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17 years Merlefest
gathers the best of the best for the largest and most diverse Americana music
festival in the country. From bluegrass (Earl Scruggs, Tony
Rice, and The Kruger Brothers), to old time music (the Reeltime
Travelers, Tara Nevins), to country (Vince Gill, Rosanne Cash, and Kelly
Willis), and everything in between (Peter
Rowan and Reggaebilly, Mark O'Connor, Nickel
Creek, Donna the
Buffalo), Merlefest covers the entire spectrum.
The annual pilgrimage to Wilkesboro, NC is in honor of Merle
Watson, son of legendary flatpicker Doc
Watson. It is an annual rite of spring and the unofficial start to the festival
season. This year's festival felt like a changing of the guard, with the emergence
of many new acoustic acts. Here's a look at some of the newer bands on the scene.
Out with the Old, in With the New
Aussie Americana invades the US in the form of the Waifs.
This band has opened up for Bob Dylan and brought its acoustic folk rock to
the Merlefest stage. Comprised of three accomplished singer/songwriters, the
music is fun, upbeat, and well crafted. Jangly guitars and gorgeous vocals make
this a band to see.
Making a splash before they even landed, Bering
Strait combine hard-driving folk rock with a touch of bluegrass. The
band has appeared on the news program 60 Minutes twice and has been nominated
for a Grammy. Their fun, upbeat music has an international flavor and was well-received
at Merlefest. The pianist possesses a beautiful voice and the banjo player is
certainly proficient. Being new to the Merlefest experience, the band was in
awe of all of their musical heroes casually strolling around the festival. The
media has lavished much attention upon Bering Strait, probably due to the novelty
of a Russian band playing country music and the fact that they are all very
striking looking Europeans. With all the publicity behind them and definite
enthusiasm for their music, Bering Strait is a band on the rise.
Musicians Making a Name for Themselves
It's slightly ironic that The
Kruger Brothers, Uwe and Jens, who hail from Switzerland, play some
of the most honest Americana music today. But play it they do, and very well.
Jens especially is lightning fast on the banjo, belting out runs so fast they
blend into one another and create a blazing tapestry of notes. Uwe has a deep
rich voice that is accented when he speaks, but he sounds like a true southerner
when he sings. Part of their popularity is their song selection; they played
numbers like "I Know You Rider" and "Tennessee Stud" they gave these traditional
selections new life. Though they've been appearing regularly at Merlefest since
1997, they recently moved to the US and released a brand new album called Choices.
Nashville's hottest session musician is Bryan
Sutton. Bryan has earned the reputation as the go-to guitarist for bluegrass.
He appeared a number of times at Merlefest, including a notable Cabin Stage
set on Friday. After playing a few solo tunes, he was joined by Tim O'Brien
and Bela Fleck, making for a mini all-star jam. The highlight was when Bryan's
father Jerry Sutton came out to play a duet. Look for Bryan's latest solo album
Bluegrass Guitar for an example of his fine flatpicking.
Adding to the buzz surrounding the old-time music this year, Tara Nevins performed three solo sets throughout the festival. Known as the fiddle player for Donna the Buffalo, Tara's real love is authentic country music. She brought the musicians who played on her solo release Mule To Ride, including the smallest banjo ever, appropriately named the "Banjuke." The set was a fantastic journey through Appalachia both old and new.
Seasoned Artists That Still Rock
Hot off Traveler, his best release in years, Tim O'Brien took the Cabin Stage with fiddle wizard Casey Driessen and played a spirited set. Songs included "Less & Less," "Family History," and "Kelly Joe's Shoes." Pete Wernick made a surprise appearance and they played their old Hot Rize classic, "Climbing up a Mountain." Tim O'Brien continues to add classics to the Americana songbook.
Gillian Welch is perhaps the best songwriter in the business today, creating brand new music that seems old as the hills. One of her three sets from the weekend contained selections from her new album, including "Look at Miss Ohio," the semi-biographical "No One Knows My Name," and "Wrecking Ball." David Rawlings displayed his passionate, one of a kind lead guitar style on "Red Clay Halo" and "Rock of Ages." Gillian and Dave performed a number of times throughout the weekend, but their best set was on the Austin Stage, with an intimate and attentive crowd.
Long Nashville's top dobro session player, Jerry
Douglas has worked on over 1,000 projects. His Main Stage set on Thursday
was fantastic, the band backing him adding a full sound to his soaring dobro.
The dobro has a range similar to a human voice, and provided the melody line during
the instrumental set. He played material off his latest CD Look Out For Hope
as well as older albums like Skip, Hop & Wobble, ending the set with a
rousing "Patrick Meets the Brickbats." His fiddle player Gabe was sporting the
first and only bluegrass mohawk I've ever seen. Leave it to Jerry Douglas to push
Perennial Merlefest favorites Donna the Buffalo is an extremely positive band of zydeco-groove musicians that's seemingly been on the road forever. As usual they got a huge crowd response and by the end of their Saturday set had everyone dancing in the aisles. They opened with "Better Days," and later got going with "Funky Side," and included an excellent "Family Picture." Loyal followers (aka The Herd) are plentiful in North Carolina and always show up in huge numbers at Merlefest.
The Creekside Stage, tucked away behind the Main Stage, is a
little slice of acoustic heaven. Playing a set on Friday was the legendary Hot
Tuna, featuring Jorma Kaukonen and Jack Casady. These two
heads from the '60s San Francisco psychedelic rock scene left all of that behind
to focus on their love of blues, country, and more traditional material. Here
at the Creekside, they performed "Keep You Lamp Trimmed and Burning" and an
old favorite "Hesitation Blues."
Celebrating his 80th birthday, the father of the five string
Earl Scruggs played a set with his Family and Friends. The North Carolina
native invented the distinctive bluegrass three-finger roll. He played with
Bill Monroe and the Bluegrass Boys before co-founding the immensely influential
group Flatt & Scruggs. Earl played admirably during the set, which featured
guest appearances by Bryan Sutton and Doc
Watson. They ran through many bluegrass classics including "Blue Moon of
Kentucky," "Long Black Veil," and the "Ballad of Jed Clampett." While Earl is
a living legend, this set was high on sentiment and low on excitement. The crowd
enjoyed seeing him, but the performances from the Family and Friends lacked
genuine energy and were fairly routine. Earl did have a chance to shine though
with a couple of slick instrumentals. Many bluegrass legends have gone "over
Jordon," (bluegrass slang for passed away) so it was good to see the likes of
Doc and Earl still playing.
With his first new studio album in five years, King of My World, Sam Bush is back on the road. Not that he needs a reason--the ubiquitous festivarian is practically a given at any major summertime musical gathering. His Friday night set on the Main Stage included "Eight More Miles to Louisville" and "They're Gonna Miss Me When I'm Gone," both staples of his set for a few years now and featured on the new album. The band's lineup has been rotating recently, but for festival energy and the comfort of hearing a familiar set, Sam Bush is king of his world.
Uniquely Merlefest
at Merlefest was the highly competitive banjo contest. When the dust settled,
William Parsons of the band Meridian
picked up first place and a brand new Earl Scruggs model banjo. Capturing third
place was a face familiar to many, Hank Smith of North Carolina-based
Barefoot Manner.
With traditional material like "Dear Old Dixie" and a highly complex original
piece "Nervous Breakdown," Hank's blazing speed and technical precision wowed
the judges. Another Friday feature is the Chris Austin Songwriting contest.
Over the years this contest has given a major boost to the careers of the winners.
Hopefully it will do the same for the winner of the bluegrass song category,
Mike Finders of Iowa City and his song "Adeline."
The Dance Tent is consistently a Merlefest favorite. It is the only place where you are actively encouraged to boogie at the festival. Each session teaches a new dance step and everyone is welcome to join in. The musicians are always excellent and everyone has lots of fun. The Reeltime Travelers were everywhere and played a set at the Dance Tent. Their traditional music served as a soundtrack to the contra lessons where strangers and friends danced arm in arm.
The man behind the festival, Americana music legend Doc Watson, is getting on in years, and decided to scale back his performances. Despite his advancing age, he remains the heart and soul of the festival. He did find time to make a guest appearance with Earl Scruggs, and also did sets with his grandson Richard Watson and longtime picking companion Jack Lawrence.
Picture yourself back in middle school and suddenly Bela Fleck
arrives to show you a thing or two on the banjo. Or maybe Tony Rice appears
to discuss the finer points of bluegrass guitar. Sound like a dream? For Wilkes
County students, it's a reality. In recent years Merlefest has sponsored an
outreach program where festival artists visit local schools to introduce them
to bluegrass and Americana music. The program helps raise musical awareness
and has created many new young fans.
