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This summer, a series of demonstrations in Paris have launched to show how to use “photonic membranes” to cool large areas of land, buildings, and, conceivably, entire cities. Designers from the international climate engineering firm Transsolar and architect Carlo Ratti have teamed up to showcase three different models of this unconventional and high-tech method of addressing climate change. You can check them out yourself from 20 August to 4 October.
The technology used in these demonstrations comes from research at Stanford University. Photonic materials have been highly effective in reflecting direct sunlight, bouncing the rays back into space as infrared light. The top of the membrane stays several degrees cooler than air temperature, creating an unprecedented shaded area for people underneath. The implications for buildings, plazas, and courtyards are seriously impressive, especially when considering how the coverings require no electricity or other resources to run.
The Paris demonstration will consist of three showcases: Greenhouse, Coolhouse, and Treehouse. Visitors can experience these interactive displays, which show how each station would feel based on humidity, wind speed, and ambient air temperature. Using the Universal Thermal Climate Index updated every 15 minutes, observers can see what each of the houses would feel like with that day’s real-time temperature and weather.
Thomas Auer, managing director at Transsolar, says, “We are particularly excited to have had the opportunity to develop this demonstrator in Paris – a city that played a big role in the development of modern greenhouses, all the way from Versailles’ spectacular glass enclosure of the 18th century.” The applications of photonic membranes are vast and could mean a lot for cities struggling with the hottest summer on record. These efforts, combined with a drastic shift toward renewable resources, could mean safer and more comfortable seasons for all.
Images via Transsolar


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VIRGINIA BEACH, Va. — About 20 children were on board a bus that went off the road and into a ditch Thursday morning in Virginia Beach.
Virginia Beach emergency dispatchers said the crash happened around 10 a.m. on Prosperity Road. The bus was near the YMCA at JT's Camp Grom, a 70-acre adventure camp that welcomes wounded veterans, Gold Star families, and adults and children with differing abilities.
A camp counselor with Families of Autistic Children in Tidewater (FACT) said none of the kids were hurt. The bus was taking the children on a field trip to JT's Camp Grom. They were able to continue on to that field trip after they got out of the bus safely.
It was not immediately clear what caused the bus to go into the ditch, but Virginia Beach police confirmed the driver had a "medical emergency" and was taken to a nearby hospital.

domain: news


The Benefits of Bubble Foil Insulation
Put the heat back where it came from.
Reflective insulation, or foil information has proven to be very useful in controlling temperatures in a home. Have you ever wondered how you could save money on how you heat and cool your home? Sure you have! Reflective insulation can do just that.
By using foil bubble insulation in spots where temperatures are difficult to control, such as attics, walls and underneath the floor. You can regulate the temperatures so that your heating and cooling systems don’t have to work so hard.
So how does foil insulation work? The foil works to reflect radiant heat rather than absorb it. Heat travels from warm areas to cooler ones by conduction, convection and radiation. Fiberglass and other mass type insulations only inhibit heat conductions and convection. They do nothing to stop “radiant” heat transfer. The foil insulation reflects radiant heat. It can help keep your home cooler in the summer and warmer in the winter. I turns your house into a thermos.
For example, the sun beats down on a home’s roof which makes the attic temperatures rise, then the heat radiates down into the house, raising the house temperature where the air conditioner has to work overtime. You can reduce attic temperatures by laying foil bubble insulation on the ceiling joists of the attic, or stapling under the roof rafters. The heat is reflected back up by the radiant insulation barrier. In the winter, radiant barrier insulation will reflect the radiant heat energy back into your home. Instead of letting is just penetrate the ceiling, floors and walls mass type insulation.
Foil insulation is required under a “staple-up” radiant floor installation. Radiant floors generate a lot of radiant energy. They are like a light bulb shining energy in all directions. We do not want 50% of that radiant energy to be lost going in the wrong direction. Most radiant floors will not work without it. So with reflective foil insulation below our radiant floors we recover that radiant energy and reflect it back into the room.
There is also Bubble Foil Bubble insulation especially designed for under concrete slabs. The bubble layers protect the foil and give the necessary air gap to allow the foil to reflect the radiant energy.
Bubble foil insulation can be put onto pipes, hot water heaters, stud walls, roofing, and basements. Foil insulation is best in instances where radiation heat is the main source for heat. Foil insulation is easy to install, fire retardant, saves energy and money, requires little maintenance, is non-toxic, is tear and puncture resistant and much more.
Contact us to learn more about radiant heat insulation and why bubble foil insulation is a smart solution for your commercial building or home.
Tagged with: Bubble Foil benefits, Bubble Foil Insulation, Bubble Foil Insulation for Walls & Concrete Radiant Insulation, Insulation Styles, Insulation Supplies Online


The Archive holds a wealth of material about the people, productions and origins of our world-renowned opera house. Come and see curated exhibitions and find out what’s new in the Glyndebourne Archive.
New Year / Royal Festival Hall 1991
Composer: Michael Tippett
Librettist: Michael Tippett
Sung In: English
Notes: Concert performance for the International Series
25 February 1991 at Royal Festival Hall, London view performance details


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By Isaac Akugizibwe
KIU, Main Campus - KIU Environment Students Association, in partnership with the End Plastic Pollution Initiative, have waged war against plastic pollution in KIU and the surrounding communities.
During the launch of the ‘Plastic-Free Campus Program’ on 10th March 2021 at KIU, metallic dustbins were donated to vendors and business owners in and around the university to mark the beginning of the war against plastic pollution.
According to Pius Mugarura, a Youth Co-ordinator and Moblizer, End Plastic Pollution Initiative, the Environmental Student’s Association of KIU was the most appropriate group at KIU that would help promote the idea of fighting plastic pollution. He says that being a student of KIU, and the fact that the End Plastic Pollution Initiative is an idea of a KIU Alumni, convinced them to launch the Plastic-Free Campus Program at the university.
The Plastic-Free Campus program is an initiative started by the ‘Break Free from Plastic’ movement, a global campaign envisioning a future free from plastic pollution.
‘’We started with Kampala International University because it is our society and it is always better to start from home and expand gradually. Also, because the idea of our initiative started with a KIU Alumni which really makes us feel at home, but most importantly it is campuses like KIU that we are targeting to work with,’’ Pius said.
He added that the environment students of KIU were very supportive and willing to do whatever it would take when the idea of fighting plastic pollution was introduced to them. He said that their support helped the initiative to successfully join the rest of the world to fight against plastic pollution in and around school premises through the ‘Plastic-Free Campus' Program.
‘’Once we got the consent of Environment Students Association of KIU, we organized a seminar where we made presentations about the Plastic-Free Campus program after which we were able to launch it for the first time in Uganda,’’ he explained.
Joyce Gwokyalya, the Youth Ambassador for the KIU Environment Students Association, expressed gratitude towards the 'End Plastic Pollution' Initiative and ‘Break Free from Plastic’ for opening their eyes and motivating them to participate actively in the conservation of the environment. She said the launch of the Plastic-Free Campus Program will help boost their visibility in the University and surrounding communities.
‘’This initiave was inspirational. It opened our eyes and now most of the members want to actively participate in the conservation of the environment. I think the launch will really help boost our visibility in the university and its neighborhood to continue promoting a plastic-free campus community,’’ Joyce admitted.
The recipients of the metallic dustbins said the Pollution-Free Campus Program will help improve sanitation at KIU and its surroundings.
Rose Namagembe, a shop attendant at KIU, said receiving a dustbin was relieving because it would help improve sanitation around her work space. Ismael Abdul, a wholesale trader operating outside KIU, said the metallic dustbin will help stop people from throwing plastic bottles into trenches around the university.
The Plastic-Free Campus Program has so far moved into 29 schools in Uganda and will continue to attract participation of other universities in the country. Schools and universities play a special role in developing the mindset, awareness and habits of future citizens and leaders.
Read more about the End Plastic Pollution Initiative - https://endplasticpollution.blogspot.com
Read more about PFC at http://breakfreefromplastic.org


