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Five questions for the Milwaukee water winner
Last night at the City of Milwaukee's 163rd birthday party at Discovery World, Milwaukee resident Roy Henning won the Milwaukee Press Club's contest to name commemorative bottles of Milwaukee water.
His name, Brew City Blue, came with the throwback slogan of "the water that made the beer that made Milwaukee famous." "His" water was bottled and distributed to the hundreds of guests at last night's party.
Henning was recognized for his efforts last night, and this morning took a few moments to talk to us about his winning water.
OnMilwaukee.com: How did you find out about the contest?
Roy Henning: I heard about the contest during my daily reading of OnMilwaukee.com.
OMC: Where did the idea for the name and slogan come from? And, you didn't design the bottle, right? Just the concept?
RH: To be perfectly honest, I came up with both the name and the slogan in about two minutes while I was working ... don't tell my boss.
I always use "Brew City" as a login name and "Blue" ... well I guess that just makes sense. The slogan kind of just popped into my head and after I submitted it, I realized I sort of ripped off Schlitz.
I actually spoke with the person in charge of the contest last night and she said they had to send a letter to Schlitz to authorize the partial use of their slogan. I didn't design the bottle or the artwork, just the concept.
OMC: Was that your first Milwaukee birthday party? What did you think about it?
RH: This was my first Milwaukee birthday party and I was very impressed with everything. Our city really seems to be moving in the right direction. Bartolotta's had amazing food and Jim McCabe did a great job providing Milwaukee brewed beer from the Milwaukee Brewing Company and The Ale House.
I was also very impressed with Discovery World. I had never been inside before and would recommend to everyone to check it out. The view from the third floor hall (Pilot House) where the party was held was absolutely amazing; downtown to the north, the Summerfest grounds and the Hoan Bridge to the south and Lake Michigan to the east.
OMC: What did you win?
RH: I won $100 gift card from the Bartolotta Restaurant Group and all the City of Milwaukee water I can drink!
OMC: Any thoughts on Milwaukee's water future and opportunities?
RH: I didn't really attend the birthday party last night thinking I was going to learn something but I was amazed that Milwaukee has become a leader in water filtration systems in both design and manufacturing.
I've been reading a lot of national articles lately about water shortages throughout the country. Many states are now looking at the Great Lakes as a resource to be tapped into, and that is why this freshwater promotion concept Milwaukee has adopted is so important.
The Great Lakes are a precious natural resource that needs to be protected at all costs; and as residents of Milwaukee and the surrounding areas we need to be more conscious of conserving what we have.
exit_320 | Jan. 30, 2009 at 4:29 p.m. (report)
Whoever designed that bottle needs graphic design lessons -- great name, HORRIBLE bottle design.
|Rate this:
Vegetable Lasagna | Jan. 28, 2009 at 5:07 p.m. (report)
He kind of looks like Aaron Rodgers.
|Rate this:
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Food and Staples Retailing
Company Overview of M & M West Coast Produce, Inc.
Company Overview
M & M West Coast Produce, Inc. engages in the retail of food and beverages. The company was founded in 2006 and is based in Salinas, California.
18911 Portola Drive
Suite C
Salinas, CA 93908-1297
United States
Founded in 2006
Key Executives for M & M West Coast Produce, Inc.
M & M West Coast Produce, Inc. does not have any Key Executives recorded.
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subset: commoncrawl


text:
This is a social-networking legal first. A British high court awarded a man named Mathew Firsht 22,000 pounds (nearly $44,000) in damages from a fake profile and group about him on Facebook, according to a report in MSNBC. The group, called "Has Mathew Firsht lied to you?", and imposter profile reportedly were created by a former school friend. The profile contained "false claims about [Firsht's] sexuality, religion and political views, the Financial Times reports. According to MSNBC, "the information stayed on the site for 16 days until Firsht's brother spotted it. Firsht alerted Facebook staff who deleted the pages and told his lawyers they had been posted on the site from a computer at Raphael's home."
Briton wins social-site libel case
By Anne Collier on July 25, 2008 in NetFamilyNews
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Safer Internet Day 2105
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Dr. Kenneth J. Meigs Jr., DO9200 SE 91st Ave Ste 220
Happy Valley, OR 97086 (5.4 miles away)
Dr. Robert P. Barlow, MD12400 NW Cornell Rd
Portland, OR 97229 (9.9 miles away)
Dr. Ruth A. Fischer-Wright, MD4015 Mercantile Dr Ste 200
Lake Oswego, OR 97035 (2.5 miles away) – More Locations
Dr. Kara M. Kassay, MD4309 Oakridge Rd
Lake Oswego, OR 97035 (2.7 miles away)
Dr. Breanna L. Percell, MD18380 Willamette Dr Ste 202
West Linn, OR 97068 (2.2 miles away)
Dr. Tanya E. Carter, DO9385 SW Locust St
Tigard, OR 97223 (5.5 miles away)
Dr. Ryan G. Scott, MD18380 Willamette Dr Ste 202
West Linn, OR 97068 (2.2 miles away)
Dr. Joyce S. Endo, MD18380 Willamette Dr Ste 202
West Linn, OR 97068 (2.2 miles away)
Dr. Marion J. Darling, MD16679 Boones Ferry Rd Ste 105
Lake Oswego, OR 97035 (3.1 miles away)
Dr. William F. McBride, MD4475 SW Scholls Ferry Rd Ste 150
Portland, OR 97225 (6.1 miles away)
Dr. Rambod Rouhbakhsh, MD412 A Ave
Lake Oswego, OR 97034 (less than 1 mile away)
Dr. Sara K. Becker, MD3033 SE Monroe St
Milwaukie, OR 97222 (2.5 miles away) – More Locations
Dr. Robert S. Rath, MD14125 SW Farmington Rd
Beaverton, OR 97005 (8.8 miles away)
Dr. Oscar R. Polo, MD5050 NE Hoyt St Ste 362
Portland, OR 97213 (7.9 miles away)
Dr. Yves A. Lefranc, MD11211 SE Sunnyside Rd
Clackamas, OR 97015 (5.8 miles away)
Dr. Rebecca M. Cleeton, DO18040 SW Lower Boones Ferry Rd
Tigard, OR 97224 (4.5 miles away)
Dr. Emily J. Puukka Clark, MD10330 SE 32nd Ave Ste 205
Portland, OR 97222 (2.7 miles away)
Dr. Anong Y. Moua, MD13021 SE River Rd Ste 3000
Portland, OR 97222 (1.2 miles away)
Dr. Katherine L. Fisher, DO9200 SE 91st Ave Ste 300
Portland, OR 97086 (5.4 miles away)
Dr. Carl M. Erickson, DO7215 SE Milwaukie Ave
Portland, OR 97202 (3.7 miles away)
* Please verify when scheduling an appointment. If you are this provider and would like to confirm what you practice, please update your free profile.
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Procedures Commonly Performed By Family Practitioners
- Pelvic Exam & Pap Smear
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- X-Ray
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text:
More Videos...
Get your Triple-X apparel, koozies, decals, graphics, DVD's & more at
Randy Johns has worked HARD all off-season to convert his classic stepside
Chevy from big block gasser to 12v Cummins with VERY high Boost in time for River Rats and Trucks Gone
Wild at Mudfest....and he got it done! Initially it was spooling to upwards
of 100lbs of Boost, but with some
tuning and fuel regulation, it's making approximately 66lbs of Boost presently for everyday use.
Along the way, he set out to complete a late 70's model Chev with a Cummins
swap as well. This was to be a 14th birthday surprise for his son
Gunnar...and one heck of a great birthday gift it was!
Sabine River Rats was the first test ride for both trucks, and they each
did really well. On this hole towards the back, Gunnar really showed what
kinda driving skills he had and let that new truck show what it's got! Way
to go Gunnar!
Sabine River Rats near Marshall, TX - Good Friday weekend - 4/18/14.
Check out Triple-X Motorsports & Outdoors on Facebook for updates on
events, action photos, videos, giveaways and lots of other cool innerwebs
Nyce1sClips - Anthony Apuzzo's 9 Second 1972 Dodge Colt @ Import Face Off New England Dragway 2012
Checkout Anthony Apuzzo's 9 Second 1972 Dodge Colt at New England Dragway
during a recent IFO event! Congrats to Anthony on his new personal best.
Checkout Nyce1s @ http://www.facebook.com/nyce1s !!
Check us out for more videos @ http://www.youtube/nyce1s !!!