Bands That Fell Somewhere in Between
Falling into the country category were Kelly Willis and
Bruce Robison, both accomplished singer/songwriters from Austin, TX.
Their easy country sound was heartfelt and genuine, and unlike the polished
country you hear on the radio, these were well crafted songs with a tinge of
The scheduling gurus at Merlefest usually recruit a big name act for Saturday night. This year Vince Gill filled the role. Many people are unaware that Vince started as a bluegrass musician and only later moved into the mainstream with the Pure Prairie League, and then into country as a solo act. His evening set on the Main Stage was slick Nashville country music all the way: pedal steel guitars, steady drums, and silly lyrics. One number that he jokingly proclaimed to be "The greatest country song I've ever written" was a sarcastic number about an annoying ex--pretty standard fare even for pop country. He redeemed himself slightly with a version of Johnny Cash's "Ring of Fire." Vince, a close friend of the Cash family, brought out Rosanne Cash to perform their 1985 top ten duet "If It Weren't for Him."
Pine Mountain Railroad is one of those bluegrass bands that puts on a polished performance while wearing suits. They have a single on the bluegrass charts with a very country influenced feel, similar to what Alabama would sound like if they played bluegrass. Their set mixed in liberal amounts of gospel and, in true Martha White tradition, included a commercial jingle for Odom's Tennessee Pride Country Sausage, the sponsors of their tour.
Straight out of a roadside Texas saloon, The Derailers play honky tonk like it was meant to be, fast and furious. Their sound features a chunky drum beat, slick guitar runs, and a full piano--something like Elvis singing honky tonk.
You Can't See It All
With over 100 performing acts and 13 stages, it's literally impossible to see
everything. Here are a few sets that I missed but heard excellent things about.
Peter Rowan & Crucial Reggae featuring the Burning Spear Horns played a set at the Dance Tent Saturday that drew rave reviews. Combine the bluegrass songbook of Peter Rowan with the dance groove of Crucial Reggae, then add the Burning Spear Horns, and it certainly sounds like a recipe for success.
Also on the forefront of the old time music revival, Old Crow Medicine Show bring a punk mentality to their sound, the Ramones of old time music, if you will.
Nickel Creek stole the festival two years ago. This time they were not as hyped, but put on a spectacular set regardless. Chris Thile is working on becoming the next Sam Bush and the next David Grisman combined.
John Cowan, the former New Grass Revival singer, brought out the surprise of the festival with a special appearance by Led Zeppelin bassist John Paul Jones for "Dazed and Confused."
Words by Anson Burtch
Photos by Gabriel Nelson (unless otherwise noted)
JamBase | America
Go See Live Music!


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Displaying Powerpoint Presentation on DIVERSITY IN LIVING ORGANISMS available to view or download. Download DIVERSITY IN LIVING ORGANISMS PPT for free.
Presentation Title : Big Idea 15: Diversity and Evolution of Living Organisms
Presentation Summary : Big Idea 15: Diversity and Evolution of Living Organisms. Description A. The scientific theory of evolution is the organizing principle of life science.
Source : http://www.coedu.usf.edu/main/departments/sped/PROPEL/documents/BigIdea15-7th.pptx

subset: commoncrawl


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King City's Latest News
December 01, 2015
Sales numbers tell the story of who are the top-selling Realtors
Unlike some other professions, in real estate the numbers are made public When looking at property sales in King City, Summerfield and the Highlands, the top-selling real estate brokers seem to…
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King City council is back to seven members
Council is more diversified with second Edgewater resident Chi Nguyen-Ventura The King City City Council gained a new member Nov. 4 when City Manager Dave Wells administered the oath of office…
November 25, 2015
Four newcomers join King City Planning Commission
King City panel finally has all the seats filled Over the years King City has sometimes had trouble filling seats on the Planning Commission, but for the latest round, there were six applicants…
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Plan would keep outsiders from parking on some streets all day long King City will be establishing residential parking zones thanks to a new ordinance taking effect Dec. 4 that the City Council…
October 29, 2015
World War II veterans get a monument of their own
Three local men are among a group of veterans that took an Honor Flight Network trip to Washington, D.C., in mid-September Crowds of people at national monuments in Washington, D.C., and at…
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SCOTTY Foundation makes a big difference in foster kids' lives
Home-grown family-run enterprise provides Christmas gifts for all the children in the families Several years ago, a 3-year-old boy in the Washington County Department of Human Services' foster…
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Washington County foster kids' giving tree season starts Nov. 15
HomeStreet Bank and King City and Summerfield civic associations take on 250 gift tags There will again be three local giving trees set up to collect Christmas gifts for children in the…
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King City starts recruitment process to find new city manager
Current City Manager Dave Wells lays out the plan that should culminate in March with a new hire King City City Manager Dave Wells got the ball rolling on finding his replacement at the Oct. 7…
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KCCA doubles its transfer fee without input from property owners
KCCA resident questions the legality of the board's vote and whether charging new buyers a percentage is legal Following in the footsteps of the Summerfield Civic Association, the King City…
October 29, 2015
KCCA Board of Directors lists its accomplishments during Oct. 13…
They include cutting costs, raising transfer fee for new property buyers and setting up email addresses for officers and clubs The Oct. 13 meeting of the King City Civic Association Board of…
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King City Expo pulls out all the stops for another successful…
More than 400 people attend to learn about local services and businesses Kathy Peper, the coordinator of the popular annual King City Expo, is thrilled at the response to this years event, held…
Oct 29, 2015
Brad Wyatt is super salesman in King City
Prestige Properties principal broker leads sales in King City for 10 years Brad Wyatt, principal broker with Prestige Properties in King City, is having a banner year. As of Oct. 9, he had sold…
Oct 02, 2015
Haggen closing Tigard, Sherwood locations
Haggen, less than a year after a spectacular burst of growth, is now in the midst on an equally incredible shrinking act. The grocery store chain announced Sept. 24 that it would close another…
Oct 02, 2015
Sen. Ginny Burdick named Democrat majority leader
Tigard's state senator has a new job. Ginny Burdick, the longtime state senator representing Tigard, King City and a large swath of Southwest Portland in the Legislature, was voted the new…


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Common Core Standards
If our government were a tree, it would be a sad one—only three branches. The three branches of the government look like this: judicial, legislative, and executive. The executive branch includes the president and his administration; the judicial includes the Supreme Court and the federal court system; the legislative branch makes laws and includes the House of Representatives and the Senate.
Up Next:Giving Power to Congress
Up Next:Branches of Government
Up Next:Bill of Rights


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Our online process saves time and allows you to pre-register at your convenience.
Our concierge can help reduce demands and stresses during your hospital stay.
For your convenience you can pay your hospital bills online and manage your account.
Take a virtual tour and experience CRH in an exciting new way.
Join a progressive, quality-focused medical staff in a favorable malpractice state.
Some people use numbers called statistics to figure out their chances of getting cancer. Or they use them to try to figure out their chance of being cured. Because no 2 people are alike, statistics can't be used to predict what will happen to one person. The statistics below describe large groups of people. They do not take into account a person's own risk factors, such as family history, behaviors, or cancer screenings. If you have questions, talk with your health care provider.
Here are some statistics about melanoma and skin cancer:
Skin cancer is the most common type of cancer.
Basal cell and squamous cell carcinomas are the most common types of skin cancer.
Melanoma is a less common type of skin cancer.
About 76,100 people in the U.S. will get melanoma this year.
Rates of melanoma have been increasing for at least 30 years.
Before age 40, melanoma is more common in women than in men. After 40, rates are higher in men than in women.
Melanoma accounts for less than 2% of all skin cancers, but it causes most skin cancer deaths. About 9,710 people will die from melanoma this year.
Source: American Cancer Society (ACS)


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Christina Aguilera performs onstage during 102.7 KIIS FM's 2014 Wango Tango at StubHub Center on May 10, 2014 in Los Angeles, California. / Kevin Winter, Getty Images For 102.7 KIIS FM's
View the full article on USATODAY.com.
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Read the original story: Christina Aguilera shares adorably sexy bump photo


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An icy line: State stops plowing at the city border, where traffic slowed to a crawl; City lifts snow emergency
10:03 PM, Feb. 15, 2014
Clinton Corners resident Dawn Hawley drove through five towns Friday morning. She had no problems ... until she reached the line that divides the Town and City of Poughkeepsie.