We provide some virtual programming needs such as bot , vps , api documentation , etc.
We make sure your satisfaction will be fulfilled with our working professionality.
API stands for Application Programming Interface, and allows developers to integrate two parts of an application or with different applications simultaneously. API consists of various elements such as functions, protocols, and other tools that allow developers to create applications.
Operating system technology and software that allows a machine with a large capacity to be divided into several virtual machines. Each of these virtual machines serves the operating system and software independently and with fast configuration.
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Immediate download of 2-track album in the high-quality format of your choice (MP3, FLAC, and more), plus unlimited mobile access using the free Bandcamp listening app.
Download includes a redone / remastered version of my cover of "Intensive Care Unit" by Renard.
Dedicated to this really awesome friend of mine who is practically always there for me. He thinks he's not cool and that he's lame and stuff but I think the exact opposite. He'll always be one the coolest guys ever, and I love him to death.
(Yes, that's my real acoustic guitar.)
Vocals have not been put in a pitch correcter or auto-tune. Recorded with software that auto-harmonizes (such as heard in Imogen Heap's "Hide and Seek").
released 19 May 2013
All Music and Lyrics © Nich Kerzman, 2013.
Written for and dedicated to David Alderete (Escar).
Artwork © David Alderete, 2013
Typography by me.
Chord help from my brother, James Kerzman.
all rights reserved
feeds for ,

domain: artistic


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Sunday January 30, 2011
Daz’s first single, produced by himself and Ivan Johnson, off his upcoming album D.A.Z., that is supposed to hit shelves in April.
Download: Daz Dillinger – D.estruction A.dds up to Z.ero
It‘s quiet in here! Why not leave a response?
Click here to cancel reply.
Mail (will not be published) (required)
Notify me of follow-up comments by email.
Notify me of new posts by email.
© 2009-2016 Copyright Blackout Hip Hop


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Following are the top grossing movies of 2013. Which ones did you see, either in theatres or on DVD? (Select all that apply)
From the 14 movies listed above, which in your opinion was the the best one?
Here are 14 more movies released in 2013. Which ones did you see?
In your opinion, what was the best movie released in 2013?

domain: entertainment


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This specific ISBN edition is currently not available.View all copies of this ISBN edition:
"synopsis" may belong to another edition of this title.
Harry Kraus, M.D., is a practicing general surgeon and the author of eight novels, including the best-selling Could I Have This Dance? and The Chairman. He received his M.D. degree from Medical College of Virginia, completed his surgical internship and residency at University of Kentucky, and formerly practiced in Virginia. Dr. Kraus is currently serving with Africa Inland Mission as a missionary surgeon at Kijabe Hospital in Kenya, where he lives with his wife and three sons. Visit his website at www.cuttingedgefiction.com.
"About this title" may belong to another edition of this title.
Book Description Hardcover. Condition: Good. No Jacket. Pages can have notes/highlighting. Spine may show signs of wear. ~ ThriftBooks: Read More, Spend Less 1.3. Seller Inventory # G0786213663I3N00

domain: medical


Who out there is lucky enough to be getting Monday off for MLK Day?! Our office is! But before we all head out for the 3-day weekend, there’s one last thing to do (duh). So let’s take a look at some of the hottest releases from the week!
Red Baraat is our one and only album stream of the week, and rightfully so. No other album can compete with what these guys are throwing down! The brass band utilizes infectious rhythms from Hip-Hop, Funk, Cumbia, and tons of other genres to create an undeniably danceable album. You’ll want to have this playing at your next party, so preview it now.
For a totally different sound (think mellowed-out Indie Rock, much in the style of some Sparklehorse tracks), try out The Black Ryder, the Aussie-based duo who’s about to rock your world. Next, there’s Jesse Harris‘ latest: a Folky, almost Jazzy frill of guitars and harmonies with pal Star Rover. Play this track in the midst of your early-morning, post-night-out haze. Then turn to Fucked Up‘s Young Guv (AKA Ben Cook) for a Funkadelic groove — surprising coming from him, right?! But SO worth the listen. Follow that up with Vision Fortune‘s Jam, completely warped and electronically heady. Top it all off with Dirty Dishes and their masterful romp of Psychedelic Alt-Rock, and you’ll be all set for a last-minute road trip with this star-studded lineup!
When you’ve got some free time for your eyes, be sure to check out this trio of music videos, starring: Malik B & Mr. Green, ft. Philly urban street scenes; Mount Eerie, ft. trippy, distorted dream images; and Magalie, ft. a totally 80s fluorescent dance-a-thon.
Stream or download, whatever you choose, but make sure you hit up all these fantabulous releases from the week. And most importantly, have a phenom weekend!
Red Baraat: Full album stream via NPR
Gaadi of Truth out January 20 on Sinj Records
The Black Ryder: “Seventh Moon” track premiere via Louder Than War
The Door Behind the Door out February 24 on The Anti Machine Machine
Jesse Harris and Star Rover: “Catch the Ash” track premiere via PopMatters
No Wrong No Right out February 10 on Dangerbird Records
Young Guv: “Wrong Crowd” track premiere via Fader
Ripe 4 Luv out March 10 on Slumberland
Vision Fortune: “Back Crawl II” track premiere via Fader
Country Music out February 9 on ATP/Recordings
Dirty Dishes: “Guilty” track premiere via Entertainment Weekly
Guilty out January 27 on Exploding in Sound
Malik B and Mr. Green: “We Gonna Make It” music video premiere via OK Player
Unpredictable out February 24 on Enemy Soil
Mount Eerie: “This” music video premiere via Stereogum
Sauna out February 3 on P.W. Elverum & Sun
Magalie: “Love Criminal” music video premiere via Vevo
Love Criminal out February 15 on Ovation Records


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Nicholas P. Negroponte, the driving force behind One Laptop Per Child—the non-profit organization that provides low-cost laptops to children around the globe—recalled the entrepreneurial obstacles he faced despite his philanthropic mission.
His presentation, jointly sponsored by the Harvard Undergraduate Women in Business and the Leadership Institute at Harvard College, provided students an opportunity to engage with a successful social entrepreneur and hear about the difficult progress Negroponte’s organization has made since its formation in 2005.
The OLPC has provided low-cost laptops to 2.5 million children in 40 countries since 2005, but Negroponte said the path has been challenging.
He said his concept of a cheap laptop was initially dismissed, but contacts he acquired while working at MIT since the late 1960s helped the project gain traction.
Still, it lacked commercial interest until word of the project began to spread.
He soon drew interest from long-established computing giants Intel and Microsoft, he said, introducing competition into the market and providing new, unforeseen challenges for his philanthropic organization.
“The real difference was between thinking about children as a mission versus a market,” Negroponte said.
He said he expected his organization’s “Mother Theresa shield”—the protection afforded by OLPC’s partnership with the United Nations and what he considered the goodwill of its cause—would help the organization avoid the corporate pressure.
In addition, Negroponte said, the OLPC met criticism on how they were verifying their project’s success, lacking evidence showing that giving a kid a laptop and walking away is a successful strategy.
Negroponte and the OLPC are now addressing this concern, he said, by implementing empirical research to see if children will learn to read—and read to learn—when given a laptop to use at their whim.
The OLPC project arose, Negroponte said, from an observation of a friend and MIT mathematician Seymour A. Papert, an early pioneer of artificial intelligence.
Papert, in his research on technology and learning, observed that programming and debugging computer software offered children incredible learning experiences.
Together, Papert and Negroponte brought this insight to Senegal and Pakistan, where they offered children the opportunity to use computers and were blown away by the pace at which they learned how to operate them, Negroponte said.
“These kids played the computers like pianos,” he said.
Audience member Tina Liu ’11, who said she is interested in a career in social enterprise, said she viewed OLPC’s story as a reality check.
“It was surprising to see a non-profit with a glorious mission meet such obstacles,” she said. “But at the same time, their success is very encouraging.”