SRT-4 Dodge Omni swap pt1 GLH SRT4 Neon
2.4 Dodge Neon SRT4 engine swap into an L body Omni GLH GLHS horizon
2.6L SRT-4 Dodge Neon 10 second drag race, Ed Bradley 10.36 @ 138mph
Ed Bradley made the trip up from FL to New England Dragway in Epping, NH to
run his low 10 second SRT-4 Neon at Import Face Off.
Making multiple 10 second passes.
New England Dragway - Epping, NH.
Import Face Off - May 20, 2012
2003 SRT-4: DCR 2.6 Stroker
500hp 1989 Plymouth Colt Dyno & Driving
http://www.1320video.com/ turbo Colt
goes for a drive, gets pulled over, makes some pulls, then goes to the Dyno to make over 500whp
on a Mustang Dyno!
57 trim SRT4
stage 4 srt4 with 57trim vs some cars civic at end is fast as fuck. all
races done on 15 lbs
Twin Turbo Supra vs SRT-4
Twin turbo Supra racing a Stage 3 SRT-4
10.00 740whp 4g63 Colt sleeper!
This 740whp Colt runs multiple 10.0 passes at the 2013 DSM Shootout!
Sponsored by FORCED PERFORMANCE
Dressed in Drag - A 1978 (or 1979) Mitsubishi Mirage / Plymouth Champ / Dodge Colt
Car-spotting in Japan: http://wasabicars.com
Wasabicars T-shirts: http://wasabicars.spreadshirt.com/
Music Credit:
Created by Dylan Benson, and used with permission and much appreciation.
Dylan Benson on Youtube: http://www.youtube.com/user/marineUSA93
Dylan Benson on the web: http://www.dbproductioncompany.webs.com/
Site: http://wasabicars.com
Facebook: http://www.facebook.com/WasabiCars
Shop: http://wasabicars.spreadshirt.com/
Srt - 4 neon versus vs turbo t bird and a few cobra terminators 600 plus wheel horsepower
Srt - 4 neon versus vs turbo t bird and
a few cobra terminators 600 plus wheel horsepower
10 Second Suzuki Samurai Drag Race! Small Block Chevy POWER! - "EL TRAUMA"
This beast ran in the 10.0 index class at MIR World Cup Finals 2012 and put
on a great show! What a fun car to watch.
World's fastest suzuki samurai?
"EL TRAUMA" runs a 10.20 !
Edward Rivera - from FL.
Maryland International Raceway
Budd's Creek, MD
1972 Dodge Colt
In this episode, we're leaving the confines of the turbo Garage to check out my buddy Nate's
wonderful and rare 1972 Dodge Colt, also known as the Mitsubishi Colt
Thank you for watching and for more fun, visit my Facebook page at:
For more about the cars that you see in and around the turbo Garage, visit www.turbomirage.com
700 + HP SRT4 Neon
Big tubo SRT4 Neon running S374 turbo,
8200 RPM redline, tuned with DiabloSport CMR software, built motor, cyl
head, custom camshafts, blah blah blah... email me if you want a list of
mods or want us to build you a car like this!
9 Second Porsche Drag Race - Small Block Ford v8 Power! 9.44 @ 153mph
Damn, a cowl hood on a Prosche looks cool.
Smooth run out of this clean porsche powered by a small block ford.
Import Face Off
New England Dragway
Epping, NH
CFM Performance 4G63 Powered AWD Dodge Colt
CFM Performance sponsored 4G63 Powered AWD Dodge Colt
Dodge Ram Crushes GT-R in 1/4-Mile Pass
Drag Racing Down Under — 1500HP VW
Wheelstand Wednesday — Compilation Video
All Show No Go Blown/Turbocharged 1969 Camaro
Which car is faster?
Similar 1/4 mile timeslips to browse:
1974 Dodge Colt GT: 8.480 @ 164.170
Chuck O, Engine: 502 wedge, Supercharger: no Turbos: no Tires: phoenix
2006 Mazda 3 : 10.150 @ 134.000
Vincent Tiaga, Engine: Mazda I4, Supercharger: na Turbos: na Tires: M&H
1991 Dodge Colt : 10.550 @ 140.000
Brad Brooks, Engine: 4G63, Turbos: 67mm Tires: MH 25x8.7x15
1989 Dodge Colt 200 GT: 10.892 @ 141.200
steph, Engine: 2.0 dohc, Turbos: twin 14g
1974 Dodge Colt GT Turbo & Nitrous: 10.980 @ 122.340
cody bishop, Engine: 340, Turbos: yes Tires: cobra
2005 Mazda 3 SP23: 11.659 @ 128.790
Kurt Scott, Engine: 2.3L, Supercharger: N/A Turbos: GT3582
2008 Mazda 3 mazdaspeed3: 11.682 @ 117.170
NJSPEED3, Engine: 2.3L mzr/disi , Turbos: T3 HTA3586 .82ar
2008 Mazda 3 MPS: 12.350 @ 119.830
RE-ACTIONENGSPEED3, Engine: mzr, Turbos: k4 hybrid
2009 Mazda 3 Speed3: 12.737 @ 108.510
Daniel Lalonde, Engine: mzrdisi 2,3, Turbos: k04 Tires: Toyo RA1 235/40/17@16psi
2008 Mazda 3 Mazdaspeed 3: 12.793 @ 106.900
driver311, Tires: m@h 24.5x8.5x15 inch slicks
2007 Mazda 3 Mazdaspeed: 12.847 @ 111.635
Dave, Engine: 2.3L MRZ DISI TURBO, Turbos: stock Tires: pontenza
2007 Mazda 3 Mazdaspeed 3: 12.906 @ 111.140
Laloosh, Tires: STREET
1989 Dodge Colt GT: 12.945 @ 112.470
Justin Crisswell, Engine: 4G63, Supercharger: n/a Turbos: 16G w/ 6cm exhaust Tires: All Weather
2013 Mazda 3 MAZDASPEED 3: 12.988 @ 107.850
Michael Vincent Sarricchio, Engine: MZR DISI, Turbos: STOCK K04 Tires: FACTORY TIRE
2007 Mazda 3 Speed3: 13.190 @ 110.670
Kyle Hasenheyer, Engine: Stock engine, Turbos: Stock K04 turbo Tires: Venezia Crusade HP
2010 Mazda 3 Mazdaspeed 3: 13.264 @ 108.230
Jimmy, Engine: Stock, Supercharger: NA Turbos: Stock Tires: G-force T/A KDW 2's
2008 Mazda 3 Mazdaspeed3 Grand Touring: 13.330 @ 107.980
James Rondepierre, Engine: 2.3L Direct Injected, Turbocharged, Intercooled, Supercharger: N/A Turbos: K04 STOCK Tires: HANKOOK VENTUS V12 EVO (STREET TIRES) (25PSI)
2007 Mazda 3 MazdaSpeed 3: 13.394 @ 104.330
2010 Mazda 3 Mazdaspeed 3: 13.485 @ 108.840
Michael Loopy, Engine: 2.3 litre, Turbos: Stock K04
2007 Mazda 3 Mazdaspeed3 GT: 13.488 @ 103.220
Matt, Engine: 2.3 MZR turbo, Supercharger: n/a Turbos: stock Tires: 245/50/16 Nitto NT555 Extreme Drag's for track onl
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text:
Wal-Mart will scale back free home shipping in Canada
The world's largest retailer will end free shipping for online orders under $50 Canadian starting April 2.
Wal-Mart will scale back free home shipping in Canada
The world's largest retailer will end free shipping for online orders under $50 Canadian starting April 2.
Color plays a key role in web design and closing the sale
The design element of color can evoke feelings, action—and help pave the way to purchase.
Sign up for IRNewsLink and get daily reports delivery to your inbox. Sign Up Now
Sponsored by 2013 Social Media 300
Before the chain's bankruptcy it was No. 154 in the Internet Retailer Top 500.
Linens 'N Things, once a major home furnishings brand online and offline that now survives only as an e-commerce site, is getting a new owner.
Hilco Global, Gordon Brothers Group and Infinity Group have sold the brand assets of Linens 'N Things to Galaxy Brand Holdings after about five years of ownership. The sale includes LNT.com, the e-commerce site of the former chain retailer of home goods and home décor. The parties did not disclose the terms of the sale.
Hilco and the others purchased Linens 'N Things in February 2009 after the retail chain declared bankruptcy in 2008. They relaunched LNT.com in spring 2009 and the brand has existed as a web-only retailer since.