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Full Paper
The Effect of He Implantation on the Tensile Properties and Microstructure of Cu/Fe Nano-Bicrystals
Article first published online: 12 OCT 2012
DOI: 10.1002/adfm.201201776
Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Advanced Functional Materials
Volume 23, Issue 10, pages 1281–1288, March 13, 2013
Additional Information
How to Cite
Landau, P., Guo, Q., Hattar, K. and Greer, J. R. (2013), The Effect of He Implantation on the Tensile Properties and Microstructure of Cu/Fe Nano-Bicrystals. Adv. Funct. Mater., 23: 1281–1288. doi: 10.1002/adfm.201201776
Publication History
- Issue published online: 7 MAR 2013
- Article first published online: 12 OCT 2012
- Manuscript Revised: 11 SEP 2012
- Manuscript Received: 28 JUN 2012
- tensile tests;
- interfacial strength;
- helium implantation;
- mechanical properties
In situ uniaxial tensile experiments on as-fabricated and helium-implanted 100 nm-diameter Cu/Fe bicrystals unearth the effect of individual face-centred-cubic/body-centred-cubic (fcc-bcc) interfaces on improving radiation-damage tolerance and helium absorption. Arrays of nanotensile specimens, each containing a single Cu grain in the bottom half and a single Fe grain on top, were fabricated by templated electron-beam lithography and electrodeposition. Helium is implanted at 200 keV to a dose of 1014 ion/cm2 nominally into the interface region. High-resolution, site-specific transmission electron microscopy (TEM) and through-focus analysis reveal that the interfaces are nonplanar and contain ≈5 nm-spaced He bubbles with diameters of 1–2 nm. Nanomechanical experimental results show that the irradiated samples exhibit yield and ultimate tensile strengths more than 60% higher than the as-fabricated ones, while they retain comparable ductility. Tensile failure always occurs gradually, along the interfaces, with no noticeable shape localization. The absence of brittle failure in He-irradiated metals might be explained, in part, by the inability of the small He bubbles to serve as sufficient stress concentrators for cracking. In addition, the non-orthogonal orientation of the interfaces with respect to the loading axes results in the development of both normal- and shear-stress components. Tensile loading along the pillar axes may cause those interfacial regions subjected to normal stresses to detach, while the inclined regions, subjected to shear, to carry plastic deformation until final fracture.


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MicroRNA is a naturally occurring class of non-coding RNA molecules that mediate posttranscriptional gene regulation and are strongly implicated in cellular processes such as cell proliferation, carcinogenesis, cell survival and apoptosis. Consequently there is increasing focus on miRNA expression as prognostic factors for outcome and chemotherapy response. Only approximately 50% of patients with bladder cancer respond to chemotherapy. Therefore, predictive markers, such as miRNAs, that can identify subgroups of patients who will benefit from chemotherapy will have great value for treatment guidance.
We profiled the expression of 671 miRNAs in formalin fixed paraffin embedded tumors from patients with advanced bladder cancer treated with cisplatin based chemotherapy. We delineated differentially expressed miRNAs in tumors from patients with complete response vs. patients with progressive disease and in tumors form patients with short and long overall survival time. Furthermore, we studied the effect of up- and down regulation of key miRNAs on the cisplatin sensitivity in eight bladder cancer cell lines with different sensitivities to cisplatin.
miRNA expression profiling identified 15 miRNAs that correlated with response to chemotherapy and 5 miRNAs that correlated with survival time. Three miRNAs were associated with both response and survival (886-3p, 923, 944). By changing the cellular level of the response-identified miRNAs in eight bladder cell lines with different cisplatin sensitivity we found that down-regulation of miR-27a, miR296-5p and miR-642 generally reduced the cell viability, whereas up-regulation of miR-138 and miR-886-3p reduced the viability of more than half of the cell lines. Decreasing miR-138 increased the cisplatin sensitivity in half of the cell lines and increasing miR-27a and miR-642 generally increased cisplatin sensitivity.
MiRNAs seem to be involved in cisplatin based chemo response and may form a new target for therapy and serve as biomarkers for treatment response.
Bladder carcinoma is the fourth most common cancer in men in the Western world. The disease is characterized by frequent recurrences and poor clinical outcome when tumors progress to invasive disease. The most prevalent histopathologic type of bladder cancer in Western countries is transitional cell carcinoma (TCC) accounting for up to 95% of all cases. About 30% of patients with TCC's either present with or develop invasion into the detrusor musculature, a prognostic indication carrying an about 50% risk of fatal outcome following development of metastatic disease dissemination. In patients with locally advanced or metastatic disease, the response rate to chemotherapy is 30-50% [1]. Presently, there are two standard chemotherapeutic regimens for advanced urothelial carcinomas: MVAC (Methotrexate, vinblastine, doxorubicin, and cisplatin) and GC (gemcitabine and cisplatin). Median survival in these patients is around 15 months, and the 5-year overall survival rate is about 15% [2]. Although the gemcitabine and cisplatin combination has a significantly better toxicity profile, both regimens still carry risk for significant toxicity and toxic deaths [3], and a substantial fraction of patients will suffer from adverse reactions without achieving clinical benefit. Early, or even pretherapeutic, discrimination between likely responders and non-responders would greatly improve selection of patients to chemotherapy and thereby benefit both groups.
Deregulation of microRNA (miRNAs or miRs) levels is associated with dysplasia and cancer, and miRNA profiles have been used to classify human cancers and predict outcome more accurately than mRNA expression profiles [4-10]. Furthermore, urinary miRNAs have been shown to be clinically useful for noninvasive bladder cancer diagnostics (miR-452 and miR-222) [9].
miRNAs are endogenous, non coding RNA molecules of approximately 19-25 nucleotides in length. Most miRNAs represses mRNA translation by blocking of translation, less frequently mRNA degradation/deadenylation, however a minor proportion of the miRNAs mediate mRNA target up-regulation [11,12]. Due to the low stringency of the required binding of 6-8 bases of the seed sequence of the miRNA to the mRNA, each miRNA can potentially interact with hundreds of mRNA targets. With the more than 1576 identified unique human miRNAs (miRBase, version 18) it is predicted that around 30% of the transcriptome is regulated by miRNAs. miRNA genes are frequently located in cancer-associated genomic regions of loss of heterozygosity, amplified regions, or fragile sites [13]. Aberrantly expressed miRNAs have been shown to be associated with many types of cancers. miRNAs can function as both oncogenes (onco-miRs) or tumor suppressors [14-16]. Therefore expression profiles of miRNAs may provide information about chemotherapy sensitivity prior to treatment and changes in miRNA expression during treatment could be a marker for chemotherapy response. Improvements in high throughput miRNA profiling have provided increasing evidence of miRNA deregulation in drug resistant and sensitive cells. Blower and colleagues investigated miRNA expression profiles in the NCI-60 cancer cell panel and showed correlation between chemotherapy potency and miRNA expression patterns [15]. Kovalchuk, Pogribny and colleagues identified 137 deregulated miRNAs when comparing doxorubicin resistant and sensitive MCF-7 breast cancer cell lines and 103 deregulated miRNAs when comparing cisplatin resistant and sensitive MCF-7 cells [17-19]. Boren and colleagues investigated 16 different ovarian cancer cell lines and found 27 (of 335 profiled) miRNAs correlated to response to one or more drugs (cisplatin, gemcitabine, docetaxel, doxorubicin, topotecan, paclitaxel) [17]. In human epithelial ovarian cancer samples Yang and colleagues found 34 out of 326 miRNAs were associated with response to platinum based chemotherapy (complete response (n = 42) and non-complete response groups (n = 27))[20].
To gain information on the potential role of miRNAs in drug response we profiled miRNA expression in bladder tumors from patients having either complete response or progressive disease after cisplatin based chemotherapy. Furthermore, we studied the effect on cisplatin sensitivity in different bladder cancer cell lines (UMUC9, UMUC14, SLT4, 253JBV, RT4, CLR2169, HT1197, 575A) by changing the levels of the miRNAs predictive of chemotherapy response. Our findings show that 15 miRNAs correlated with response to chemotherapy, and 5 miRNAs were associated with survival time. Three miRNAs were associated with both response and survival (886-3p, 923, 944). By changing the cellular level of the treatment-response associated miRNAs in eight bladder cell lines with different cisplatin sensitivity we found that down-regulation of miR-27a, miR296-5p and miR-642 reduced the cell viability, whereas up-regulation of miR-138 and miR-886-3p reduced the viability of more than half of the cell lines. Decreasing miR-138 increased the cisplatin sensitivity in half of the cell lines and increasing miR-27a and miR-642 increased cisplatin sensitivity.