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For the second straight season, the Rockford IceHogs pulled off a two-game sweep in the play-in series to advance to take on the regular-season champs in the Central Division Semifinals. Last season they beat the Texas Stars in two games but were beaten soundly in a three-game sweep at the hand of the eventual Calder Cup champion Chicago Wolves. This time around, they swept the Iowa Wild with back-to-back overtime wins for a rematch against the Stars.
The IceHogs should be more competitive against Texas than they were versus Chicago a year ago. Both teams are loaded with AHL veterans and like to play an up-tempo game. Rockford added more veterans this season, like David Gust, Bret Seney, Luke Philp, and Rocco Grimaldi, just for a series like this.
“They’ve been massive,” head coach Anders Sorensen told us on a recent episode of the CHGO Blackhawks podcast. “Having those guys in the locker room helps the younger guys. It helps the Reichels and the Vlasics. We as coaches can get nagging at times, so sometimes hearing it from a teammate, especially an older guy, can create a certain culture. When you look at the habits of Gust, Philp, or Seney – their habits are excellent in terms of the way they play. That rubs off on everybody else.”
The Stars won the Central Division by earning 92 points, 13 more than the IceHogs. However, the IceHogs won five of their eight regular-season meetings with Texas.
“That’s definitely better than going 0-8 against them,” IceHogs forward Buddy Robinson said on the CHGO Blackhawks podcast. “We know they are a good team. They are at the top of the division for a reason. To know we are right there with them and played good games against them all year, we are confident.”
As mentioned earlier, both teams like to move quickly up the ice and play aggressively when they have the puck. This likely means the two netminders will be the most important players for both sides. Texas goaltender Matthew Murray (not that Matt Murray) has been terrific since locking down the starter’s crease. He went 18-10-3-2 with a 2.37 goals-against average (GAA) and .911 save percentage (SV%).
Arvid Soderblom will draw the start for Game 1 of the series. It’s been an up-and-down season for the second-year pro. After playing well in his stint with the Chicago Blackhawks, he struggled when he returned to Rockford before an injury sidelined him for three weeks. While his 2.92 GAA and .905 SV% on the season don’t jump off the page, he is currently playing his best hockey of the season. In his 10 starts, Soderblom has a 2.20 GAA and .917 SV%.
“Arvid had a little meat on his bones after playing for us last year,” Sorensen said. “It was a tough season to start. He didn’t do as well up here as he or anyone wanted. When he got back to us, he had to battle through a major injury, which he’s never had to do before. The way he’s playing right now, he’s back to where he was last year. He’s been awesome.”
Due to the AHL’s unorthodox playoff format, the Stars may have a disadvantage despite winning the division. They haven’t played in 12 days as the rest of the Central waited for the IceHogs and Wild to duke it out. In addition to the extended time off, they will have to play the first two games of this series on the road due to travel costs. This hardly seems like a reward for finishing in first place, as there is a chance they could be facing elimination before they even step foot on home ice. The IceHogs won three of the four contests at the BMO Center during the regular season.
“It’s a little blessing that we got a couple of playoff games under our belt already,” Robinson said. “We’re feeling good. We’re feeling what it’s like to be in tight games. We are going to carry that over into Friday. Hopefully, we jump on them early because we’ve had a couple of games to get into playoff hockey.”
This series will ultimately come down to the goaltenders and the veterans. Stars captain Curtis McKenzie will be one to watch out for. He has been to the Calder Cup Finals twice, with the Stars team that beat the IceHogs in the 2015 Western Conference Finals and again with the Wolves in 2019. He had a six-game point streak before being held off the scoresheet in the regular-season finale. McKenzie is a warrior, and you aren’t going to show him anything he hasn’t seen before. For the IceHogs, Gust is picking up where he left off last postseason, with a goal in each game against Iowa.
There are some youngsters to watch as well. Of course, all eyes will be focused on Lukas Reichel, who is looking for his first point of the playoffs. Defenseman Alex Vlasic has two assists, including one on Robinson’s series-clinching goal against the Wild. Isaak Phillips and Michal Teply both have a goal and an assist so far. For Texas, Mavrik Borque finished his regular season scorching hot with seven goals and 11 points in his last seven games.
The IceHogs have an advantage early with the first two games at the BMO Center and the Stars not playing in nearly two weeks. If they can make the most of these factors, they have a real shot to pull off an upset and move on to the Central Division Finals against either the Milwaukee Admirals or Manitoba Moose.
GAME 1: Friday, Apr. 28 @ IceHogs, 7 p.m. CT
GAME 2: Sunday, Apr. 30 @ IceHogs, 4 p.m. CT
GAME 3: Wednesday, May 3 @ Stars, 7 p.m. CT
GAME 4*: Friday, May 5 @ Stars, 7 p.m. CT
GAME 5*: Saturday, May 6 @ Stars, 7 p.m. CT
Regular Season Team Stats
Regular Season Team Leaders
|Rocco Grimaldi – 33
|Riley Barber – 32
|Grimaldi – 40
|Will Butcher – 37
|Grimaldi – 73
|Barber – 64
|Luke Philp – 11
|Barber – 9
|Grimaldi – 23
|Butcher – 20
|Hunter Drew – 149
|Riley Tufte – 90
Photo credit: Brad Repplinger (Rockford IceHogs)
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Glynn K. Maxwell June 21, 1943- February 15, 2021
Once upon a time there was a dog named Peewee. And Peewee had some dog friends and they were Blackie, Brownie, Spot and sometimes Rex……
Thus began many a bed time story from my dad. As many of you know, our dad was a storyteller. Spend any time with him, or in his office, and the majority of the time you would would come away with a story about something. A lot of the time, you would get a quick lesson about the Civil War, the cold war, World War 2, or even a story or two about Grant Parish. So, in the spirit of my dad, I’m going to tell you a story.
Glynn Keith Maxwell was born on June 21, 1943 at the crossroads of Hwy 123 and Hwy 8. At some point, Papaw Bill and Mamaw Chloe moved dad and his brothers,Larry and Billy to Dry Prong where his adventures with Peewee, a chihuahua mix, were born. He attended school in Dry Prong, and as he liked to say, he gave his teachers some grief. I have a feeling he got in trouble for too much talking. As a teacher myself, that does not surprise me one bit. Dad played basketball for Dry Prong High School, and loved to regale us with the tales of playing on the court at the Dry Prong gym. He loved to tell the stories of how the Dry Prong Wildcats were the best in the state and how they would run opponents ragged because the Dry Prong court was longer than regulation high school courts at that time.
After graduation, Dad would go on to attend Northwestern State University and then enlisted in the US Air Force where over his four years, his time would include a very close encounter with JFK and Jackie at Carswell AFB about an hour or so before JFK was assassinated. When I asked him what stuck out to him the most, he said, JFK had on the most beautiful pin striped suit and a country boy, I thought it was the most beautiful suit that I had seen.” After a posting in Pakistan at a base ‘that was there, but wasnt’, Dad returned home and would eventually marry the girl next door, our mom, Robbie Creed and have four children.
During the 80s, dad served as Mayor of Dry Prong for two terms, with one of the highlights being the invitation to the White House to meet then President Reagan and VP Bush after he and the entire town council switched to the Repulican Party. Something that got Dry Prong quite a bit of press at the time. It was also during this time that he co-founded KVDP, Dry Prong’s radio station.
Dad’s storytelling and writing abilities would serve him well throughout the years. It made people feel at ease no matter if they were in his office at Southern Funding, or if he was interviewing them for the Dry Prong Village Voice or the Colfax Chronicle. He loved to write about the Civil War, as many of you know. He wrote and spoke extensively about the Civil War as it related to Central Louisiana.
He could talk your ear off about the Red River Campaign, and as kids, we can attest to reading pretty much every single historical marker that we came across. I attribute my knowledge of seemingly useless information from some of our ‘rides’ that are a family tradition. Sometimes those “Maxwell shortcuts” would lead to an adventure that we would tell stories about years later.
I am sure that all of you have stories to tell about my dad, and I’m sure that we have heard stories (all good, don’t worry!) about ya’ll. We would love for you to tell us those stories.
In the spirit of dad, I will ask you to do something. Tell your stories to your kids and grandkids and your great grand kids. Ask your relatives to tell stories. My students love when I tell them stories of how I grew up in Louisiana.When our loved ones are gone, these stories become treasured memories.
As for Dad, I’m sure he’s up there talking Mom’s ear off and telling stories to Uncle Bob, Uncle Larry and Pawpaw Bill and Mamaw Chloe.
Can you do me a favor? If you see a little redheaded Clark that looks like me, running around up there, can you tell them a Peewee story for me? Love you.
Beautifully written. So sorry for your loss.