Galaxy Brand Holdings is a brand development company majority-owned since May by private equity and asset management firm The Carlyle Group. Galaxy's other brand holdings include shoe brands Avia and Nevados.
"We plan to leverage [Linens 'N Things'] heritage to connect with consumers by offering Linens 'N Things branded products, and by re-establishing the brand as a premier home destination both in-store and online," says Abe M. Hidary, vice president of corporate development at Galaxy Brand Holdings.
Before its 2008 bankruptcy, Linens 'N Things was No. 154 in the Internet Retailer Top 500 Guide, with 2007 web sales of $75 million, according to an Internet Retailer estimate.
Training, signage and motivation are all critical elements to making in-store pickup work. If done ...
Posted 03/13/15
With holiday promotions starting as early as September, now is the time to assess the ...
Posted 03/12/15
If there's something you've bought offline that you weren't able to buy online, we're probably thinking about that."


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Mass Protest against Monopolies Sweep Bulgaria's Capital
Tens of thousands hit the streets in Bulgaria's capital Sofia on Sunday against the excesses of power and heating utilities in a continuation of recent wave of protests over high electricity and heating bills.
The rally was staged at Bulgaria's Economy Ministry, with participating protesters of all ages, including mothers with young kid.
The protesters were chanting "mafia" under the windows of the ministry building blockading traffic on the Rakosvki Str.
The demonstrators demand the nationalization of Bulgaria's three power utilities – CEZ, EVN, and Energo-Pro, with the National Electric Company NEK assuming their responsibilities, the publication of all secret contracts in Bulgaria's energy sector, holding those who signed secret energy contracts responsible, abolishing NEK's obligation to buy out any produced electricity, as well as individual contracts with Sofia heating utility Toplofikatsiya that can be terminated on a month's notice.
- » French Officials Deny Video Footage Found at Germanwings Plane Crash Site
- » Bulgarian Cabinet Allocates BGN 14 M for Natural Disaster Relief
- » Minor Earthquake Hits Southern Bulgaria
- » Sofia Declared Best Bulgarian City to Live in for 2014
- » Another Six Pelicans Die at Bulgaria's Srebarna Nature Reserve
- » Bulgarian National TV Begins Preparations for Hosting Junior Eurovision


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Rivlin urges Netanyahu to expedite forming coalition
Netanyahu on Iran deal: Israel not 'burying head,' will do what is needed to secure its future
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Israel has second-lowest abortion rate in Europe, continues to drop
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US model adapted by the National Poison Control Center 'boosts saving of lives and money'
First-ever effort between hospital and taxi company to ensure newborns are taken home safely
Study: Israeli patents in US jump 21%
It's Passover cleanup time in Israel and e-waste recycling is on the rise
American-Israeli team secures US gov't grant to build Burundi's first solar field
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Damian Marley to serenade Rishon
Cinematic reality
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Play it once again, Sam
Enormous Belgium showdown for Israel
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Israel increases budget for 2016 Rio Olympics
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In 'major victory to Arab residents,' 2,200 homes approved in east Jerusalem
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PLO member accuses Israel of war crimes over Har Homa
Sources: US may push renewed discussion of the Saudi peace initiative
Israeli Arabs mark 39th Land Day with protests
Official report: Dutch royal family owns stolen Nazi painting
Anne Frank may have died earlier than previously believed, new research shows
'Biden told US Jews that Israel - not America - guaranteed their security'
Virginia State Bar cancels Jerusalem seminar, cites 'discriminatory' border policies
Changed date of death shows Janusz Korczak was killed in Treblinka
Head of Gaza reconstruction committee quits Palestinian unity government
US says ready to pursue Iran nuclear talks past midnight deadline if needed
West hopes for a breakthrough as deadline arrives with Iran
Can the Americans walk away from talks with Iran?
As nuclear talks near deadline, Khamenei aide warns of West's 'deceptive tactics'
No to a UN-imposed settlement
Whatever Iran gets in nuclear talks, Saudi Arabia wants
Real differences
March 31: Dealing with Iran
The Green Knesset: A vision of Tikkun Olam
The blood libel Haggada
The turning point
Travel tech start-ups
On wings of carbon
Veterans: On target
Fusing modern and traditional in the kitchen
Book Review: Perceptive Haggada
Hot off the Arab press
Let's cooperate
Shabbat screenings
Passover perspective
On the field and in court
Happily Eva After: Times, they are a-interestin'
Music and the mamaloshen
What's the catch?
Kayaking for gold on the Kinneret
Why Tunisia?
Palestine – words matter but their meaning matters more
A nuclear deal with Iran – would it stick?
When words matter and when they don't
'מְלָגוֹת 'צוּק אֵיתָן
עוֹלִים חֲדָשִים
כּוּלָם רוֹצִים לִחְיוֹת
יִשְׂרָאֵל בְּמַבָּט פָּנוֹרָמִי
Getting a young start
A culinary festival starring the women next door
Global girl power
Apartheid South Africa: Israel edition
Sa'ar says national unity government is 'still on the table'
Israeli parties prepare legislative demands for Netanyahu's new coalition
With full term possible, Netanyahu may outlast Ben-Gurion (and Obama)
Arab sector turnout for recent elections reached 63.5%, polling data shows
Joubran: Polls have to include parties that don't pass threshold
'The Resistance' against Pope Francis
Israeli doctors save life of Iraqi Christian toddler who fled ISIS
Christian Friends of Magen David Adom donate ambulance in Jerusalem
Christians celebrate Palm Sunday in the Holy Land
Pope Francis pays tribute to Christians persecuted by ISIS during Palm Sunday mass
Green Leadership Conference in Hamalachim-Shahariya Forest
Scenic Lookout in Memory of Lt-Gen. Amnon Lipkin Shahak
Following in the Footsteps of Meir Har-Zion
Celebrating the 100th Birthday Anniversary of Sculptor Ahiam Shoshany
French Students Get to Know the Real Israel
Serbian PM Plants a Tree in the Grove of Nations
Sharing and Learning - German Forestry Students Intern with KKL-JNF
Israel and Finland Connect through the Environment
New Bio-filters Purify Surface Runoff Water for Reuse
Future Tour Guides Discover the Negev with KKL-JNF
The Shamir Drilling Project
Graduation Ceremony for Rosh HaAyin's Volunteer Firefighters
From Sunny Israel to Cold Canada
Walking for Water with KKL France in the Mountains of Eilat
JNF Ontario Community Mission Tours the Western Negev
Celebrating 50 Years of German-Israeli Diplomatic Relations
The Negev Conference on Agricultural Research and Development
Arava Open Day: Food Security for Israel and the Entire World
Living Green in Israel: the Contest and the Winners
Addressing Climate Change Conference
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Outdoor Fitness Israel: An Innovative Fitness Program Developed By UK Olim
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Starting Your Own Business in Israel: Real Life Examples
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How Women Can Have it All in Israel
The Eisenberg's Long Journey Home
Why Retirees are Thriving in Israel
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Make yourself at home… in Israel
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Diplomacy & Politics
07/22/2012 04:29
Netanyahu 370.
(photo credit:Moshe Milner/GPO)
Prime Minister Binyamin Netanyahu is scheduled to appear on CBS and Fox news programs Sunday to link Iran to last week's terrorist attack in Burgas and push home the point that if this is how Tehran acts without nuclear weapons, imagine what its behavior will be with them.The interviews are part of a stepped-up diplomatic campaign following the Bulgarian attacks to get the world not only to condemn Iran for the attacks, but also to take measures against Iran for its support and involvement in international terrorism.According to Netanyahu's spokesman Mark Regev, the Iranian government's denial of involvement in terrorism, including in the attack in Burgas Wednesday that killed five Israelis and a Bulgarian bus driver, "simply have no credence whatsoever."Regev said that a terrorist involved in the attack on the wife of an Israeli diplomat in New Delhi in February was first recruited by the Iranian embassy. He also said that three Iranian terrorists were arrested in Thailand for an attack that went awry there in February, and two other Iranians were arrested last month with explosives in Kenya.In addition, operatives of Hezbollah, which he dubbed Iran's terrorist "sub-contractor," have been arrested both in Cyprus and Thailand. Over the last year Iran has been behind some 20 terrorist initiatives, he saidOne diplomatic official said Israel was in "conversations at both working levels and the highest levels" with governments around the world to share the facts it has regarding Iran and Hezbollah's involvement both in the Bulgaria attack and in the others over the last year.Efforts to get the international community to take action against Iran for its sponsorship of terrorism picked up support from The Washington Post's editorial page Saturday, when the paper wrote an editorial headlined "Holding Iran accountable for terrorist attacks.""The bombing of a bus in Bulgaria filled with Israeli tourists on Wednesday underlines the grim fact that Iran is waging a war of terrorism. Using the territory of countries across the world, working sometimes through proxies like Lebanon's Hezbollah and sometimes with its own forces, Tehran has been intentionally targeting not just diplomats of enemies such as Israel and Saudi Arabia but also civilians," the paper wrote."If Iran suffers no consequences from its acts of terrorism, they will continue," the paper continued. "The Security Council should review the abundant evidence of involvement by the Revolutionary Guard and Hezbollah in this year's attacks and punish both those groups as well as the Iranian government with sanctions."