Patient samples
The study included a total of 30 patients receiving first line platinum containing combination chemotherapy for locally advanced (T4B, N2-3) and/or metastatic (M1) transitional cell carcinoma (TCC) at the Department of Oncology, Aarhus University Hospital, Denmark, in the 10-year period between 1991 and 2001. All patients had histologically confirmed transitional cell carcinoma of the urothelial tract (urethra, bladder, ureters or renal pelvis). All patients received cisplatin-based combination chemotherapy schedules (Additional file 1: Tables S1 and Additional file 2: Table S2) and were included in either one of four phase II protocols, one phase III protocol, or were treated with the standard regimen at the relevant time (MVAC or GC). Patients were treated to a maximum of 6 cycles unless progression or unacceptable toxicity appeared. Patients receiving standard treatment were prospectively followed under the same conditions as patients in protocols [21]. Patients presenting with locally advanced disease, obtaining a significant partial or complete response to chemotherapy, were offered consolidating surgery or, more often, radiotherapy when applicable [21].
Additional file 1. Table S1. Response cohort - patient characteristics and treatment regimens.
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Additional file 2. Table S2. Survival cohort - patient characteristics and treatment regimens.
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Cell culture
Bladder cell lines UMUC9, UMUC14, SLT4, 253JBV, RT4, CLR2169, HT1197, 575A [22] (provided by Professor Dan Theodorescu, University of Colorado Comprehensive Cancer Center) were re-authenticated via STR analysis using the Cell-ID-system (G9500, Promega, Nacka, Sweden), products were analyzed using an Applied-Biosystems 3130 Genetic Analyzer. No Mycoplasma contamination was detected using nested PCR-based Mycoplasma detection. SLT4, 253JBV, RT4, CLR2169 cells were cultured in DMEM + with L-glutamine (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin (Gibco) and 100 U/mL streptomycin (Gibco). UMUC9, UMUC14, 253JBV, HT1197, 575A cells were cultured in MEM with L-glutamine (Gibco, Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 1 mM Sodium Pyruvate + 0,1 mM nonessential amino, 10% fetal bovine serum (Gibco), 100 U/mL penicillin (Gibco) and 100 U/mL streptomycin (Gibco). All cells were maintained in a humidified atmosphere at 37°C and 5% CO2.
Transient transfection
miRNA molecules (Ambion, Austin, TX, USA) and LNA knockdown molecules (Exiqon) were reverse Transfected using Lipofectamine 2000 (Invitrogen) according to manufacturer's guidelines. Non-targeting miRNA (Ambion, Austin, TX, USA) and non-targeting LNA molecules (Exiqon, DK) were used as controls. In short 4x103 cells in 100 μL culture medium were added to wells of 96 well plates containing 50 μL miRNAs or LNA antagonism giving a final concentration of 20 nM and 40 nM respectively and then incubated at 37°C and 5% CO2. Cisplatin (Sigma Aldrich, Germany) was added 48 h post transfection in experiments studying the effect of chemotherapy, by first removing 50μL from each well and then adding 100μL new medium containing cisplatin at 2 times the desired concentration and then incubated for an additional 48 h at 37°C and 5% CO2. Finally viability was measured using the MTT assay (see section: Cell viability assay). mercury LNA™ microRNA inhibitors (EXIQON) used: hsa-miR-27a (410167-00), hsa-miR-138 (412560-00), hsa-miR-296-5p (410171-00), hsa-miR-642 (410409-00), hsa-miR-886-3p(410445-00) and mercury LNA™ microRNA inhibitors negative control A (199004-04). The miRNA molecules used for transfection are chemically modified double-stranded RNA molecules designed to mimic endogenous mature miRNA molecules: hsa-miR-27a (PM10939), hsa-miR-138 (PM11727), hsa-miR-296-5p (PM10609), hsa-miR-410 (PM11119), hsa-miR-642 (PM11477), hsa-miR-886-3p(410445-00), Pre-miR™ miRNA Precursor Molecules Negative Control #1(AM17110). Prior to miRNA transfection experiments we performed transfection with a cy3 labeled siRNA molecule to monitor successful uptake under fluorescence microscope.
Isolation of RNA
Total RNA was isolated from formalin-fixed paraffin embedded (FFPE) tissues specimens as follows. Haematoxylin- and eosin-stained (HE) sections of the FFPE blocks were examined and areas with carcinoma cells circled and the section was photocopied onto transparent paper. Using this as a guide, an appropriate area of the block was sampled using a disposable punch biopsy needle (1.5 mm, Miltex GmbH, Germany). The sample cylinder was dispensed and minced into small pieces with a scalpel. Total RNA (range 1200 ng – 2300 ng) was then prepared using the miRNeasy® FFPE Kit (Qiagen, Germany), essentially as specified by the manufacture. The miRNeasy FFPE kit is specifically designed for FFPE material and contains reagents to reverse formalin cross linking of RNA. Furthermore the kit is developed to allow purification of all usable RNA larger than 18 nucleotides.
Cell line RNA was isolated from 6-well and 24-well plates using RNeasy spin columns (Qiagen, Hilden, Germany), as recommended by the manufacturer. All RNA samples were quantified using an Infinite® 200 PRO NanoQuant spectrophotometer (Tecan, Switzerland).
miRNA profiling using Taqman Micro fluidic cards
Quantification of miRNAs from tissue samples was carried out by a two-step procedure using the Taqman LDA Human microRNA Panel v3.0 (Micro fluidic card, Applied Biosystems, Foster City, CA) according to the manufacturer's protocols. Taqman LDA Human microRNA Panel v3.0 was designed using Sanger miRBase v14. Briefly, 500 ng of total RNA was used for multiplexed RT reactions followed by pre-amplification. Cycling conditions were chosen according to the manufacturer's protocols. The ABI7900 HT platform with TLDA upgrade was used and data was analyzed using RQ Manager Software provided by Applied Biosystems.
Quantitative real-time PCR
Quantification of miRNAs by Taqman real-time PCR was carried out as described by the manufacturer (Applied Biosystems). Briefly, 500 ng of template RNA was reverse transcribed by using the Taqman MicroRNA Reverse Transcription Kit and the multiplex RT primer pools containing miRNA-specific stem-loop primers (Applied Biosystems). Diluted RT product (1.5 μL) was introduced into the 20 μL PCR amplifications, which were incubated in 384-well plates on the ABI 7900HT thermo cycler (Applied Biosystems) at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Gene expression was normalized using expression of miRNA-193b. Taqman® MicroRNA Assays (Applied Biosystems, Foster City, CA) used: hsa-miR-27a (Assay ID000408), hsa-miR-138 (Assay ID002284), hsa-miR-193a-5p (Assay ID 002281), hsa-miR-193b (ID 002367), hsa-miR-296-5p (Assay ID 000527), hsa-miR-410 (Assay ID001274), hsa-miR-492 (Assay ID001039), hsa-miR-639 (Assay ID001583), hsa-miR-642 (Assay ID 001592), hsa-miR-886-3p(Assay ID002194), hsa-miR-944 (Assay ID002189).
Cell viability assay
The viability of sub-confluent cells was analyzed by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The assay was performed in 96 well plates with 200μL/well. 100 μL culture medium was carefully removed and 25 μL MTT solution was added (1 g MTT (Sigma M5655) dissolved in 200 mL D-PBS.) and stored shielded from light 1.5 h at 37°C and then 100μL solubilization (50% dimethylformamide, 20% SDS) buffer was added and left protected from light overnight. Readout was done using a micro plate reader (Lab systems Multiscan MCC/340) at 540 nm. Absorbance at 692 nm was used as reference.
Ethics statement
All patients gave their written informed consent, and the study was approved by the Central Denmark Region Committees on Biomedical Research Ethics (1994/2920).