domain: library


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ORIGINAL RESEARCH article
LINC00518 Promotes Cell Proliferation by Regulating the Cell Cycle of Lung Adenocarcinoma Through miR-185-3p Targeting MECP2
- Department of Thoracic and Cardiovascular Surgery, The Sixth Affiliated Hospital of Nantong University, Yancheng, China
Previous studies have shown that long intergenic non-protein coding RNA 00518 (LINC00518) are essential for the cell growth and metastasis of human cancer. However, the role of LINC00518 in lung adenocarcinoma (LUAD) is still unknown. This research put emphasis on the function of LINC00518 on the cell growth of LUAD. The lncRNA, miRNA and mRNA expression were measured by using qRT-PCR. Protein levels were measured by using Western blotting. CCK-8, colony formation assays and transwell assay were performed to evaluate the cell proliferation ability and invasion. Bioinformatic analysis and luciferase reporter assays were chosen to confirm the mechanism of LINC00518 in LUAD. We found that LINC00518 was highly expressed in LUAD specimens and the high-expression was negatively correlated with the overall survival rates. This finding was also proved in the LUAD cell lines. Through a series of in vitro and in vivo experiments, we proved that LICN00518 promoted the cell growth of LUAD by regulating the cell cycle. Moreover, LICN00518 upregulated the expression of MECP2 by mutagenesis of miR-185-3p. The results suggested that LICN00518 could be used as a survival indicator and potential therapeutic target for LUAD patients.
Lung cancer still remains the highest rates of morbidity and mortality among all malignancies (1). At present, more than 800,000 new cases of lung cancer are reported annually all over the world, of which 700,000 are fatal (2). Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer, accounting for 85% of lung cancer, and consists of three main subtypes: lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LSCC), and large cell carcinoma (3). In addition, LUAD accounts for the majority of NSCLC. At present, the main effective treatment methods for lung cancer are: surgery, radiotherapy and chemotherapy, immune and targeted therapy (4, 5). Despite these recent advances in the treatment of lung cancer, the 5-year survival rate is still not satisfactory (6). Therefore, early diagnosis and molecular targeted therapy are critical to enhance the prognosis of patients with LUAD.
Long intergenic noncoding RNAs(lincRNAs)have been highlighted as new regulatory molecules characterized by cell-type specific expression and subcellular compartment localization (7). LincRNAs play a critical role in the pathogenesis and development of cancer by interacting with cancer related genes (8, 9). Previous studies have shown that LINC-PINT promotes cell proliferation through EZH2 and predicts poor prognosis in clear cell renal cell carcinoma (10). In addition, Linc-ROR targets FGF2 to regulate HASMC proliferation and migration via sponging miR-195-5p (11). LINC00518 was found to be highly expressed in many human cancers including gallbladder cancer and colorectal cancer and acted as a functional player in tumor cell proliferation, migration and invasion (12, 13). Nevertheless, the specific role of LINC00518 in LUAD needs more complete exploration.
In this manuscript, the authors first discovered that LINC00518 was highly expressed in LUAD tissues and cell line. In vitro and in vivo experiments have demonstrated that LINC00518 promoted cell proliferation by regulating the cell cycle of LUAD through miR-185-3p targeting MECP2. The LINC00518/ miR-185-3p/MECP2 axis may have therapeutic potential for lung cancer tumorigenesis and development.
Materials and Methods
Patients and Tissue Samples
Since 2019, 42 LUAD patients were selected form The Sixth Affiliated Hospital of Nantong University. This research was approved by the Ethics Committee of Yancheng Third People’s Hospital. Patients who received preoperative radiotherapy or chemotherapy, and those who had a history of other cancers were excluded from the study. All patients voluntarily participate in this study.
Cell Culture and Transfection
SPCA1, H1299, PC9, A549(LUAD cells), 16HBE (human bronchial epithelial) cells and HEK293T cells were purchased from the Chinese Academy of Sciences (Shanghai, China) and were cultured in DMEM medium (Gibco, NY, USA), with 10% fetal bovine serum (Gibco, NY, USA), and placed in a 37 °C incubator containing 5% CO2. LINC00518 siRNA, LINC00518 overexpression plasmid, miR-185-3p mimics, miR-185-3p inhibitor, NC were purchased from Genechem (Shanghai, China). Transitory transfection was performed using lipofectamine 3000 (Invitrogen, USA) in accordance with the manufacturer’s instructions.
Isolation of Total RNA and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was isolated from LUAD tissues or cells using Trizol reagent (Invitrogen, USA). Reverse transcription was performed according to the instructions of Prime Script RT reagent (Takara, Kusatsu, Japan). QRT-PCR was conducted by using SYBR Green Master Mix I (Takara, Kusatsu, Japan) on an ABI 7500 Fast Real Time PCR System (ABI, CA, USA). miRNA samples were calibrated with U6, lncRNA and mRNA samples were calibrated with GAPDH. Relative quantification was performed by using the 2-ΔΔCT method. For miRNAs, primer probes were designed and synthesized by Gemma (Shanghai, China). For mRNA, Primer 5.0 (Gemma, Shanghai, China) was used to design the gene primers. All the oligonucleotide sequences used in the experiment are shown in Table 1.
RNA Binding Protein Immunoprecipitation (RIP)
The EZMagna RIP kit (Merck, Darmstadt, Germany) was used to perform RIP analysis according to the manufacturer's protocol. First, the RIP lysis buffer was used to lyse HEK293T cells. And then the lysate products were Incubate with magnetic beads at 4°C for 6 hours that were pre-conjugated with anti-Ago2 or anti-IgG antibody. Protease K was used to obtain purified RNA by eliminating proteins. Finally, the mRNA level was determined by qRT-PCR.
Colony Formation Assay
The LUAD cells of each group in the logarithmic growth phase were inoculated on a 35mm petri dish (Corning, NY, USA) with 10 mL of 37°C pre-warmed culture medium and cultured for 2 weeks. Then, the petri dish was stained with crystal violet staining solution (Beyotime, Shanghai, China), and colonies with ≥10 cells were counted under the microscope with low magnification. After all preparations, three independent experiments were carried out.
Cell Counting Kit-8 (CCK-8) Analysis
Stably transfected LUAD cells were incubated in 96-well plates (Corning, NY, USA) and then stained with 10 μl of CCK-8 solution (Dojindo, Tokyo, Japan) for 2 hours at 37°C. Each absorbance (A450) was measured at 450 nm using a spectrophotometer. Each sample was run in triplicate.