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What happens when psychiatric hospitals disappear
Hurricane Irene wiped out Vermont's last-standing psychiatric facility in 2011, making the state the only one in the country that now goes without any government-operated psychiatric beds. Bloomberg's Tom Moroney looks at what that means on the ground:
The number of patients held in Vermont emergency rooms for want of a bed has spiked. This year through April, 49 mentally ill patients were held for as long as two days, up from 18 in the same period last year, according to the state Department of Mental Health.
When the state hospital was open, the private Fletcher Allen facility typically turned away about 15 mentally-ill patients a month, said [Robert] Pierattini, the chief psychiatrist [at the private hospital in Burlington]. Just after the closing, that number hit 40 a month. It peaked at 130 early this year, and has since returned to 40 or so this spring, he said.
Some patients end up handcuffed to hospital beds, Moroney reports, for as long as two days as they wait for a transfer to a psychiatric facility. The changes aren't unique to Vermont; During the recession, 3,144 psychiatric beds were eliminated across the country in 2009. Those funding decisions have consequences: One 2009 study that looked at the elimination of psychiatric beds during the 1980s in California found the elimination of one public bed per 100,000 people was associated with an increase of 0.25 suicide each year.
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Good news, you can find the song "love runs out" on the Now that's what I call music 52 disc.
This album does not include the very popular song "Love Runs Out".
It is like they took off the song off the album to generate more money on online sales.
Only the album available online includes the song "Love Runs Out"
It took me a few listens before I really liked this cd. Now I think it's great
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The UFC 151 PPV event that was scheduled for September 1st has caused a huge tidal wave of changes going forward in regards to fights being moved to other events. The UFC has already announced several of them via the official UFC Twitter account and I'm sure there's more to come. So below you'll find a complete list of the changes thus far and this post will be updated as information comes in. Read More »
McGregor says he "bent Aldo over"
Remember that slap incident between Jose Aldo and Conor McGregor? The description has changed from McGregor slapping Aldo's back, to his neck, to his backside, and now McGregor says he straight up "bent him over". This incident happened during the UFC 189 championship world-tour's London stop and here's Conor's official story… "I heard I slapped him …
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We reported eary this morning that Vitor Belfort would be fighting Jon Jones in September for the UFC light heavyweight title instead of Lyoto Machida. Machida stated that he needed more time in preparation. Read More »
Lyoto Machida out; now it's Jon Jones vs. Vitor Belfort on Sept. 22
Current UFC light heavyweight champion Jon Jones will now face former light heavyweight champion Vitor Belfort at UFC 152 on Sept. 22 in Toronto, Canada. Read More »
Bellator 73 weigh-in results
The Bellator 73 weigh-ins have been completed from Tunica, Mississippi in preparation for Bellator's Friday night event at Harrah's Tunica, which features the Light Heavyweight Tournament Finals, as well as the Bantamweight Tournament Finals. Read More »
Pro MMA Now Radio welcomes Joseph Benavidez, Chris Weidman, Nate Landwehr and Josh Ward tonight at 10 p.m. ET
Pro MMA Now Radio returns tonight at 10 p.m. ET with UFC flyweight contender Joseph Benavidez and XFC 20's Josh Ward and breaking UFC news. Read More »
UFC 151:Dan Henderson is shocked that Jon Jones wouldn't step up and fight
We reported earlier that Dan Henderson had to pull out of his scheduled title fight against Jon Jones at UFC 151 due to a knee injury. We now know that Jon Jones will now face Lyoto Machida on September 22nd. Read More »
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UFC 151 cancelled: Jones to fight Machida on Sept. 22; White rips Jones camp (updated)
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Three bouts added to UFC on FX 5 in Minnesota
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The selection for this month is Londoners : the days and nights of London now -- as told by those who love it, hate it, live it, left it, and long for it. (2011), by Craig Taylor.
The West Portal Book Club meets the 3rd Thursday of every month from 2:00 pm until 3:30 pm.
Our reading choices alternate between fiction and nonfiction. New members are always welcome. Want to know what we're reading next? Call 355-2886
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The search found 321 results in 0.186 seconds.
Under Republican leadership, a Senate committee on high-skill guest worker visas focuses on outsourcing and job losses, not the need for more high-skill workers.
If ratified, the agreement will raise pay, improve health care coverage, subsidize health insurance for family members, and provide tax-free funding for child care.
As oil prices drop, a once red-hot employment market cools.
Research shows that many members of groups underrepresented in academic science seek careers that express values beyond pure research.
As the economy recovers, unemployment rates are falling but most salaries are falling, too.
As a training-reform effort by four scientific leaders slows, science trainees may already be voting with their feet.
French geneticist Jérôme Lejeune may have missed out on the Nobel Prize, but now he's nominated for an even higher honor.
A reanalysis of an oft-repeated claim highlights the need for better understanding of job creation.
In Australia, too, prognostications of imminent science-related workforce shortages are unreliable.
As members of Congress renew the push to raise high-skill immigration quotas, a report predicts a dramatic fall in the wages of new computer science graduates.
An analysis of a 1960s Italian experiment shows that STEM-workforce expansion can have unintended consequences.
The National Academies' new report details why doing a postdoc is generally a bad idea.
The former Princeton University president has been fighting for the interests of early-career scientists for more than 20 years.
By the end of the Ph.D., women from underrepresented groups were far more attracted to nonresearch careers.
NSF's annual report on new doctorate recipients contains mostly bad news for early-career scientists.
For those who follow postdoc issues, the new National Academies proposals sound familiar. But maybe things will turn out differently this time.
The realities of women's careers in academic science have changed. The discussion about them should change, too.
A postdoc-organized conference in Boston yields a consensus statement on "what the postdocs are worried about."
A federal judge says that tech workers can challenge a Bush-era immigration order that allows foreign STEM students to work an extra 19 months.
An immigration expert says the executive action will hurt U.S. tech workers, but for scientists the impact seems benign.
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Gastric cancer is the fourth most common cancer in the world and the second most prevalent cause of cancer related death. The development of gastric cancer is mainly associated with H. Pylori infection leading to a focus in pathology studies on bacterial and environmental factors, and to a lesser extent on the mechanistic development of the tumour. MicroRNAs are small non-coding RNA molecules involved in post-transcriptional gene regulation. They are found to regulate genes involved in diverse biological functions and alterations in microRNA expression have been linked to the pathogenesis of many malignancies. The current study is focused on identifying microRNAs involved in gastric carcinogenesis and to explore their mechanistic relevance by characterizing their targets.
Invitrogen NCode miRNA microarrays identified miR-449 to be decreased in 1-year-old Gastrin KO mice and in H. Pylori infected gastric tissues compared to tissues from wild type animals. Growth rate of gastric cell lines over-expressing miR-449 was inhibited by 60% compared to controls. FACS cell cycle analysis of miR-449 over-expressing cells showed a significant increase in the sub-G1 fraction indicative of apoptosis. ß-Gal assays indicated a senescent phenotype of gastric cell lines over-expressing miR-449. Affymetrix 133v2 arrays identified GMNN, MET, CCNE2, SIRT1 and CDK6 as miR-449 targets. Luciferase assays were used to confirm GMNN, MET, CCNE2 and SIRT1 as direct targets. We also show that miR-449 over-expression activated p53 and its downstream target p21 as well as the apoptosis markers cleaved CASP3 and PARP. Importantly, qPCR analyses showed a loss of miR-449 expression in human clinical gastric tumours compared to normal tissues.
In this study, we document a diminished expression of miR-449 in Gastrin KO mice and further confirmed its loss in human gastric tumours. We investigated the function of miR-449 by identifying its direct targets. Furthermore we show that miR-449 induces senescence and apoptosis by activating the p53 pathway.