Statistical analysis
Selection of differentially expressed miRNAs were based on t-test statistics (p < 0.05) and simultaneously on assessment of false discovery rates (FDR) to correct for multiple testing. We used Significance Analysis of Microarrays (SAM) software for estimating FDR. In the analysis of treatment response we used a maximum of 10% in FDR as cutoff and in the analysis of survival we used a maximum of 50% in FDR as cutoff. FDRs for the individual miRNAs are reported.
miRNAs differentially expressed between chemotherapy responders and non-responders
We profiled the expression of 671 miRNAs in tumors from patients with advanced bladder cancer subsequently treated with cisplatin based chemotherapy (Table 1 and Additional file 1: Table S1). Seven patients experienced progression of disease (PD) and eight patients experienced complete responses (CR) according to the RECIST criteria [23]. Differentially expressed miRNAs (PD vs.CR) are listed in Table 2. The expression levels of the 15 top ranked miRNAs in the different tumors samples are shown in Figure 1A. Data was normalized using miRNA-193b as this was identified as the most stably expressed miRNA across all samples by Norm Finder [24]. Different normalization approaches showed highly similar results (Additional file 3: Table S3). As a technical evaluation of the Taqman Human Array MicroRNA Cards we selected the 5 top ranked miRNAs from the analysis (miRNA-642, 492, 27a, 296-5p, 944, 193a-5p) and performed single-plex qRT-PCR. The qRT-PCR fully confirmed the Taqman Human Array MicroRNA results (Additional file 4: Table S4). Interestingly, the analysis showed that the majority of top ranked miRNAs had a reduced expression in the CR group compared to the PD group. The chromosome location of the 15 differentially expressed miRNAs (Table 2) shows that the unidirectional expression pattern is not due to regional epigenetically silencing of the miRNAs.
Additional file 3. Table S3. miRNA normalization. Normalization of miRNA expression is challenging and therefore we chose to make 4 different normalizations prior to analysis of differentially expressed miRNAs (PD vs.CR) and compare the results. Normalizes used were mammalian U6 (MammU6) and RNU48 RNAs that is often used as normalizes, miR-193b selected using the Norm Finder program, and finally quintile normalization using the qpcrNorm program (Mar J et al. Data-driven Normalization Strategies for qPCR Data. Technical Report, 2008). The results show a large degree of agreement between normalizations. We argue that the Norm Finder approach is the best normalize approach for our data because quintile normalization is better suited for large gene sets and miRNA-193b is more stable than MammU6) and RNU48 across the samples (data not shown).
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Additional file 4. Table S4. Technical validation of miRNA profiling. As a technical evaluation of the Taqman Human Array MicroRNA Cards (LDA analysis) the 6 top ranked miRNAs from the Array analysis were determined using real-time q-RT-PCR (n = 3) (singleplex) and compared to the MicroRNA card analysis. miR-193b expression was used as normalize.
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Table 1. Patient characteristics and clinical parameters
Table 2. miRNAs differentially expressed between patients with progressive disease and complete response
Figure 1. Heatmaps of miRNA expression patterns. miRNA expression is depicted according to (A) treatment response and (B) overall survival. Yellow; upregulation of the gene, blue; downregulation, black; median expression, grey; missing value.
miRNAs differentiating short and long overall survival time
We also delineated miRNAs that correlated with survival time. We compared tumors from patients with short survival (SS, 15 patients, overall survival < 1 year) to tumors from patients with long survival (LS, 15 patients, overall survival 1 > year) patient survival cohort. Patient survival cohort contained the 15 patients from response cohort plus 15 additional patients (Table 1, Additional file 2: Table S2). Differentially expressed miRNAs (SS vs.LS) are listed in Table 3 and expression levels are shown in Figure 1B. Again Norm Finder was used to calculate the best normalize miRNA from the dataset (miRNA-324-3p). Other normalization approaches were applied as before with highly similar results (Additional file 5: Table S5). As expected we found an overlap of differentially expressed miRNAs between the analyses performed for patient response cohort and patient survival cohort (miRNA-886-3p, miRNA-923 and miRNA-944).
Additional file 5. Table S5. miRNA normalization. Tree different normalizations prior to analysis of differentially expressed miRNAs (SS vs.LS) were performed to inspect the robustness of the analysis. Normalizes used were mammalian U6 (MammU6), miR-324-3p selected using the Norm Finder program and miR-193b. The results show a large degree of agreement between normalizations.
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Table 3. miRNAs differentially expressed between patients with short and long survival
Cisplatin sensitivity of bladder cell lines
In order to investigate the ability of the differentially expressed miRNAs from patient response cohort to predict the sensitivity to cisplatin based chemotherapy we exposed a range of bladder cancer cell lines (UMUC9, UMUC14, SLT4, 253JBV, RT4, CLR2169, HT1197, 575A) to cisplatin. These cell lines have previously been documented to exhibit differing sensitivity to cisplatin. Above the cell are listed from high to low cisplatin sensitivity. The concentration of cisplatin required to inhibit the cell growth by 50% (GI50) during 48 h incubation in our laboratory was calculated from dose response curves (Figure 2). The studies were conducted with equal cell seeding density to avoid any confluences-related difference in effect observed on cell viability. Next we measured the expression of miRNA-27a, miRNA-138, miRNA-193a-5p, miRNA-296-5p, miRNA-410, miRNA-492, miRNA-642, miRNA-886-3p, miRNA-923 and miRNA-944 using qRT-PCR in the 8 cell lines (Figure 3). We observed no association between cisplatin GI50 and miRNA expression in the cell lines.
Figure 2. Single agent dose-dependent cytotoxicity induced by cisplatin. Human bladder cancer cells UMUC9, UMUC14, SLT4, 253JBV, RT4, CLR2169, HT1197, 575A were seeded in 96 well plates and treatment with the indicated cisplatin concentration started 48 h after seeding and continued for 48 h. Cell viability was assessed with MTT-assay. Panel A: cell viability cisplatin dose response curve. Panel B: Cisplatin concentration killing 50% of the cells after 48 h relative to non-treated cells (GI50, Growth inhibition 50%). GI50 was estimated based on two independent experiments. Each drug concentration was tested in six individual wells.
Figure 3. Expression of mature miRNA in bladder cell lines. Expression of mature miR-27a, miR-138, miR-193a-5p, miR-296-5p, miR-410, miR-492, miR-642, miR-886-3p, miR-923 and miR-944 in bladder cell lines 253JBV, UMUC9, UMUC14, SLT4, 575A, RT4, CLR2169 and HT1197 were determined using real-time Q-PCR (n = 3). miR-193b expression was used as normalizer. Cell lines are listed according to Cisplatin GI50 sensitivity.
Cisplatin sensitivity following miRNA knock down
To further investigate the role of the identified miRNAs in treatment response and survival time of cancer patients, we knocked down five of these key miRNAs (miRNA-27a, miRNA-138, miRNA-296-5p, miRNA-642, miRNA-886-3p) using LNA-anti-miRs. 48 hours after transfection the cells were incubated with cisplatin (GI50) for another 48 hours followed by viability analysis. The experiments were conducted in all eight bladder cancer cell lines to avoid any single cell line-dependent phenomenon and to enable drawing of more general conclusions. miRNA down regulation was verified by qRT-PCR following knockdown using two LNA-anti-miRs (Additional file 6: Figure S1). First, the effect of miRNA knock down was measured using MTT assays (Figure 4A). The viability of the cell lines per se was clearly reduced following down-regulation of miR-296-5p and miR-642, and reduced to some extent following down-regulation of miR-27a and miR-886-3p. Next, the effect of miRNA-knock down on cisplatin sensitivity was measured by incubating Transfected cells with the estimated GI50 concentration of cisplatin. Cisplatin was added 48 h post transfection and incubated for additional 48 h followed by MTT viability measurement (Figure 4B). This revealed no clear correlation between cisplatin sensitivity and miRNA knock down, however down-regulation of miR-138 increased the cisplatin sensitivity of four of the eight tested cell lines by approximately 20% (Figure 4B). The cellular response in cisplatin treated cells to miR-296-5p and miR-642 transfection was more difficult to interpret as these miRNAs already showed reduced viability following the miRNA transfection.
Additional file 6. Figure S1. miRNA down-regulation in bladder cell lines. LNA based antagonism were used to silence miR-27a and miR-138 in the bladder cell lines 253JBV and CLR2169. LNA knockdown molecules were reverse Transfected (n = 3) and after 48 h incubation culture media, with or without cisplatin (GI50), was added to the cells (n = 4). Expression of mature miR-27a and miR-138 was determined using real-time Q-PCR (n = 3). LF2000 designate controls using the transfection reagent alone. The 2-ΔΔCT method was used for relative quantification with miR-193b expression normalize.