First collect the cells in each group, centrifuge and suspend them in 0.3ml of PBS containing 10% calf serum, then add 0.7ml of absolute ethanol to fix the cells and place them at -20°C for 24 hours. Discard the supernatant and resuspend the cells with 1ml PBS, and then centrifuge to wash the cells once. After discarding the supernatant, 100μL RNase A was added to the cells, and then placed at 37°C for 30 minutes. Then, 400 μL of propidium iodide (PI) was added to the cells and incubated in the dark for 10 minutes. Finally, the cell cycle was detected by flow cytometry (BD Biosciences, Detroit, USA).
Luciferase Report Analysis
The 3'-UTR sequence of MECP2 and LINC00518 containing the binding site of miR-185-3p were inserted into the promoter vector (Promega, Madison, USA). HEK293T cells were seeded in six-well plates (Corning, NY, USA) and transfected with related oligonucleotides and luciferase reporter plasmids by the Lipofectamine 3000 (Invitrogen), and transfected cells were obtained after 48 hours. Relative luciferase activity was evaluated using a luciferase assay kit (Promega).
Total protein was isolated from cells by using RIPA Lysis Buffer (Beyotime, Shanghai, China). BCA Protein Assay Kit was used to evaluate the protein concentration. The extracted protein was separated by 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, CA, USA). After blocking with 5% nonfat dry milk for 1 hours, the membrane was cultured with specific primary antibody at 4 °C overnight. The membrane was later cultured with HRP-conjugated secondary antibody (1:1000) for 2 hours at room temperature. An enhanced chemiluminescence (ECL, Rockford, USA) detection system was applied to detect protein blots.
5-Ethynyl-2′-deoxyuridine (Edu) assay
Cells are seeded into 96-well plates (5×104 cells/well). Add DMEM (10% FBS) containing 100uLEdU (50μmol/L) to each well and incubate for 2 hours. After washing the cells with PBS, fix them in 4% paraformaldehyde for 15 minutes. 1x ApolloR reaction mixture (400μL) was add to react with EdU (RiboBio, China) at room temperature for 30 minutes, and infiltrate with 0.5% TritonX-100 for 10 minutes. Then the cells was washed with PBS, and incubated with Hoechest33342 (500μL) at room temperature for 30 minutes. After washing the cells with PBS, observe the cells through a fluorescence microscope and take pictures. The mean number cells of each sample in the three fields were counted under the microscope.
Animal experiments were approved by the Ethics Committee of Yancheng Third People’s Hospital. si-LINC0051 cells transfected with lentivirus were injected into 4-week-old nude mice (five mice per group). Tumor volume were measured every 4 days. 24 days after Injecting, the mice were euthanized, and then the tumors were resected for weighting. PerkinElmer IVIS Spectrum (Xenogen, CA) was used for in vivo imaging.
All specimens were fixed with formalin and embed them in paraffin. After the specimen was sectioned, it was deparaffinized with xylene and then hydrated with alcohol. Then the section of specimens were incubated with primary antibodies Ki-67 and P-Akt (Abcam, USA) at 4°C overnight, and then HRP-conjugated secondary antibodies were added and incubated at 4°C for 1 hour. Subsequently, DAB developer was added for dyeing and counterstained with hematoxylin. The images were taken by fluorescent microscope. Three visual fields were selected for each group.
All experimental data were obtained using SPSS 23.0 and GraphPad software 7.0. The P-values were analyzed by performing Student’s t-test, one-way ANOVA and Spearman’s test. P < 0.05 was considered statistically significant.
LINC00518 Was Over-Expressed in LUAD Tissues and Cell Lines
Through the TCGA database, we found that compared with normal lung tissues, the expression of Linc00518 in cancer tissues of LUAD patients was significantly increased (Figure 1A). In addition, data from the TCGA dataset showed that patients with high expression of LINC00518 had poorer overall survival (Figure 1B). Due to the differential expression of LINC00518 in the database, we used qRT-PCR to detect the expression of LINC00518 in tumor tissues and adjacent tissues in 42 pairs of LUAD samples. The data showed that LINC00518 expression was significantly higher in LUAD tumor tissues compare with normal lung tissue (Figure 1C). In addition, the expression of LINC00518 in a series of human LUAD cell lines, were further verified this finding. The expression of LINC00518 in LUAD cell lines (SPCA1, H1299, PC9, A549) was significantly higher than that of 16HBE (Human bronchial epithelial cells) (Figure 1D). As shown in Table 2, in clinical samples, patients with larger tumor size and late pathological stage (TNM stages) have higher LINC00518 levels. Above all, LINC00518 may play an important role in the proliferation of LUAD cells.
Figure 1 LINC00518 was over-expressed in LUAD tissues and cell lines. (A) The expression level of LINC00518 in LUAD tumor tissues and normal tissues form the TCGA database. (B) Survival rate of patients with high and low LINC00518 expression. (C) LINC00518 expression in LUAD tumor tissue compared with normal adjacent tissue. (D) Expression level of LINC00518 in human bronchi Epithelial Cells (16HBE) and LUAD cell lines(SPCA1, H1299, PC9, A549). Data are represented as mean ± SD. (*P < 0.05, **P < 0.01, ***P < 0.001).
Table 2 The Correlation between LINC00518 Expression and Clinicopathological Features in 42 LUAD Patients.
LINC00518 Promotes LUAD Cell Proliferation
In order to study the role of LINC00518 in LUAD cells, we performed RNA interference (small interfering RNA (siRNA)) technology to transfect A549 and PC9 cells respectively, and upregulate LINC00518 by transfected pcDNA-LINC00518 in SPCA1 as overexpression group. Through qRT-PCR analysis, we found that the expression of LINC00518 in A549 and PC9 cells transfected with si-LINC00518 was significantly inhibited, while the expression was increased significantly by transfecting with pcDNA-LINC00518 in SPCA1 cells (Figure 2A). Through the CCK-8 assay, compared with the NC group, the cell proliferation of the si-LINC00518 group was significantly reduced, while enhanced LINC00511 expression promoted the proliferation of SPCA1 cells. (Figure 2B). The results indicated that down-regulate the expression of LINC00518 can inhibit the proliferation of LUAD cells. The colony formation experiments also showed that the number of colonies formed by A549 and PC9 cells in the si-LINC00518 group were significantly less than that in the NC group, while enhanced LINC00511 expression produced the opposite effect (Figure 2C). Similarly, Edu assays showed the same results that LINC00518 affect LUAD cell proliferation (Figure 2D). Through flow cytometry analysis, we explored the effect of LINC00518 on the cell cycle of LUAD. As shown in Figure 2E, the down-regulation of LINC00518 caused an increase in the proportion of G1 in A549 and PC9 cells, while the proportion of cells in the S phase decreased. These findings indicated that the expression of LINC00518 accelerates the proliferation of LUAD cells by extending the proportion of S phase and reducing the proportion of G1 phase. In addition, we measured the protein level of p-Akt, P-Erk and cyclin D1 in the LUAD cell lines by Western blotting (Figure S1). By inhibiting the LINC00518 expression, the protein levels of proliferation-associated protein (p-Akt, P-Erk and cyclin D1) were significantly reduced. The opposite result was obtained after overexpressing LINC00518. Through the above series of in vitro experiments, we confirmed that the overexpression of LINC00518 can promote the proliferation of LUAD cells.