Gastric cancer is among the five most common cancers in the world and the second most prevalent cause of cancer-related deaths [1]. It is mainly, but not exclusively, caused by H. Pylori infection [2] as not all of H. Pylori infected persons develop tumours [3]. Other factors involved in the development of gastric cancer include the degree and type of the inflammatory response [2] as well as the levels of the Gastrin hormone [4,5]. Several studies have shown that both hypergastrinemia [6,7] and the lack of Gastrin [5] contribute to the pathogenesis of gastric cancer. Achlorhydria is a common feature of mouse models prone to developing metaplasia and cancer [6,8,9]. Gastrin knockout mice are achlorhydric [10], favouring a bacterial gastric overgrowth [11,12], and chronic bacterial gastric infections lead to gastric metaplasia which may progress into gastric cancer [6,12].
Since their discovery microRNAs, have been found implicated in a very wide range of normal and pathological processes [13]. MicroRNAs exert their regulatory functions posttranscriptionally by binding to partly complementary sequence motifs predominantly in the 3' UTR of target mRNAs resulting in mRNA destabilization and translational repression [14]. From a biological point of view, microRNAs are challenging objects to study as they regulate cohorts of target genes, which are not readily identified. From a therapeutical point of view, microRNAs are highly interesting as several studies have demonstrated the power of microRNAs as biomarkers and initial preclinical studies have established that microRNAs may be therapeutically targeted in vivo [15].
Profiling studies have evidenced microRNA deregulation in a broad spectrum of diseases including all major cancers [16]. MicroRNAs likely affect tumourigenic processes at two levels. Firstly, several studies have established pro-oncogenic or tumour-suppressive roles of individual microRNAs firmly linking these to cancer etiology as exemplified by miR-155, miR-10b and miR-21 [17-19]. Secondly, the microRNA regulatory system per se appears to have tumour suppressive functions as genetic ablation of key microRNA biogenesis factors, such as Dicer, strongly increase cancer susceptibility [20] and loss of function mutations have been identified in important microRNAs processing factors in human tumours [21-23].
In this study, we address the importance of microRNAs in gastric cancer taking advantage of the Gastrin knockout mouse model and H. pylori infection of wild type mice. We identify miR-449 as significantly down-regulated or lost in mouse models of gastric cancer as well as in primary human gastric tumours. Identification of mRNA targets reveals that this microRNA likely exerts tumour suppressive functions through the concerted regulation of a cohort of cancer-associated cell-cycle regulators including MET, GMNN, CCNE2, SIRT1, and HDAC1.
Three different age groups (12-16 weeks, 1 year or 1½ years) of wild type (wt) or Gastrin knockout (KO) mice were used. All mice were on a mixed 129/SvJ, C57BL/6J background, backcrossed at least four times to C57BL/6J [12]. The mice were kept under specific pathogen-free conditions and monitored according to the Federation of European Laboratory Animal Science Associations recommendation [24] with 12 h light, 12 h dark cycles.
H. pylori infection
C57BL6/J mice (n = 10) were inoculated with a non-mouse-adapted clone of H. Pylori strain 67:21, originally isolated from an antral biopsy obtained from a Swedish female with gastric ulcer. The strain is VacA+ and contains the entire Cag pathogenicity island (PAI) with genetic stability in the Cag PAI [25]. The mice were inoculated every second day (three times) during a 5-day period. DNA was extracted and analyzed for the presence of helicobacter species using a semi-nested polymerase chain reaction-denaturing gradient gel electrophoresis assay, specific for the genus helicobacter, as described previously [26]. A matched group of uninfected C57BL6/J mice were used as controls.
The stomachs of all mice were dissected into fundus and antrum prior to RNA extraction. All animal experiments were approved by the Danish Animal Welfare Committee (2005/562-40) and the Danish Forest and Nature Agency (20010077355/6).
Mice antrum sections
Mice were sacrificed by cervical dislocation. The antrum was removed, washed gently in ice-cold PBS, frozen in liquid nitrogen and stored at -80°C until RNA extraction.
Clinical samples analyses
Biopsies from gastric cancer and the adjacent normal tissues were obtained from patients undergoing surgery for gastric cancer at the Department of Gastrointestinal Surgery, Rigshospitalet. The inclusion took place in July to December 2008 and all patients provided signed, informed consent (Ethical committee approval H-B-2008-049) and Danish Data Protection Agency (2008-41-2138). The biopsies were placed in RNAlater (Ambion) in the operating room and subsequently frozen at -80°C until RNA extraction.
RNA extraction and qPCR analyses
RNA was extracted using TRIzol (Invitrogen) according to manufacturer. miRNA expression profile was assessed using Taqman miRNA assays (Applied biosystems) for hsa/mmu-miR-449a and b, hsa/mmu-miR-34a, b and c and rnu44 or hsa/mmu-miR-191. Primer sequences for Affymetrix targets validation are listed in additional file 1, table S1.
Additional file 1. Figure S1 - miR-449 is down-regulated in Gastrin knock out mice compared to wild type. qPCR analysis of miR-449 expression in Gastrin knock out gastric tissues compared to relative expression in wild type gastric tissues, miR-449 is significantly down-regulated (p = 0.04) in Gastrin knock out tissues compared to wild types. Figure S2 - miR-449 inhibits cell proliferation in human gastric cancer cell line MKN74. Visual inspection of human gastric cancer cell line (MKN74) upon miR-449 re-introduction (lower panel) showing a decrease in cell proliferation as well as a senescent like phenotype compared to scrambled RNA control (upper panel) Figure S3 - miR-449 induces cell death in human gastric cancer cell line MKN74. FACS cell cycle analysis of MKN74 cell line upon miR-449 re-introduction (right histogram) showing an increase in the sub-G1 fraction indicative of cell death compared to scrambled RNA control (left histogram), table showing percentage of cell accumulation in G1 fraction 48 hours post miR-449 re-introduction followed by cell shift to the sub-G1 fraction 72 hours post transfection compared to RNA scrambled control. Figure S4 - miR-449b and miR-34a induce highly correlated expression changes. A - Chart showing significant down-regulation (p < 1.2e-70) of mRNAs with predicted miR-449 seed match in their 3'UTR (red line) compared to mRNAs lacking the seed match (black line). B - Chart showing high correlation of expression changes upon re-introduction of miR-449b or miR-34a into SNU638 cells with a Pearson's correlation coefficient of r = 0.94, p = 0. C - Word analysis showing shared miR-449b/34a seed site correlating with gene down-regulation. Figure S5 - miR-449 expression is p53 independent. A -Western blot showing p53 induction in HCT116 wild types and p53 knockouts using 5FU. B - qPCR analyses of miR-449a and miR-449b post p53 induction. No significant change is observed. Table S1 - primer sequences. Table S2 - list of genes deregulated upon miR-449 re-introduction. Affymetrix top down-regulated genes upon miR-449 re-introduction into SNU638 cells.
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Cell culture
SNU638 and MKN74 were grown in RPMI-1640 (Gibco) with 10% FBS (Hyclone), 100U/ml penicillin and 100 μg/ml streptomycin (Invitrogen) and incubated at 37°C in 5% CO2. HCT116 cells (wt and p53-/- were grown in McCoy's 5A (Gibco) with 10% FBS (Hyclone), and 100U/ml penicillin and 100 μg/ml streptomycin (Invitrogen) and incubated at 37°C in 5% CO2. HEK293 and MEF cells (wt and p53-/-) were grown in DMEM (Gibco) with 10%FBS (Hyclone), 100U/ml penicillin and 100 μg/ml streptomycin (Invitrogen) and incubated at 37°C with 5% CO2.
miRNA precursors and siRNA
miRNA precursors were purchased from Ambion, hsa-miR-449a (PM11521), hsa-miR-449b (PM11127) and hsa-miR-34a (PM11030).
Cell growth analyses
SNU638 cells were seeded in 24-well plates and transfected the following day with 50nM miRNA duplex or siRNA using Lipofectamine 2000 (Invitrogen). Cells were fixed at indicated time points in 4% paraformaldehyde, stained in a 0.1% crystal violet solution, and resuspended in 10% acetic acid. Sample absorbance was measured at 620 nm.
Cell cycle FACS analyses
SNU638 and MKN74 cells were seeded at 2 × 106 cells per 10cm plate and transfected with 50nM miRNA duplex (Ambion) using Lipofectamine 2000 (Invitrogen). Cells were harvested 48 and 72 hours post-transfection, stained for DNA content using propidium iodide (PI) and analyzed on a FACS Calibur flow cytometer (Becton-Dickinson). Briefly, cells were harvested by trypsinization and washed once with PBS before fixing over night in 70% EtOH. To stain the DNA, cells were pelleted, re-suspended in 100 μl EtOH and stained for 1 hour with 300 μl PI solution (0.05mg/ml PI, 20 μg/ml RNAse A in 0.1%BSA).