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Figure 4. miRNA down-regulation in bladder cell lines. LNA based antagomirs were used to silence miR-27a, miR-138, miR-296-5p, miR-642 and miR-886-3p in the bladder cell lines 253JBV, UMUC9, UMUC14, SLT4, 575A, RT4, CLR2169 and HT1197. LNA knockdown molecules were reverse transfected at 20nM (n = 8), and after 48 h incubation culture media, with or without cisplatin (GI50), was added to the cells (n = 4). The viability of the cells was determined after 48 h incubation (96 h post transfection) by MTT-assay. LF2000 designate controls using the transfection reagent alone. A: Relative cell viability expressed as the viability of the antagomiR transfected cells normalized to the negative control molecule for each cell line. B: Relative cell viability expressed as the viability of the antagomiR transfected cells treated with cisplatin (GI50) compared to the antagomiR transfected cells without cisplatin normalized to the negative control molecule for each cell line. (#: the high reduction of cell viability imposed by the antagomirs prohibits precise measure of effect of cisplatin treatment during miR down regulation).
Cisplatin sensitivity following miRNA knock in
We also analyzed the effect of miRNA overexpression using synthetic precursor miRNAs (miRNA-27a, miRNA-138, miRNA-296-5p, miRNA-642, miRNA-886-3p, miRNA-944). First, we measured the effect of miRNA overexpression using MTT assays (Figure 5A). Cell viability after miRNA transfection varied extensively, however especially knock in of miR-138 and miR-886-3p had a highly deleterious effect on cell viability in half of the analyzed cell lines. Next we measured the effect of miRNA knock in on cisplatin sensitivity by incubating transfected cells with the estimated GI50 concentration of cisplatin. Cisplatin was added 48 h post transfection and incubated for additional 48 h followed by MTT viability measurement (Figure 5B). This revealed that miR-27a and miR-642 overexpression increased the cisplatin sensitivity in most of the cell lines. The effect of miR-138 and miR-886-3p overexpression on cisplatin sensitivity cannot be determined due to the highly reduced viability by the miR alone.
Figure 5. miRNA up-regulation in bladder cell lines. miRNA molecules were used to up-regulate the levels of miR-27a, miR-138, miR-296-5p, miR-642, miR-886-3p and miR-944 in the bladder cell lines UMUC9, UMUC14, SLT4, 253JBV, RT4, CLR2169, HT1197 and 575A. miRNAs molecules were reverse transfected at 40 nM (n = 8) and after 48 h incubation culture media with or without cisplatin (GI50) added to the cells (n = 4). The viability of the cells was determined after 48 h incubation (96 h post transfection) by MTT-assay. LF2000 designate controls using the transfection reagent alone. A: Relative cell viability expressed as the viability of the miRNA transfected cells normalized to the negative control molecule for each cell line. B: Relative cell viability expressed as the viability of the antagomiR transfected cells treated with cisplatin (GI50), compared to the antagomiR transfected cells without cisplatin, normalized to the negative control molecule for each cell line. (#: the high reduction of cell viability imposed by the precursor miRs prohibits precise measure of effect of cisplatin treatment during miR down regulation).
Here we performed miRNA expression profiling of tumors from patients with advanced bladder cancer that showed progressive disease, or complete response, following cisplatin based chemotherapy according to the RECIST criteria [23].
The miRNA profiling identified a number of down-regulated miRNAs that were correlated with chemotherapy response. Among these were miRNA-27a (3.5 fold), miRNA-296-5p (7 fold), miRNA-642 (14 fold), miRNA-886-3p (4.3 fold). Mature miR-27a and miR-886-3p transfection reduced the viability of the tested cell lines marginally; however the cisplatin sensitivity was not increased. Knock in of miRNA-296-5p, miRNA-642 resulted in reduced viability of all eight cell lines tested but the cisplatin sensitivity was unaltered (Figure 4A3B). miR-27a is part of the miRNA cluster miR-23a ~ 27a ~ 24-2 and functions as an oncogene in gastric cell lines (MKN45, MGC803) and the tumor suppressor Prohibitin is up regulated both at the transcriptional and translational level by antimiR-27a [25,26]. miR-27a has been shown to bind directly to the mRNA coding for the p44 subunit of general transcription factor IIH (TFIIH) during G2-M transition resulting in destabilizing the TFIIH complex and modulate the transcriptional shutdown during the G2-M phase [27]. Over expression of miR-23a ~ 27a ~ 24-2 sensitized HEK293T cells to TNF-α cytotoxicity suggesting it to be of value in cancer therapy [28,29].
Among the identified miRNAs, mir-138 was the only miRNA up-regulated more than 2 fold (3.8 fold) in the survival group (Table 2). In leukemia cell line HL-60/VCR miR-138 was demonstrated to significantly down-regulate the expression of P-glycoprotein, Bcl-2, and the transcription of the multidrug resistance gene 1 (MDR1) [30]. In human osteosarcoma cell line, U2OS MiR-138 was shown to directly target the histone γH2AX 3'-UTR and reduce histone γH2AX expression, and induce chromosomal instability after DNA damage. MiR-138 over expression enhanced cellular sensitivity to cisplatin and reintroduction of histone γH2AX in miR-138 overexpressing cells attenuated the sensitization to cisplatin [31]. miR-138 is inversely correlated with excision repair cross-complementing rodent repair deficiency, complementation group 1 (ERCC1) expression in the A549/DDP multidrug-resistant human lung adenocarcinoma cells. Furthermore, the enforced increase of miR-138 levels in the A549/DDP cells down-regulates expression of ERCC1 and increases sensitivity of the drug-resistant cancer cells to cisplatin by inducing apoptosis [32]. Based on this we would expect increased cisplatin sensitivity when overexpressing mature miR-138. This was only observed in three of the eight tested bladder cell lines (UMUC14, RT4, CLR2169, Figure 5B), however overexpression of miR-138 in itself reduced the viability markedly of half of the cell lines (Figure 5A), and so the effect of subsequent cisplatin exposure is difficult to interpret. Down-regulation of miR-138 alone did not affect viability; however it increased the cisplatin sensitivity of four of the eight cell lines by approximately 20% (UMUC9, UMUC14, 253JBV, HT1197, Figure 4B). This is particularly interesting because LNA-anti miR-138 has minimal effect on cell viability by itself (Figure 4A), making cisplatin sensitivity comparisons more robust.
Overexpression of the ABCB1/MDR1 gene coding for the transmembrane transporter P-glycoprotein (P-gp) is a common course of multi drug resistance (MDR). P-gp confers resistance to a broad range of structurally diverse chemotherapeutic drugs by contributing to resistance phenotype by drug efflux from cancer cells [33]. A number of miRNA profiling studies have implicated miRNAs in the MDR phenotype. miR-27a and miR-451 were up-regulated in MDR ovarian cells (A2780DX5) and cervix carcinoma cells (KB-V1). Transfection with antagomirs and mature miR-27a correlated with MDR1/P-gp expression [34]. In esophageal squamous cell carcinoma down-regulation of miR-27a confer sensitivity of both P-gp related (Vincristine (VCR), Adriamycin (ADR)) and non P-gp related drugs (5-Fluorouracil (5-Fu), Cisplatin (CDDP)) [35]. miR-27a is up-regulated in pediatric B-ALL and act as a suppressor of the Tumor suppressor FBW7. FBW7 act as a negative regulator of proliferation by facilitating proteasome degradation of cyclin E, c-Myc, c-Jun and Notch1 [13,36]. Mir-27a down-regulation inhibits proliferation of gastric MKN45 cells in vitro and decreases expression of P-gp and confers increased sensitivity against VCR, ADR, 5-Fu and CDDP [25,26]. miR-886 previously proposed to be a vault RNA, a component of the vault complex implicated in cancer drug resistance, was recently shown neither to be a genuine pre-miRNA nor a vault RNA [37]. miR-886 binds directly to PKR (Protein Kinase RNA-activated) and silencing of miR-886 activates PKR and its downstream pathways, eIF2α phosphorylation and the NF-κB pathway, leading to impaired cell proliferation [37]. MicroRNA miR-886-5p inhibits apoptosis of cervical cancer cells (H8, an HPV16-immortalized human cervical squamous epithelial cell line) by down-regulating the production of Bax [38]. miR-193a-3p and miR-492 was up-regulated in bladder cancer cells compared to normal urothelium [39,40].
In conclusion, we have identified miRs that alter the viability of bladder cancer cells in vitro, and that seem to impact on the cisplatin sensitivity in vitro. This was shown by both increasing and reducing the miR level in the cells. In clinical samples several miRs were related to Cisplatin response, however may require a larger data set for confirmation. Interestingly several of these seem to impact on molecules relevant for chemo response, however, the network of microRNA interactions is extremely complex and thus requires further study to reveal alterations of importance for the clinical management of patients.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
IN designed experiments, performed experiments, interpreted results and drafted manuscript. LD conducted statistical analysis and made critical revision to manuscript. MA provided tumor samples and made critical revision to manuscript. KBD, DT, TFØ, MSO, AH and MB designed experiments, interpreted results and made critical revision to manuscript. All authors have read and approved the final manuscript.