Figure 2 LINC00518 affects LUAD cell proliferation by regulating the cell cycle. (A) A549 and PC9 cells were transfected with si-LINC00518 respectively, while SPCA1 cells were transfected with pcDNA-LINC00518. The results were validated by qRT-PCR.NC and blank were used as controls. (B) CCK-8 assay was applied to detect the proliferation of A549,PC9 and SPCA1 cells transfected with si-LINC00518 or NC. (C, D) Colony formation analysis and Edu assays in A549, PC9 and SPCA1 cells transfected with si-LINC00518,pcDNA-LINC00518 or NC. (E) Cell cycle distributions were analyzed in A549, PC9 and SPCA1 cells, respectively by Flow Cytometry. All data are expressed as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
Knockdown of LINC00518 Inhibits LUAD Tumor Growth In Vivo
We further evaluated the effect of LINC00518 on the growth of LUAD tumors in vivo by inoculating A549 cells transfected with si-LINC00518 into nude mice. The results showed that the low expression of LINC00518 can significantly reduce tumor growth in vivo (Figures 3A-C). At the same time, the LINC-00518 mRNA level of tumor tissues of nude mice which inoculated with A549 cells transfected with si-LINC00518 was significantly decreased (Figure 3D). The results of IHC staining showed that the expression of Ki-67 and p-Akt is consistent with the results of LINC00518 promoting the proliferation of LUAD cells (Figure 3E). These results indicated that LINC00518 promotes tumor formation in vivo.
Figure 3 Knockdown of LINC00518 inhibits LUAD tumor growth in vivo. (A) Comparison of the effects of transfection with si-LINC00518 or NC on tumor growth in nude mouse models. (B, C) Analysis of tumor size and weight in different groups. (D) LINC00518 expression changes were identified by qRT-PCR. (E) The expression of Ki-67 and P-Akt were detected by IHC of sections from the xenografts (Scale bar, 50 μm). *P < 0.05, **P < 0.01, ***P < 0.001.
LINC00518 Serves As Competing Endogenous RNA for miR-185-3p in LUAD
Previous studies have shown that LINC00518 plays a role in tumors through the ceRNA mechanism. We used Starbase to predict the LINC00518/miRNA interaction. Finally, we selected miR-185-3p as the research object. QRT-PCR nuclear and cytoplasmic fractions showed that LINC00518 was mainly located in the cytoplasm of LUAD cells (Figure 4A). This indicates that LINC00518 may have an effect on post-transcriptional epigenetic regulation. Next, we confirmed that both wild-type and mutant-type LINC00518 have multiple binding sites with downstream miR-185-3p (Figure 4B). We also found that miR-185-3p expression was decreased in 42 paired LUAD tumor tissues and LUAD cell lines (Figures 4C, D). In the luciferase reporter assays, we found that the luciferase activity of the LINC00518-WT was significantly reduced by transfecting with miR-185-3p mimic (Figure 4E). The results of the RIP assay further verify the direct interaction between miR-185-3p and LINC00518. Compared with IgG antibody, the relative RNA expression levels of miR-185-3p and LINC00518 were significantly increased in the immunoprecipitation formed by the Ago2 antibody (Figure 4F). In addition, we also found that the mRNA expression levels of LINC00518 was negatively correlated with that of miR-185-3p by qRT-PCR (Figure 4G). All results indicated that LINC00518 may act as the ceRNA for miR-185-3p in LUAD.
Figure 4 LINC00518 serves as competing endogenous RNA for miR-185-3p in LUAD. (A) The subcellular position of LINC00518 on the cytoplasm or nucleus were analyzed by qRT-PCR. 18S and U6 acted as the cytoplasm and nuclear control. (B) The putative binding site of miR-185-3p on LINC00518, and LINC00518 MUT. (C) The expression level of miR-185-3p in LUAD tissues and adjacent tissues were detected by qRT-PCR. (D) The expression levels of miR-185-3p in H1299, PC9, A549, SPCA1 and 16HBE were detected by qRT-PCR. (E) Luciferase assay of HEK293T cells transfected with LINC00518 WT or LINC00518 MUT reporter together with miR-185-3p or NC. (F) RIP assay was used to further verify the direct interaction between miR-185-3p and LINC00518. (G) A negative correlation was found between the mRNA level of LINC00518 and miR-185-3p in 42 LUAD tissues. Data are represented as mean ± SD. (*P < 0.05, **P < 0.01, ***P < 0.001).
LINC00518 Regulate LUAD Cell Proliferation and Cell Cycle by Inhibiting miR-185-3p Expression
Transfection of si-LINC00518 in A549 and PC9 cells can significantly upregulate the expression of miR-185-3p. While transfecting with miR-185-3p inhibitors, the promotion effect of si-LINC00518 on miR-185-3p expression can be significantly reversed. On the contrary, miR-185-3p expression was down-regulated by overexpressing LINC00518 in SPCA1 cells (Figure 5A). CCK-8 assay and colony formation assays showed that knockdown of LINC00518 inhibited the proliferation of LUAD cells, and this inhibition was rescued by co-transfection with miR-185-3p inhibitor (Figures 5B, C). By flow cytometry analysis, we found that si-LINC00518 co-transfected with miR-185-3p inhibitor can significantly decrease the ratio of G1 and increase the ratio of S-phase cells in A549 and PC9 cells (Figure 5D). All these results demonstrated that LINC00518 promoted LUAD cell proliferation and regulate cell cycle by down-regulating miR-185-3p expression.
Figure 5 LINC00518 regulate LUAD cell proliferation and cell cycle by inhibiting the miR-185-3p expression. (A) The expression levels of miR-185-3p in LUAD cells following transfection with NC, LINC00518 siRNA, LINC00518 siRNA plus miR-185-3p inhibitor, pcDNA-LINC00518 or pcDNA-LINC00518 plus miR-185-3p mimics. (B, C) By CCK-8 assay and colony formation assays were used to detect the cell proliferation ability after transfecting A549, PC9 and SPCA1 cells respectively. (D) Cell cycle distributions were analyzed in transfected LUAD cells by Flow Cytometry. *P < 0.05, **P < 0.01, ***P < 0.001. Data are represented as mean ± SD. (*indicates P < 0.05, **P < 0.01).
MECP2 Is a Target of miR-185-3p Regulated by LINC00518 in LUAD Cells
Starbase were also used to predict the putative targets of miR-185-3p. The 3’-UTR of MECP2 has the same binding sites that LINC00518 combined with miR-185-3p (Figure 6A). By searching the TCGA database, we found that MECP2 was highly expressed in LUAD samples (Figure 6B). The expression of MECP2 detected by IHC from the HPA database demonstrated the consistent results (Figure 6C). The luciferase activity of the MECP2-WT was significantly reduced by transfecting with miR-185-3p mimic, but not that of the mutant plasmid (Figure 6D). In addition, through qRT-PCR and West blotting, relative mRNA and protein levels of MECP2 were obviously reduced by transfecting with si-LINC00518 (Figures 6E, F). These results confirmed that MECP2 is a target of miR-185-3p and LINC00518 regulate the expression of MECP2.