Senescence analyses
SNU638 cells were seeded at 400.000 cells per 6-well-plate and transfected with 50nM miRNA duplex (Ambion) using Lipofectamine 2000 (Invitrogen). Four days post-transfection, cells were washed in PBS and fixed for 5 minutes at room temperature in 2% formaldehyde/0.2% glutaraldehyde. Cells were washed twice in PBS pH6.0 before being stained with fresh senescence associated β-Gal stain solution (1mg/ml 5-bromo-4-chloro-3-indolyl-βD-galactoside (X-Gal), 0.12mM K3Fe[CN]6, 0.12mM K4Fe[CN]6, 1mM MgCl2 in PBS pH6.0) overnight at 37°C without CO2 supply. Cells were washed once in PBS (pH6.0) and observed under the microscope.
Antibodies and western blot analyses
SNU638 were seeded at 2 × 106 cells per 10 cm plate, transfected twice on two successive days with 50nM miRNA duplexes using Lipofectamine 2000 according to manufacturer (Invitrogen). Cells were harvested by trypsinization, washed once with PBS and lysed in RIPA buffer (150mM NaCl, 0.5% Sodium Deoxycholate, 0.1% SDS, 1% Igepal, 50mM Tris-HCl pH8, 2mM EDTA) supplemented with 1mM DTT, 1mM Pefabloc, 1mM NaV3, 10mM NaF and 1X complete mini protease inhibitor cocktail tablets. 25 μg of protein/lane were resolved on 4-20% NuPAGE Bis-Tris gels (Invitrogen) and transferred to a nitrocellulose membrane. Primary antibodies used were MET (Cell Signal 4560), MYC (Cell Signal 9402), GMNN (Santa Cruz Sc-53923), VCL (Sigma V9131), TP53 (Santa Cruz Sc-126), CDKN1A (Santa Cruz Sc-6246), CDK6 (Santa Cruz Sc-177), HDAC1 (Santa Cruz Sc-7872), CCNE2 (Cell Signal 4132), TUBB (Abcam ab11304), PARP (Cell Signal 9542) and Cleaved CASP3 (Cell Signal 9661).
Microarray analyses
Small RNAs (< 200 nt) were isolated with Invitrogen PureLink miRNA Isolation Kit from fundic and antral tissue from 1) Gastrin KO mice and age and sex matched C57BL6/J control mice, and 2) C57BL6/J mice infected with H. Pylori and uninfected age and sex matched C57BL6/J control mice, (n = 4 for each group). The quality of isolated small RNAs was determined using the Small RNA Assay on an Agilent Bioanalyzer. 500ng of small RNA was labelled with Genisphere FlashTag Kit and hybridized to Invitrogen NCode Multi-Species miRNA Microarray V2 in a Maui hybridization station. Processed slides were scanned in an Agilent DNA microarray scanner. Resulting images was analyzed and ratio of median normalized using GenePix Pro 6.0. Four biological replicates were used for each comparison. Samples were hybridized to four arrays in a dual colour dye swap microarray experimental design. BRB ArrayTools were used for fold change and statistical calculations. Selected miRNA data from the array analysis were validated using TaqMan real-time PCR miRNA assays. Data will be deposited at ArrayExpress upon acceptance.
mRNA arrays
SNU638 were transfected with 50 nM of miR-34a or miR-449b duplexes with siGLO siRNA used as negative control. Total RNA was extracted 24 hours post-transfection using TRIzol reagent. Affymetrix microarray analysis (HG-U133 Plus 2.0 human) was performed at the Microarray Center, Rigshospitalet, Copenhagen University Hospital. Experiments were run in either triplicates or quadruplicates. Data will be deposited at ArrayExpress upon acceptance.
Vectors construction and reporter assays
The 3'UTRs of HDAC1, SIRT1, MET, GMNN and CCNE2 holding miR-449 binding sites were cloned downstream of the luciferase reporter in pMIR-REPORT vector system (Ambion). Quickchange site-directed mutagenesis kit (Stratagene) was used to induce two point mutations into the seed region. Mutagenesis primers sequences are listed in Additional file 1, table S1.
HEK293 cells were seeded in 96 well plates and transfected with 20nM miRNA precursor or scrambled siRNA control, 20-50ng of luciferase vector (pMIR-report) and 5ng of renilla vector (pRL-TK) using lipofectamine 2000 (Invitrogen). Cells were harvested 24 hours post transfection and luciferase activity was measured using Dual-Glo luciferase assay (Promega).
Microarray analysis
Microarray expression data was processed using the 'affy' package in BioConductor [27]. Probe set intensities were summarized and quantile normalized using the BioConductor RMA and VSN packages. Differential expression was determined per probeset using a t-test. Probe sets were mapped to Ensembl transcripts (version 49) using mappings provided at BioMart. Probesets that mapped to two different Ensembl genes were discarded.
Evaluating global down-regulation of microRNA target genes
The 3'UTRs, 5'UTRs and coding sequences of the transcripts were scanned for matching 6mer, 7mer and 8mer miRNA seed sites (complementary to position 2-7, 2-8, and 2-9 of the miRNA). Global analysis of miRNA target down-regulation was evaluated using the longest 3'UTR sequence per gene to avoid bias introduced by genes with many transcript isoforms. We discarded transcripts with 3'UTR sequences shorter than 50 nt. To globally evaluate if miRNA target genes were down-regulated after miRNA transfection, we tested the null hypothesis that the expression change distribution of miRNA targets (having a 7mer target site) was equal to the distribution of all expressed genes without predicted target sites using the non-parametric Wilcoxon rank-sum test. A similar approach was used to evaluate down-regulation of genes with miRNA target sites in coding regions and 5'UTRs of mRNAs.
Exhaustive statistical assessment of words correlated with down-regulation
We used a previously published non-parametric rank-based statistic to exhaustively assess the correlation of word occurrences in 3'UTRs and the change in gene expression after miRNA transfection [28,29]. Genes were sorted by expression change induced by transfection of miR-34a or miR-449b, and the correlation with down-regulation was tested for all words of length 5-7 (N = 21 504).
Statistical tests
Students t-test with Welch's correction.
miR-449b is down regulated in the antrum of both Gastrin KO mice and H. pylori infected mice
Gastrin knockout mice are achlorhydric with a tendency for developing antral hyperplasia and gastric adenomas over time (figure 1) [6,12]. In order to identify microRNAs deregulated during the development of gastric cancer, we examined miRNAs expression profiles in gastric neoplasias from Gastrin knockout mice using miRNA microarrays. As shown in table 1, 20 microRNAs were significantly deregulated in the knockout mice compared to wild type littermate controls, with three miRNAs differing more than two fold, miR-7 being up-regulated and miR-709 and miR-449b being down-regulated in the antrum of Gastrin knockout mice compared to wild types. To further confirm miR-449 deregulation during gastric cancer development, we examined its expression in wild type mouse antrum tissues infected with H. Pylori. Interestingly, miRNA arrays demonstrated a specific down-regulation of miR-449b in H. Pylori infected mice (table 2 and Additional file 1, figure S1).
Figure 1. Old Gastrin knockout mice develop gastric adenomas. Antrum sections isolated from 12-16 weeks old mice (left panel), 12 to 18 months old mice (middle panel) and older than 18 months mice (right panel), from wild type (upper panel) and Gastrin knockout mice (lower panel). Sections show adenoma development in antrum tissues in the Gastrin knockouts compared to the wild types.
