168:3235-3241. PubMed Abstract | Publisher Full Text
Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, Geuze HJ: B lymphocytes secrete antigen-presenting vesicles.
J Exp Med 1996, 183:1161-1172. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Thery C, Regnault A, Garin J, Wolfers J, Zitvogel L, Ricciardi-Castagnoli P, Raposo G, Amigorena S: Molecular characterization of dendritic cell-derived exosomes. Selective accumulation of the heat shock protein hsc73.
J Cell Biol 1999, 147:599-610. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Mears R, Craven RA, Hanrahan S, Totty N, Upton C, Young SL, Patel P, Selby PJ, Banks RE: Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.
Proteomics 2004, 4:4019-4031. PubMed Abstract | Publisher Full Text
O'Neill HC, Quah BJ: Exosomes secreted by bacterially infected macrophages are proinflammatory.
Sci Signal 2008, 1:pe8. PubMed Abstract | Publisher Full Text
Denzer K, Kleijmeer MJ, Heijnen HF, Stoorvogel W, Geuze HJ: Exosome: from internal vesicle of the multivesicular body to intercellular signaling device.
J Cell Sci 2000, 113(Pt 19):3365-3374. PubMed Abstract | Publisher Full Text
Huber V, Filipazzi P, Iero M, Fais S, Rivoltini L: More insights into the immunosuppressive potential of tumor exosomes.
J Transl Med 2008, 6:63. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Xiang X, Poliakov A, Liu C, Liu Y, Deng ZB, Wang J, Cheng Z, Shah SV, Wang GJ, Zhang L, et al.: Induction of myeloid-derived suppressor cells by tumor exosomes.
Int J Cancer 2009, 124:2621-2633. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Solinas G, Marchesi F, Garlanda C, Mantovani A, Allavena P: Inflammation-mediated promotion of invasion and metastasis.
Esser J, Gehrmann U, D'Alexandri FL, Hidalgo-Estevez AM, Wheelock CE, Scheynius A, Gabrielsson S, Radmark O: Exosomes from human macrophages and dendritic cells contain enzymes for leukotriene biosynthesis and promote granulocyte migration.
J Allergy Clin Immunol 126:1032-1040.
1040 e1031-1034
Wang HW, Joyce JA: Alternative activation of tumor-associated macrophages by IL-4: priming for protumoral functions.
Cell cycle 2010, 9:4824-4835. PubMed Abstract | Publisher Full Text
Loke P, Nair MG, Parkinson J, Guiliano D, Blaxter M, Allen JE: IL-4 dependent alternatively-activated macrophages have a distinctive in vivo gene expression phenotype.
BMC immunology 2002, 3:7. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text
Cao B, Guo Z, Zhu Y, Xu W: The potential role of PDGF, IGF-1, TGF-beta expression in idiopathic pulmonary fibrosis.
Chinese medical journal 2000, 113:776-782. PubMed Abstract | Publisher Full Text


text:
The environment
The company encourages measures which promote recycling and reduced energy consumption. It is the companys policy to:
- minimise the extent of the environmental impact, where reasonably practicable.
- conserve energy through minimising consumption and maximising efficiency.
- promote efficient purchasing which will both minimise waste and allow materials to be recycled
- adopt efficient waste-management strategies which reduce the amount of waste going to landfill or other disposal sites.
- seek to minimise any emissions or effluents which may cause environmental damage.
Over the past 12 months, the company has complemented its policy with several initiatives, including areas around energy-efficiency, recycling, ethical working and health and safety.
The company recognises that energy consumption is unavoidable, but understands that we have a responsibility for those resources which we use and that good environmental management is an essential part of being a responsible business.
In the last year, we have continued to work with Carbon Statement to measure each pubs carbon footprint. From March 2014, ISTA have introduced a weekly carbon emissions report for each pub, detailing the amount of carbon dioxide (CO2) emitted by each pub, through energy usage and waste disposal, as well as the CO2 emission reduction through recycling. We also comply with the governments Carbon Reduction Commitment (CRC) Energy Efficiency Scheme, which started in April 2010.
The company also aims to ensure that, when new pubs are developed or current ones upgraded, equipment and processes are introduced to minimise ongoing energy consumption.
Greenhouse gas (GHG) emissions
The table below show the companys annual CO2 emissions
|GHG Emissions
|GHG Emissions
|Scope 1
|Tonnes CO2e
|Scope 2
|Tonnes CO2e
|Tonnes CO2e / £m Revenue
- Conversation factors for electricity and gas are those published by the Department for Environment, Food and Rural Affairs
- Reported data is in respect of the year ended 31 March 2014 to align to the period under which carbon emissions are reported
- Refrigerant emissions from our pub are not reported as they are not material
Sustainability reduce, reuse, recycle
The company aims to reduce the amount of waste going to landfill and other disposal sites, through a combination of packaging reduction, reusing packaging and the recycling of waste products.
The company continues to work with suppliers to reduce packaging materials brought into pubs. This leads to fewer deliveries and reduces the number of road miles required.
Where possible, unwanted equipment is transferred among pubs by our distribution fleet, so that it can be used elsewhere.
The company has a dedicated supply chain for its food, bottled drinks and non-consumable products, so that material can be returned to the companys recycling operation, reducing the required number of road miles.
During the financial year 2013/14, the pubs segregated and returned 9,179 tonnes of waste to our distribution centre, an increase of 11% on the previous year. This included 49 tonnes of aluminium, 5,534 tonnes of cardboard and paper, 2,979 tonnes of cooking oil, 458 tonnes of plastic and 160 tonnes of steel.
A food waste recycling trial has commenced in 103 pubs.
Our supply chain
Roll cages, used to deliver products to our pubs, have been redesigned to reduce the noise created during deliveries.