Figure 6 MECP2 is a target of miR-185-3p regulated by LINC-00518 in LUAD cells. (A) The binding sites of miR-185-3p on the 3′-UTR of MECP2. (B) Relative expression of MRCP2 in LUAD tissues compared with adjacent normal tissues from the TCGA database. (C) The expression of MECP2 were detected by IHC from the HPA database. (D) Luciferase assay of LUAD cells transfected with MECP2-WT or MECP2-MUT reporter together with miR-185-3p or NC. (E, F) Relative mRNA and protein levels of MECP2 in A549, PC9 and SPCA1 cells transfected with NC, si-LINC00518 or pcDNA-LINC00518. Data are represented as mean ± SD. (*indicates P < 0.05, **P < 0.01).
Among all malignant tumors, the incidence and mortality of lung cancer are still at the forefront. Although immunization and targeted therapy have achieved satisfactory results in the diagnosis and treatment of lung cancer, more effective target genes are still needed for early diagnosis and efficacy prediction of lung cancer.
More and more studies have shown that LncRNA plays an important role in the biological process of tumors, such as cell proliferation, differentiation, migration and apoptosis (14, 15). Previous research has shown that Linc-DYNC2H1-4 promotes EMT and CSC phenotypes by acting as a sponge of miR-145 in pancreatic cancer cells (16). Chen et.al reported that Linc-RoR can promote proliferation, migration, and invasion via the Hippo/YAP pathway in pancreatic cancer cells (17).In this study, we investigated the expression pattern and clinical significance of LINC00518 in LUAD tissues by searching the microarray data set published in the TCGA database, and found that the expression of LINC00518 in LUAD tissues and cell lines were significantly up-regulated and the increased LINC00518 expression may be related to a poor survival rate. Besides, by detecting our 42 LUAD samples, we found that the high expression of LINC00518 is significantly related to the advanced TMN stage and lymph node metastasis of LUAD tumors, which indicates that LINC00518 is related to the development of LUAD tumors. Further, We found that knocking down LINC00518 significantly inhibited LUAD cell proliferation and tumor growth in vivo. LINC00518 can also accelerate the proliferation of LUAD cells by extending the proportion of S phase and reducing the proportion of G1 phase. These results indicate that LINC00518 may be an oncogene of LUAD and can be used as an independent potential prognostic biomarker for LUAD patients.
Many studies have proven that lncRNA can exert its function by regulating the expression of miRNA (18–20). For instance, it has been reported that LINC-smad7 promotes myoblast differentiation and muscle regeneration via sponging miR-125b (21).Hao et.al found that LINC-PINT suppresses tumor cell proliferation, migration and invasion through targeting miR-374a-5p in ovarian cancer (22).Previous study has proved that LINC00518 serves as a sponge for the miR-204-5p in melanoma (12). According to the subsequent bioinformatics analyses and luciferase reporter assays, we predicted that the LINC00518 may have a strong interaction with miR-185-3p.In this study, we first proved that miR-185-3p was down-regulated in LUAD tumor tissues and cell lines. We also found that the luciferase activity of the LINC00518-WT was significantly reduced by transfecting with miR-185-3p mimic, and the expression of miR-185-3p is negatively correlated with the expression of LINC00518 in LUAD tissues. Through a series of functional experiments, we found that the effect of LINC00518 in promoting the proliferation of LUAD cells can be reversed by transfection of miR-185-3p mimics. All these results demonstrate that LINC00518 promotes LUAD cell proliferation by down-regulating miR-185-3p expression.
Many studies have shown that miRNAs function by inhibiting the expression of target mRNA. There are reports that MECP2 promotes breast cancer cell proliferation and cell cycle progression and inhibits cell apoptosis (23, 24). MECP2 was also reported as a target of miR-638, facilitated gastric cancer cell proliferation and may serve as a potential target for gastric cancer therapy (25). In our study, MECP2 was selected as a direct target of miR-185-3p through bioinformatics analyses and luciferase reporter assays. We found that the MECP2 expression was up-regulated in LUAD tumor tissues. Through a series of in vitro experiments, we proved that LINC00518 expression has a positive correlation with the expression of MECP2. These findings demonstrated that MECP2 is a target of miR-185-3p regulated by LINC00518 in LUAD cells.
In conclusion, our results prove that LINC00518 promotes cell proliferation and tumor growth by regulating the cell cycle of LUAD through miR-185-3p targeting MECP2 in vitro. In addition, over-expression of LINC00518 is related to the poor prognosis, advanced TMN stages and tumor size of LUAD patients. These data highlight the importance of LINC00518 in the progression of LUAD, indicating that LINC00518 may be a key predictor of LUAD poor prognosis and a potential therapeutic target.
Data Availability Statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession numbers can be found in the article/Supplementary Material.
The studies involving human participants were reviewed and approved by the Ethics Committee of Yancheng Third People`s Hospital. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individuals for the publication of any potentially identifiable images or data included in this article.
YZ and JS contributed to the construction of the subject. ZS and HC are responsible for the collection of samples. XH and JW contributed to data analysis and interpretation. XH is responsible for writing, reviewing and/or revising the manuscript. All authors contributed to the article and approved the submitted version.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fonc.2021.646559/full#supplementary-material
Supplementary Figure 1 | The protein levels of p-Akt, p-Erk and cyclin D1 in A549, PC9, SPCA1 cell lines transfected with si-LINC00518 or NC were determined by Western blots separately. GAPDH was used as a control.
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Keywords: NSCLC, LINC00518, cell cycle, MECP2, miR-185-3p
Citation: Han X, Wu J, Zhang Y, Song J, Shi Z and Chang H (2021) LINC00518 Promotes Cell Proliferation by Regulating the Cell Cycle of Lung Adenocarcinoma Through miR-185-3p Targeting MECP2. Front. Oncol. 11:646559. doi: 10.3389/fonc.2021.646559
Received: 27 December 2020; Accepted: 22 March 2021;
Published: 15 April 2021.
Edited by:Yueming Sun, Nanjing Medical University, China
Reviewed by:Shen Yihui, Fudan University, China
Yu Feng, The First Affiliated Hospital of Soochow University, China
Copyright © 2021 Han, Wu, Zhang, Song, Shi and Chang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Yajun Zhang, email@example.com
†These authors have contributed equally to this work