Table 1. miRNAs deregulated in Gastrin knockout mice
Table 2. miRNAs deregulated in H.Pylori infected tissues
miR-449 inhibits cell cycle progression and induces senescence
Having demonstrated down-regulation of miR-449 expression in gastric cancers we wanted to examine the effect of re-expressing miR-449 in gastric cancer cell lines. Interestingly no noticeable expression of the miR-449 family was detected across a panel of gastric cell lines including SNU638, SNU5, SNU216, SNU601 and MKN74 sustaining the notion of miR-449 having tumour-suppressive functions (data not shown). The miR-449 family consists of miR-449a and b in humans and miR-449a, b and c in mice. Interestingly, they share the same seed sequence as the miR-34 family and are hence expected to regulate overlapping cohorts of target genes (figure 2a). To assess the function of miR-449 in gastric cell lines we re-introduced miR-449b in SNU638 and MKN74 cells. Compared to negative control microRNAs, re-introduction of miR-449b strongly affected the proliferation of SNU638 cells (figure 2b) and visual inspection of the cells indicated induction of apoptosis and cellular senescence (figure 2c, and Additional file 1, figure S2). Flow cytometric analysis of propidium iodide-stained cells transfected with miR-449b showed a G1 accumulation 48 hours after transfection, followed at 72 hours post transfection by an accumulation of cells in the sub G1 fraction suggestive of cell death (figure 2d). The induction of cellular senescence was confirmed by acidic beta gal staining using miR-34a as a positive control microRNA (figure 2e). To rule out cell line-specific effects, the functional consequences of miR-449 re-introduction in terms of cell cycle arrest were verified in MKN74 cells (Additional file 1, figure S3). Thus, re-introduction of miR-449 negatively affects proliferation of gastric cancer cell lines concomitant with the induction of senescence and apoptosis in concordance with miR-449 having tumour suppressive functions.
Figure 2. miR-449 is part of the miR-34 family and inhibits cell proliferation. A - miR-449 is part of the miR-34 family and is evolutionarily conserved. B - miR-449 re-introduction into human gastric cell lines (SNU638) inhibits cell proliferation (red line) compared to a scrambled control (blue line) and miR-146 control (black line). Error bars represent S.D. C - Visual inspection of cell proliferation inhibition and senescence-like phenotype upon miR-449 re-introduction into SNU638 cells (lower panel) compared to scrambled transfection control (upper panel). D - FACS cell cycle analysis showing sub-G1 accumulation of SNU638 cells 72 hours following miR-449 re-introduction (right histogram) compared to a scrambled transfection control (left histogram) The table shows cell accumulation in G1 fraction upon miR-449 re-introduction compared to scrambled RNA control at 48 hours post transfection, followed by a shift to the sub-G1 fraction at 72 hours post transfection indicative of cell death. E - Senescent phenotype of SNU638 cells upon miR-449 and miR-34a positive control re-introduction shown by acidic β-gal assay compared to RNA scrambled control.
The joint seed sequence of miR-449b and miR-34a induce highly correlated expression changes
To characterize the transcripts controlled by miR-449 and to see if miR-449 regulates different transcripts than miR-34a, SNU638 cells expression profiles were examined 24 hours post transfection of miR-449b or miR-34a and differentially expressed transcripts identified. We found that mRNAs with predicted miRNA target sites (7 mer seed site) in the 3'UTR were significantly down-regulated compared to mRNAs without predicted target sites after transfection of miR-449b (p < 1.2e-70, two-tailed Wilcoxon rank-sum test), (Additional file 1, figure S4a). mRNAs having predicted miRNA seed target sites in coding regions were also found to be significantly down-regulated (p < 9.9e-25), while mRNAs with sites in 5'UTRs were only marginally down-regulated (p < 5.3e-2). The expression changes induced by transfection of mature miR-449b and miR-34a were highly correlated despite divergence of the mature sequences outside the seed region (Pearson's correlation coefficient R = 0.94, p = 0), (Additional file 1, figure S4b). We exhaustively evaluated all oligonucleotides (words) of length 5-7 for correlation with down-regulation after miR-449b and miR-34a transfection (see methods). Consistent with many previous studies, this analysis revealed the shared miR-34a/449b seed site as the 3'UTR word most correlated with down-regulation in the two experiments (Additional file 1, figure S4c).
miR-449 regulates numerous cell cycle controllers
The expression profiles were used to identify differentially expressed transcripts in cells transfected with miR-449b or controls (Additional file 1, table S2). A pathway activation analysis based on the differentially regulated transcripts demonstrates that miR-449 mainly controls transcripts coding for proteins involved in cell damage responses, cell cycle control, inflammation and cancer pathways (figure 3a). Focusing on a set of putative target genes with well-established roles in tumourigenesis, we confirmed down-regulation by miR-449 of met proto oncogene (MET), cyclin dependent kinase 6 (CDK6), geminin (GMNN), myelocytomatosis viral oncogenes homolog (MYC), sirtuin 1 (SIRT1) and histone deacetylase 1 (HDAC1) at the transcript level (figure 3b). Western blot analyses confirmed the ability of miR-449 to down-regulate MET, GMNN, MYC, SIRT1, cyclin E2 (CCNE2) and HDAC1 at the protein level to an extent similar to that achieved by re-introduction of miR-34a (figure 3c). For a subset of target genes including MET, GMNN, CCNE2, SIRT1 and HDAC1, we confirmed direct interaction of miR-449 with the target gene 3' UTR using luciferase assay (figure 3d).
Figure 3. miR-449 targets cell cycle controller genes. A - Ingenuity Pathway Analysis (IPA) of deregulated genes upon miR-449 re-introduction into SNU638 cells showing enrichment for the gene categories cancer, cell death and cell cycle pathways among others. B - qPCR validation of Affymetrix arrays showing down-regulation of MET, CDK6, GMNN, MYC and HDAC1 upon miR-449 re-introduction compared to scrambled RNA controls. C - Western blot validation of down-regulated genes upon miR-449 re-introduction into SNU638 cells compared to scrambled RNA controls. Vinculin (VCL) and tubulin beta (TUBB) were used as loading controls D - verification of direct and functional target binding using luciferase constructs holding wild type 3'UTRs and mutated 3'UTRs (two mutations in miR-449 binding site), * indicates statistical significance in luciferase expression between wild type 3'UTRs transfected with miR449a/b compared to RNA scrambled control, # indicates statistical difference in luciferase expression between wild type 3'UTRs compared to mutant 3'UTRs transfected with miR-449a and miR-449b. "ns" not significant p value > 0.05, "*" or "#" significant 0.01 < p value < 0.05, "**" or "##" very significant 0.01 < p value < 0.001, "***" or "###" extremely significant p value < 0.001
Hence, miR-449 directly targets cell cycle regulator genes consistent with a tumour suppressor function and with the cell cycle arrest observed upon miR-449 re-introduction into cancer cell lines.
miR-449 induces p53 expression but is not regulated by p53
As miR-34a was previously found to function downstream of p53 [30-33], we analyzed if also miR-449a/b were linked to p53. This was furthermore spurred by the presence of a putative p53 binding site 10 kb upstream from human miR-449 (data not shown). We therefore induced p53 by DNA damage using UV or 5-fluorouracil (5FU) in four different systems, HCT116 and MEF wild type and p53 knockout cells (Additional file 1, figure S5a). However, no significant change in miR-449 expression was detected after p53 pathway activation (Additional file 1, figure S5b). In conclusion, we found no evidence that miR-449 is a transcriptional target of p53. On the other hand, we found that miR-449a/b is able to induce activation of p53, activation of p53 response genes such as p21 and the induction of apoptosis as evidenced by cleavage of caspase 3 (CASP3) and poly (ADP-ribose) polymerase 1 (PARP) (figure 4). Towards understanding the mechanism by which miR-449 does this we examined the effect of miR-449 on SIRT1 and HDAC1. SIRT1 and HDAC1 are deacetylases which inhibit, among others, the activation of p53 and miR-34 has been shown to repress SIRT1[34]. We validated the specific binding of miR-449 to SIRT1 and HDAC1 using 3'UTR luciferase assays (figure 3d).
Figure 4. miR-449 activates the p53 pathway. Western blot analysis showing an increase of the p53 protein upon miR-449 and positive control miR-34a re-introduction into SNU638 cells compared to RNA scrambled control as well as an activation of the p53 downstream target p21 and apoptosis markers cleaved CASP3 and PARP. Vinculin (VCL) and tubulin beta (TUBB) were used as loading controls.
Hence, we speculate that miR-449 induces apoptosis by inhibiting the histone deacetylase HDAC1 and SIRT1 leading to the p53 pathway activation thus the induction of apoptosis markers cleaved CASP3 and PARP.
miR-449 is down-regulated in human gastric cancers
To evaluate the importance of miR-449 in human malignancies we next examined the expression of miR-449 in 10 gastric cancer biopsies. Importantly, we found both miR-449a and b to be significantly down-regulated or absent in 8 out of 10 primary gastric cancers. Furthermore, the expression of miR-449a and b seem to be co-regulated (figure 5a). We did not find any correlation between the reduction in miR-449 expression and clinical characteristics of the cancer (figure 5b). Analyses of the genomic DNA from the tumours found no evidence for loss or hyper-methylation of the miR-449 loci using methylation-specific melting curve analysis (MS-MCA) indicating transcriptional down-regulation of expression (data not shown). Hence, in agreement with data from two mouse models of gastric inflammation and hyperplasia, the expression of miR-449 is down-regulated in human gastric cancers.