We thank Professor Dan Theodorescu, University of Colorado Comprehensive Cancer Center for providing Bladder cell lines UMUC9, UMUC14, SLT4, 253JBV, RT4, CLR2169, HT1197, 575A. We thank Pamela Celis, Susanne Bruun, Gitte Høj, Lisbet Kjeldsen and Rudolf Jung for excellent technical assistance. Grant support: The John and Birthe Meyer Foundation; Fritz, Georg, and Marie Cecilie Glud's Foundation; The Oticon Foundation; The University of Aarhus; The Danish Cancer Society, Furthermore, the research leading to these results has received funding from the European Community's Seventh Framework program FP7/2007-2011 under grant agreement n° 201663.
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Can Tech Ruin the Kim Pham Murder Trial?
By Charles Lam
While the county is best-known nationwide in sex terms for Octomom and her clan, the siliconed silliness of South County's Real Housewives and our porno starlets, those of us who live here know that local sexiness exists outside these stereotypes—and outside Imperial Showgirls or the Pump Room. It's in our law-school deans, our burlesque queens whom we remember more for their humble personalities than their impossibly small waists, our accessible surf stars and charity-supporting manly men. Sure, there are some snarky picks on the list (the picture of the Chapman University Undie Run-loving Christian is as great a summation of Orange County as anything not named Arrested Development), but we truly mean it when we say most of the following folks are as wholesomely sexy as anything in Orange County right now.
Click here to see who made the list!
Click here for a bonus slideshow of cover model Erica Hosseini. And go here for more photos of the OC Roller Girls.
This article appeared in print as "Good, Clean Fun: Don't believe the stereotypes! Meet the real Sexiest People of OC."
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Key things you need to know aboutDr. Sivanesan:
Dr. Sivanesan's Experience
Research training, expertise and qualifications
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- Acne Surgery
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Conditions Dr. Sivanesan Treats
- Abscess
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- See all 30 conditions
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Hospital Quality
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- Centrastate Medical Center
- 901 W Main St
- Freehold, NJ 07728
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Likelihood of recommending Dr. Sivanesan to family and friends is 4.2 out of 5 5 1 20
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1989 Chevrolet S-10 • 180,000 miles
I have a 89 s10 2wd with the 4.3l. It cranks over but will not start. My question is, is it possible for the fuel pump to work sometime but not other or is it the relay?
December 3, 2012.
December 3, 2012.
Could be engine compression, fuel and spark problem-Go to this link for more information: http://www.2carpros.com/articles/car-cranks-but-wont-start
Dec 3, 2012.
Dec 3, 2012.
I have a 1996 Saturn SL1. The engine has a misfire and alternates between running fine for a few weeks and then running rough for a few weeks. When the car is running rough it has less ...
3 answers • 1996 Saturn SL1 • 183,173 miles
Can a compression test be done if engine will not turn? Car will not start. Mechanic wants to tow car to his workshop and have compression checked. All parts under bonnet have been ...
2 answers • 2000 Ford Ranger
In completing a dry compression test my results were as follows: 1 - 140 2 - 10???? 3 - 100 4 - 170 I repeated the test twice cranking the car for 5 seconds on each cylinder...
7 answers • 1996 Toyota Paseo • 165,000 miles
I just started the car and put it in drive and took off when i heard a puff noise and the car stalled. no it wont turn over. everything electrical works but it sound like no compression...
2 answers • 1998 Plymouth Breeze • 100,000 miles


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Riley Hotel Group Official Management Company for Comfort Suites Twinsburg
The Riley Hotel Group out of Medina, Ohio has taken over as the hotel management company for the Comfort Suites in Twinsburg, Ohio.
Twinsburg, Ohio, January 09, 2014 - (PressReleasePoint) - The Riley Hotel Group out of Medina, Ohio has taken over as the hotel management company for the Comfort Suites in Twinsburg, Ohio.
"Our Ohio portfolio continues to grow and improve and we're thrilled to now include Comfort Suites Twinsburg," said Riley Hotel Group President Joe Moffa. "This hotel is the epitome of what all our hotels are about – the utmost in customer service and providing an outstanding experience for business and pleasure travelers alike."
The hotel boasts in-room refrigerators, microwaves, coffee makers and hair dryers; electronics charging devices, including AC and USB outlets, are provided in all guest rooms. Wireless high-speed Internet, local calls, and a newspaper on weekdays are all provided to every guest free of charge.
Guests can also take advantage of the indoor pool and fitness center as well as the complimentary daily hot breakfast.
The Comfort Suites Twinsburg is conveniently located near several local points of interest, including the Geauga Lake & Wildwater Kingdom, Aurora Farms Premium Outlets, and the Rock and Roll Hall of Fame and Museum.
Riley Hotel Group is a top 100 full-service hotel management corporation that currently manages 14 properties throughout the United States, with several more to be announced in the coming months.
"The Twinsburg Comfort Suites is delighted to now be a part of the Riley Hotel Group," said Comfort Suites General Manager Lindsay Halkovics. "With many award-winning hotels in their portfolio, we are eager to become one of them as well. We look forward to 2014 with all the positive changes coming our way."
For more information, visit www.rileyhotelgroup.com or http://www.comfortsuites.com/hotel-twinsburg-ohio-OH271
About Riley Hotel Group:
Established in 2003, Riley Hotel Group is a premier luxury development and management company dedicated to helping hotel owners provide superb levels of service. Riley provides lucrative partnerships to investment groups, developers, owners, and operators. The company's portfolio includes independent and franchised hotels in Michigan, Ohio, Georgia, and Wyoming. Riley offers real estate brokerage, hospitality consulting, creative design, employee training, sales and marketing, and joint venture opportunities. For more information, visit www.rileyhotelgroup.com
About Joseph Allen Agencies:
With over 40 years of combined experience, the principals at Joseph Allen specialize in design, marketing and public relations for a diverse portfolio of clientele. At JAA, they place a significant focus on appealing to human emotion and psyche. They can handle all aspects of design and marketing needs through their internal staff, as well as through their incomparable mix of strategic partners. For more information, visit www.josephallenagencies.com
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text: > View Product Details Please select the size of the product. Please enter valid quantity. BCBGMAXAZRIA rio red jersey 'Kian' side zipper dress overview details Retail: $168.00 Reduced: $44.97 25% Off : $33.73 color : We're sorry. This style is sold out. Rio Red Wiggle into this chic little number from BCBGMAXAZRIA and let the evening begin! Color: Rio Red Lightweight jersey knit Boat neck Short sleeves Ruched skirt with decorative side zipper Unlined Contemporary fit Size S measures approximately 22'' long from natural waist; may vary by size Self: 95% Acetate / 5% Spandex; Lining: 90% Polyester / 10% Spandex; Hand Wash; Imported; style#320193201


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November 26, 2015
NASHVILLE, Tenn. (AP) -- Researchers at Tennessee State University will demonstrate how to turn oilseed crops into biodiesel with a new mobile lab at the school's Small Farm Expo.
The demonstration takes place on Thursday from 8 a.m. to 3 p.m.
According to the school, the mobile biodiesel lab is the showpiece of the University's alternative fuels program.
After the expo, researchers will be taking the lab across the state to demonstrate the process to farmers.
Jason de Koff is assistant professor of agronomy and soil sciences at TSU's College of Agriculture, Human and Natural Sciences. He says a typical farm uses 2 to 6 gallons of diesel per acre every year. But if farmers devote a portion of an oilseed crop to biodiesel, they would not have to buy diesel.
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I tend to be independent, like the outdoors, gardening, hunting, fishing, woodworking, hiking, beekeeping.
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63 • Emeryville, CA • Man
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Working full-time for now. Like to spend time in the Rocky Mountains where I have a 2nd home and am restoring a old homestead orchard plus learning about and doing permaculture.
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There are a lot of good books out there, but my last favorite one is the Resilient Gardener.
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Spending time in the outdoors, fresh homegrown food, exercise.
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If Dorna DO introduce a rev-limit that renders pneumatic valves unnecessary (they are uber-expensive for not much in the way of a power gain or faster lap-time - CRT's and WSBK prove that beyond reasonable doubt), then HRC will have a ready-developed alternative in the 'customer' bikes' motor, along with heaps of set-up data.