text:
|Get 1 Year of unlimited Apress for $199
|Instant access to all available titles and new releases
Related Titles
- Full Description
Promptly growing demand for telecommunication services and information interchange has led to the fact that communication became one of the most dynamical branches of an infrastructure of a modern society. The book introduces to the bases of classical MDP theory; problems of a finding optimal САС in models are investigated and various problems of improvement of characteristics of traditional and multimedia wireless communication networks are considered together with both classical and new methods of theory MDP which allow defining optimal strategy of access in teletraffic systems. The book will be useful to specialists in the field of telecommunication systems and also to students and post-graduate students of corresponding specialties.
- Table of Contents
Table of Contents
- 1. Two
- Dimensional Traffic Models of Integrated Voice/Data Cellular Wireless Networks 2. Performance Analysis of Call Handling Processes in Buffered Cellular Wireless Networks 3. QoS Optimization Problems in a Cellular Wireless Networks 4. Models of Multi
- Rate Systems with Inelastic Calls 5. Models of Mixed Multi
- Rate Systems 6. Parametric Optimization Problems in a Multi
- Rate Systems 7. Markov Decision Processes (MDP) Approach in Optimization Problems for Multi
- Rate Systems
- Errata
Please Login to submit errata.No errata are currently published


text:
Center for Healthy Communities
CalFresh recipients impacted by the devastating fires in Northern California counties are eligible for "replacement" benefits from their counties.
Like our Facebook page!
Click here to like us and stay up to date with recent events and publications!
Recent NSPR News Coverage
Read about how Colusa City Schools Have Kids Eating Their Fruits And Veggies
Read about how North Valley Food Service Gets A Healthy, Tasty Start To The New Year
$350 EASY GAP Grant
California State University's Agriculture Research Institute has funded the CHC for its three-year Enhancing Agricultural Safety Year-Round.
Mindful Eating for Kids
The CHC has received a $149,890, two-year grant from the U.S. Department of Agriculture to study mindful eating among children and families.
CNAP is now CHC
The Center for Nutrition and Activity Promotion Center at Chico State is now the Center for Healthy Communities. It provides programs and services in 19 Northern California counties.
Support the CHC through Annie B's
Each $30 gift made to the CHC through the Annie B's campaign provides one month of services per classroom.


text:
NOVATO, Calif., Nov. 30 /PRNewswire-FirstCall/ -- BioMarin Pharmaceutical Inc. (Nasdaq: BMRN) announced today that the Food and Drug Administration (FDA) has granted orphan drug designation for 3,4-diaminopyridine (3,4-DAP), amifampridine phosphate, for the rare autoimmune disease Lambert Eaton Myasthenic Syndrome (LEMS). 3,4-DAP has previously received orphan drug designation in the E.U. Also, in October 2009, the Committee for Medicinal Products for Human Use of the European Medicines Evaluations Agency adopted a positive opinion recommending approval of amifampridine phosphate for LEMS. If approved by the European Commission, amifampridine phosphate will be the first approved treatment for LEMS, thereby conferring orphan drug protection and providing ten years of market exclusivity in Europe.
"With its experience in the development and commercialization of orphan drugs, BioMarin is well-positioned to seek U.S. registration of amifampridine phosphate for LEMS, a serious and debilitating autoimmune disease often associated with small cell lung cancer," said Jean-Jacques Bienaime, Chief Executive Officer of BioMarin. "We look forward to meeting with the FDA in early 2010 to determine the necessary regulatory path for amifampridine phosphate in the U.S. We are also preparing to launch the product in Europe in the first quarter of 2010, and will also evaluate the best development strategy for amifampridine phosphate in other indications in the U.S. and Europe."
About LEMS
Lambert Eaton Myasthenic Syndrome (LEMS) is a rare autoimmune disease with the primary symptoms of muscle weakness. Muscle weakness in LEMS is caused by autoantibodies to voltage gated calcium channels leading to a reduction in the amount of acetylcholine released from nerve terminals. The prevalence of LEMS is estimated at four
|SOURCE BioMarin Pharmaceutical Inc.
Copyright©2009 PR Newswire.
All rights reserved