Figure 5. miR-449 is down regulated in human gastric cancers. qPCR analysis (upper panel) showing down-regulation of miR-449 expression in 8 gastric cancer tissues compared to miR-449 expression in sample-matched controls (dotted line). U44 was used as endogenous control. Table showing clinical information of patients (lower panel). "ns" not significant p value > 0.05, "*" significant 0.01 < p value < 0.05, "**" very significant 0.01 < p value < 0.001, "***" extremely significant p value < 0.001
Gastric cancer is a highly lethal malignancy with more than 21,500 new cases each year in the United States alone [35]. The disease is often detected late and the 5-year survival rate is consequently below 20% [36]. It is therefore important to understand the etiology and progression stages of the disease. The importance of the bacterial, environmental and host genetic risk factors in gastric carcinogenesis have been studied, however less is known about the molecular progression of the disease [37,38]. Among others, p53 pathway inactivation is reported in 30-60% of gastric cancers [39,40] and recent studies suggest H. pylori direct modulation of the p53 gene or its downstream targets [41]. Another common alteration in gastric cancer is the perturbation of the cell cycle control via the over expression of Cyclin E1, which is associated with tumour aggressiveness and lymph node metastasis [42,43]. In contrast to our current understanding of the role of tumour suppressors and cell cycle factors, the knowledge about the post transcriptional changes affecting gene expression in gastric cancer is still incomplete.
Using the Gastrin knockout mouse model for gastric cancer [6,12], we identify 20 deregulated microRNAs of which 3 were more than 2-fold deregulated relative to their levels in normal gastric mucosa. As infection with H. pylori has been causally linked to gastric cancer [44,45], we next examined changes in microRNA expression levels following infection of wild type mice with H. Pylori. Interestingly, miR-449b was the only miRNA significantly deregulated in both mouse models. To establish a causal relationship between miR-449 deregulation and cancer-relevant parameters, such as cell cycle regulation, apoptosis and senescence, we over-expressed miR-449 in gastric cancer cell lines and observed a significant down-regulation of proliferation coupled with up-regulation of the acidic beta-gal senescence marker and induction of apoptosis. Importantly, analyses of primary gastric tumours from patients clearly documented a tumour-specific down-regulation of miR-449 also in humans. Previously, a number of studies have demonstrated deregulation of miRNA in gastric cancer [46-50] and the importance of the entire microRNA system has been documented in both gastric and colon cancer by the presence of cancer-specific mutations in the key RISC components AGO2 and TNRC6A [51]. Furthermore, microRNAs are likely of important prognostic value in gastric cancer [52-54]. The present study represents the first report demonstrating cancer-related down-regulation of miR-449 in both mouse models for gastric cancer and in primary human gastric tumours. Beside gastric cancer, the expression of miR-449 has also been found to be reduced in several cell lines [55] and in prostate cancer, where it was found to target HDAC1 and induce growth arrest following over-expression in prostate cancer cells [56]. In contrast, the expression of miR-449 has been reported to be increased in endometrioid adenocarcinoma [57] and melanoma in young adult patients [58]. The expression of miR-449 was also increased in skeletal muscle damage and regeneration [59].
To unveil molecular links between the loss of miR-449 and cancer progression or initiation we experimentally identified a number of direct mRNA targets using transcriptional profiling and extensive bioinformatics analysis. A series of the putative miR-449 targets were subsequently validated at endogenous level using western blotting and quantitative PCR and their direct regulation by miR-449 was established using heterologous reporter constructs and binding site-specific mutation studies. Prominent validated targets include CDK6, MYC, CCNE2, MET and GMNN and we furthermore validated the regulation of HDAC1 and SIRT1 also in gastric cancer cells. Hence, the cancer-specific loss or down-regulation of miR-449 in gastric cancer can likely be explained by the connection to key cell cycle regulators. During the search for miR-449 targets we also identified several growth factors (AREG, and KITLG) and growth factor receptors, such as MET, as targets. This suggests that deregulation of miR-449 not only leads to deregulated control of cell cycle proteins but also of growth factors and their receptors. Thus, another important property of miR-449 could be as a regulator of signals important for growth and migration/invasion.
Interestingly, miR-449 was recently shown to operate under the control of E2F1 [55,60]. This is highly interesting as it places miR-449 at a key node in a feed-back loop in which E2F1 activates the transcription of miR-449 that in turn targets CDC25A and CDK6. Reduced levels of CDC25A and CDK6 decreases the phosphorylation of pRB and subsequently inhibits the release of E2F1 [55,60,61]. Hence, aside from the pro-oncogenic effects of up-regulation of MYC, MET, CCNE2 and other direct targets, loss of miR-449 may result in increased E2F1 activity.
Many of the direct mRNA targets for miR-449 identified in this study are also targets of miR-34a and miR-449 and miR-34a belong to the same family of miRNAs as they share the same seed sequence. In accordance, miR-34a has been demonstrated to act as an important tumour-suppressor miRNA and reduced expression of miR-34a has been reported for neuroblastoma [62] and many other cancers [63]. In addition, the miR-34a gene is activated by p53 following DNA damage [30-33] and by ELK1 following over-expression of activated B-RAF [64].
In the present study, transcriptional profiling demonstrated that over-expression of miR-449 or miR-34a results in identical transcriptome changes. While this is underlining the dominant importance of the seed sequence in this type of over-expression study it also points to a limitation in the experimental approach. While miR-449 was clearly down-regulated or lost in the analyzed mouse tumour samples no clear tendency for loss or down-regulation of miR-34a was observed (data not shown). This indicates that these microRNAs may have more deviant functions in vivo than suggested by the over-expression studies.
Finally, we examined the relationship between the p53 tumour suppressor and miR-449. As miR-34a has been firmly placed downstream from p53 [30-33] it was relevant to test if the same was the case for miR-449. In agreement with other studies [55,60], we did not observe a p53-dependent regulation of miR-449 in gastric cancer cells as well as in primary human and mouse fibroblasts. However, in agreement with previous findings for miR-34a, we find that miR-449 regulates the expression of p53 [31,32] as over-expression of miR-449 resulted in a potent up-regulation of p53 subsequently resulting in activation of p21 and induction of apoptosis markers, such as cleaved CASP3 and PARP as previously reported [60].
In summary, we have found that miR-449 may act as a tumour suppressor and is lost in gastric cancer. Its re-introduction into cancer cell lines leads to inhibition of cell proliferation by targeting different cell cycle regulators. We also found that re-introduction of miR-449 induces senescence and apoptosis. Hence, this study further underlines the importance of miRNAs in cancer and points to an important function for miR-449 in gastric cancer.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
TB performed cell cycle and senescence studies, targets validation and direct targets detection studies, p53 activation studies and miR-449 expression studies, conducted data analyses, contributed in designing the study and in writing the manuscript. EF performed the miRNA array, target identification experiments, growth assays and direct targets detection studies. miR-449 expression studies, conducted data analyses and contributed in writing the manuscript. AJ performed the bioinformatics on target identification, contributed in writing the manuscript. AK supervised the bioinformatics on target identification, contributed in writing the manuscript. LB collected the normal and tumour patient samples. CH performed the bio-informatical analysis of the miRNA arrays. KG performed the methylation assays. BF performed the pathologicalscoring of the pathological samples. AHL participated in designing the study and in writing the manuscript. LFH participated in designing the study and in writing the manuscript. All authors have read and approved the final manuscript.
We thank Mikkel Klausen for technical assistance and Disa Tehler for her comments on the manuscript. Work in the authors' laboratories is supported by The Danish National Research Foundation, The EC FP7 ONCOMIRS consortium (grant agreement number 201102. This publication reflects only authors' views. The commission is not liable for any use that may be made of the information herein), The Novo Nordisk Foundation, The Lundbeck Foundation, The Danish Cancer Society and the Danish National Advanced Technology Foundation.
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Last summer we stayed at this newly remodeled 3 bedroom condo for the 4th of July week. Having a condo right on a private beach in Cabana Row made us feel like we were at a private resort. The pool area and balcony allowed us to enjoy being outside in the heat. The accommodations were spacious, with a full kitchen and laundry in the unit. We didn't even have to bring our beach chairs, as they were supplied. This was a friendly family atmosphere, so we could enjoy our peaceful surroundings. We have already booked for 2015!