Which would mean that HRC get a data and development advantage that no other Mfr or Team could hope to have, meaning that HRC have the best chance for success in MGP.
They can use their chequebook to sidestep any attempts to take the chequebook out of the equation and level the playing field. Or am I just a cynic? Time will tell.


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Irwin to apply for loan to refurbish Main Street theater
Irwin officials will apply for a loan, seeking up to $150,000 as matching funds to rehabilitate the long-vacant Lamp Theatre.
The money would serve as a match to $250,000 in state funds and $250,000 in county funds, council President John Cassandro said.
Council approved the motion 6-0 on Wednesday. Councilwoman Joanna Jordan was absent.
Cassandro emphasized that council is not accepting money, nor are they making a commitment to refurbishing the 1937 theater — just considering it.
"We need to apply for the money," Cassandro said.
Council is not obligating funds, member Bob Wayman said.
"We are only making application to determine the viability or eligibility to receive funds," he said.
Cassandro said council is looking into buying the theater, which has sat vacant along Main Street since 2004. The Westmoreland Cultural Trust owns the theater.
Mike Langer, cultural trust president, said bids to rehabilitate the theater came in between $300,000 and $400,000 higher than expected, and the project stalled.
Cassandro said Irwin officials have met with cultural trust officials.
Council is taking the project one step at a time, he said.
"We're working on financing. We're working on acquisition of the building, even at this point beginnings of operating the theater — but none of that may ever happen," Cassandro said.
He called the Lamp a "historic landmark" and "an important part of our history."
"Council ... is very committed to economic redevelopment of our downtown, and the Lamp Theatre is a part of that," borough manager Mary Benko said.
Relight the Lamp, a community group dedicated to reopening the theater, will continue to be a partner in the project, Cassandro said.
For more information about the Lamp, visit: relightthelamp.wordpress.com.
Rossilynne Skena is a staff writer for Trib Total Media. She can be reached at 724-836-6646.
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Most-Read Westmoreland
- School lunch group hopes to revise rules it calls impractical, too restrictive
- Western Pa. students bristle at changing menu choices
- Keystone Bakery closes Greensburg store
- Puppies' eyes glued shut, South Huntingdon animal shelter says
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I'm afraid this thread is likely to be gender-specific...
As many gentlemen here will know, there's a growing charity fundraising lighthearted event - grow a moustache in November. A short google will inform.
I think the rule is, start clean-shaven on Nov 1. Just hacking back the existing fronds apart from the upper lip doesn't qualify. Then the bushy and sculpted growth is viewed for judgement at the end of the month.
Any past participants care to comment? Any new participants care to commit?
I'm in the land of proud upper lip stuff at the moment, so I might!


text: CVSROOT: /home/cvs Module name: gravity2 Changes by: richard 00/01/17 01:59:17 Modified files: src : graphics_ggi.cc graphics_gtkgl_win.cc graphics_gtkgl_win.h physics.cc universe.cc universe.h Log message: Added frame numbering system. Use it to ensure that each frame is only drawn once, by making universe->draw() return the frame last drawn, which should be passed in as a parameter to the next draw call. Want to implement an option to make sure that each frame _is_ displayed once, if desired. This will require some more re-organisation. This may cause problems with the GTKGL system, if universe->draw is called due to expose events, as well as by idle cycle calls. Need option to ask to redraw current frame.


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So bummed. I was so hopeful. I mean I swear I thought I was pg. You spend your whole life trying not to get pregnant, you just think hey, all you need to do is have sex and ditch the pill!
And to top it off I woke up this morning from the most vivid dream where I tested and got a BFP... how cruel is that!
Also kind of wondering what I can attribute my impressive gassiness too... ARG.. ok vent over! I hope everyone else has much better luck than me! Babydust for everyone!


text:
By On the occasion of our 20th anniversary
Why Do Mexis Like Wrought-Iron Fences?
By Gustavo Arellano
By R. Scott Moxley
By Alfonso Delgado
Women For: OC are Stitching and Bitching
By Courtney Hamilton
Santa Ana River Ready for Its Renaissance
By Joel Beers
By Peter Maguire
Can Tech Ruin the Kim Pham Murder Trial?
By Charles Lam
It was the inaccuracy of the slogan's claim that created the problem. There was no proof that Long Beach's streets were any less dangerous or hostile than any other place in America. The proclamation somehow incited memories—or perceptions, anyway—of a Long Beach style of civic leadership out of touch with basic responsibilities, not to mention constituents . . . and how about honesty?
On top of that, rather than being in council chambers at 5 o'clock to gavel the meeting to order, Mayor Bob Foster was outside the entrance to City Hall, milling around with the rest of the elected officials.
All of them appeared to be blowing it off. Each was paired with a bicycle, a prop for the portrait a photographer was nearly ready to shoot, and as a bank of lights washed the plaza in a movie-premiere glow, they chatted and posed for snapshots. Hanging above them—freshly bolted into City Hall's concrete wall over the weekend—was a sculpture of an iconic pennyfarthing bicycle, rendered by artist Patrick Vogel from parts culled from the police department's stolen-bike graveyard. It was underlined by a shiny metal banner engraved with those same eight words: "Long Beach, The Most Bicycle-Friendly City In America."
In quick succession, the photos were taken and the council members returned to their chamber to begin plowing diligently through the entire agenda, continuing long into the night. But the scene endured. It prompted online comments about unsavory ways city officials can play the system or constituents for perks. It revived memories of the Queen Mary operator who curried favor with officials by providing them with free use of the ship's facilities, usually for political fund-raisers, but also for private parties. It ignited criticism of the Aquarium of the Pacific's rotating offer of access to City Council members, who can cozy up to constituents by providing free admission to residents of their districts.
But bad impressions went even deeper than that, creating the sense that this slogan had become the latest in the long list of those employed over the years as synonyms for Long Beach. City leaders have always longed for a larger-than-life image. Evidence of their desperate search goes back at least to 1897, when a 63-foot finback whale strayed into shallow surf. A few of the locals killed it, picked its carcass clean and displayed the skeleton downtown at Lincoln Park, nicknaming the trophy "Minnie the Whale"; the editor of a local paper proclaimed, "Long Beach is now in possession of [a] wonder of the world." For most of the time since, Minnie's bones have been in the Los Angeles Natural History Museum storage facility in Vernon.
That's led to a lot of nicknames over the years: Queen of the Beaches, Iowa-By-the-Sea, Queen City, the International City, Many Unique Neighborhoods and One Great City, and now the one the bicycle folks just got.
Gandy concedes that Long Beach is absolutely not the Most Bicycle-Friendly City In America and insists deception was never the intention.
"The supporting theory is that it is an aspiration—the direction the city wants to go," he says. "I come from Austin, which in the early 1990s passed a resolution claiming it was the Live Music Capital of the World. That wasn't exactly the case at the time, but it certainly came true—Austin City Limits and South By Southwest are just the most obvious proof."
The payoff, Gandy says, has already begun for Long Beach's Bicycle Friendliness, attracting business partners, donations, volunteers and energy.
"It's served as an interesting magnet for unexpected opportunities, businesses stepping forward to ask what they can do to support us," he says. "That's the magic of that big, hairy goal."
He makes a case for the bicycle project based on reasons ranging from environmental benefits to the generation of business and tourism to improving Long Beach's profile as a great city. The Most Bicycle-Friendly City In America designation may be magical, but it doesn't seem quite so imaginative when you realize the City Council's unanimous vote to claim it as Long Beach's brand came only six months after its unanimous vote in late 2007 to market Long Beach as the Aquatic Capital of America.
But approving the aquatic title didn't require the City Council to look any further than its pitchmen, the passel of old, rich, yacht-club members so influential they arranged for the city's Department of Parks, Recreation and Marine to prepare their PowerPoint presentation. Most were friends of Chris Pook, founder of the Long Beach Grand Prix, and some were board members of the Long Beach Sea Festival that Pook had been running for the city—in fact, running without a contract, thanks to then-city manager Jerry Miller, who disobeyed explicit orders from the council to do that favor.
The Aquatic Capital of America designation was such a done deal that the council seemed ready to ignore the most obvious problem with the name—the then-raging controversy over the Long Beach Breakwater, which had made the city aquatically famous for turning its waterfront into a bedpan.
Finally, council member Rae Gabelich made a delicate attempt. "I would think this is a wonderful thing for us to strive to become," Gabelich began, before cautioning that the title might come across as a little "self-serving" and suggesting that using it to market the city "would be a little disingenuous" at the moment.
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