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If Charles does continue to advance and ends up on the cover, he says it will be a dream come true for a guy who spends all of his free time talking noise to teammates while on his PlayStation 3.
"I'm a huge gamer, that's all that I do," Charles explains. "I'm on that 'NBA 2K11' right now, and you know I can't stop playing 'Madden.' I play a lot of fighting games, I play a lot of racing games. I don't really go out when I'm in Kansas City, so I just stay at the house and play video games.
"I started playing 'Madden' when I was a little kid, then I played a lot when I was in high school. I remember when the PS2 came out, 'Madden' is all I played, and now on the PS3, it's changed so much, it's crazy. Everything is like real life."


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About this event
In Africa, 27 countries have mandates to fortify wheat flour, 10 of the 27 also for maize flour. Six countries in this region fortify more than half of their industrially milled wheat flour, and 2 industrially milled maize meal, even though it is not mandatory. In addition, many countries also fortify cooking oil, sugar, and salt as part of their comprehensive nutrition strategy.
Proving compliance with legislative tools and label declarations will always be difficult using purely analytical tools. Monitoring of fortified foods, however, is proving to be a challenge both technically and administratively with a noticeable lack of trust in the system and, unfortunately, between the stakeholders – this is a global problem and one that is enhanced by a lack of understanding of the nuances in fortification. One approach has been the move towards ‘assisting to comply’ rather than ‘enforcing compliance’, though some regulators see this as a conflict of interest, and one of the objectives of this webinar is to identify areas in which the global expertise of AOAC could be directed, if permitted, within the Region. Another objective is to provide an opportunity for us to share experiences and lessons learnt in the ‘real world of Africa’ and help one another.
Not sure about the start time for the webinar in your country of residence? Just click here to convert the time from 15h00 GMT+2: https://www.thetimezoneconverter.com/
For any registration assitance, please contact our secretariat through firstname.lastname@example.org
Owen is the President of the Sub-Saharan Africa Section of the AOAC INTERNATIONAL. He also works as a Scientist at the Nestlé Institute of Food Safety and Analytical Sciences, Nestlé Research in Lausanne, Switzerland.
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domain: geography


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Nintendo has revealed that Animal Crossing will be getting the Lego treatment. You can check out the teaser below:
The teaser showed off multiple Animal Crossing characters as Lego minifigures (but with unique heads comparable to the old Fabuland theme). From left to right, the characters are: Rosie, Bunnie, Marshal, Kapp’n, Fauna, Julian, Isabelle, and Tom Nook.
Nintendo is no stranger to Lego, as we have gotten a wide variety of Lego Mario sets in the past few years. It was not confirmed whether Animal Crossing would be sets or individual minifigures. However, we did see some references to the series such as a lego balloon present flying up into the air.
EDIT (10/10/2023): Nintendo has released a new video showcasing the figures with their corresponding Lego sets. You can watch it below:
Additionally, the sets themselves are now viewable on the Lego website. The sets are as follows:
- Julian's Birthday Party ($14.99)
- Bunnie's Outdoor Activities ($19.99)
- Kapp'n's Island Boat Tour ($29.99)
- Isabelle's House Visit ($39.99)
- Nook's Cranny & Rosie's House ($74.99)
The Lego Animal Crossing sets will release in March 2024.
Author’s Note: As someone who has recently gotten back into Lego after a 10+ year hiatus, this is a dream come true for me. I’m definitely going to try and collect all of these. What Animal Crossing villagers do you hope get Lego-fied? And which other Nintendo series would you like to see have their own Lego sets? Let us know in the comments below!
Thumbnail Graphic: @Zerp
Social Media: @Zerp

domain: hobby


Pasadena motorcycle officer injured in crash
Updated 8:00 pm, Wednesday, August 31, 2011
An off-duty Pasadena police officer was injured Wednesday when he lost control of his motorcycle while escorting an oversized truck in southeast Houston.
The officer, Ben Hickman, was eastbound on the Central Bridge off Lawndale Street when he lost control of his motorcycle about 3 p.m. while trying to pass the truck carrying a 155,000-pound load, Houston police officer C. Lofland said.
HPD spokesman John Cannon said the officer narrowly missed hitting a curb, causing him to lose control.
"Once he noticed he did not have sufficient clearance (of the curb), he laid his bike down," Cannon said.
Hickman injured his left shoulder, police said. He was listed in good condition at Memorial Hermann, a hospital spokeswoman said.


New Communication and Media Arts Major Now Available
The number of majors now offered is increasing this fall. The communication and theatre arts department will offer three distinct majors, allowing students to specialize in theatre arts, communications and media arts, or speech and theatre education.
The new majors benefit students who want to study specific areas of communications or theatre.
“Our students still have a common communication and theatre arts core, but they can now focus on their interests,” said Dr. Raymond Anderson, associate professor of communication arts. “The new majors and their concentrations allow us to offer the students classes that and give more depth in this particular area.”
The newly formed communication and media arts major will offer concentrations in public relations, cinema production, multi-media production, communication arts and journalism, while speech and theatre education will continue to offer two concentrations: communication arts or theatre arts. Each concentration requires courses from multiple departments, yielding an interdisciplinary approach to communications and theatre.
Vice President of Academic Affairs Dr. Gregory Kerr said, “We used the strengths from different departments to come together in a meaningful way. We also built the curriculum to include the CCCU BestSemester programs at the LA Film Institute, the Nashville Contemporary Music Center, and the Washington Journalism Center. These off campus programs meet course requirements for their designated concentrations and are exciting opportunities to offer our students.”
Anderson helped develop a media program at George Fox University and has already taken significant strides with the program at Sterling College. In addition to developing the new majors and concentrations, he was the sponsor for the first Sterling Festival of Film held in April. The festival showcased 15 student-created films.
Kerr said, “We are thrilled with the talent that our faculty bring and think that the changes really set us apart from other institutions.”


text: History, Tyrolean traditions, and a magical Christmas environment await at the Kufstein Christmas Market hosted in this medieval fortress. Dating back to the 13th century, the Kufstein Fortress, set against the backdrop of the Austrian Alps, may now be easily accessed with the castle's Panoramabahn. Take a trip back in time as you walk the historic corridors of the fortress while visiting the numerous exhibitions. Located atop one of the magnificent fortress towers is the annual Christmas Market. Here, guests may discover over 40 unique stands with goods from local craftsmen and artists.

domain: travel


MA, March 20, 2008 -Boston Applied Technology, Inc. (BATi),
a leading manufacturer of ultra-fast photonics devices, successfully
exhibited at the Optical Fiber Communication Conference and Exposition
(OFC) at the San Diego Convention Center, San Diego, CA, February
showcased its nano-second speed, low-loss optical devices based
on the revolutionary electro-optical material OptoCeramicTM.
These included BATi's newly developed NanonaTM ultra-fast optical
switch and also the high speed VOA, micro-VOA and solid-state polarization
days more and more business applications require high speed communications."
observes Run Zhang, VP of engineering, "our ultra-fast devices
can perfectly meet the speed requirements of our customers."
BATi expects that the speed and rugged design of its photonics devices
will enable many business applications.
Applied Technologies, Incorporated (BATi) was formed in 2002 by
the key management and technical team formerly with Corning Applied
Technologies, a subsidiary of Corning Incorporated. With many years
of development effort and a group of industry pioneers, BATi has
established a unique leading position in designing, manufacturing
and marketing a variety of light and polarization management components
for the telecommunications and instrumentation markets.


Sir Desmond Swayne, who was Parliamentary Private Secretary to Mr Cameron for 7 years, has been speaking as the Conservative leader hands his resignation to the Queen.
Mr Cameron exited the famous black door of 10 Downing Street with his family for the final time on Wednesday telling the gathered media that he was leaving the country "much stronger" and the economy "immeasurably stronger" after his six years in office.
He paid tribute to Samantha, who he described as "the love of my life" who had "kept me vaguely sane".
Mr Cameron concluded: "It has been the greatest honour of my life to serve our country as Prime Minister over these last six years and to serve as leader of my party over 11 years.
Mr Swayne, was at Downing Street for Mr Cameron's last evening there.
He said: "He certainly didn't want to go but he's been remarkably cheerful about it in the last few weeks.
"I went to dinner at Downing Street last night and it was a tremendously cheerful and optimistic affair, there was no tears or regrets or remorse at all."
Earlier on Wednesday, David Cameron hosted his last ever Prime Minister's Questions in the House of Commons.
He called on his successor to stay as close to the European Union as possible.
Opposition leader Jeremy Corbyn praised him for his role in changing the law on gay marriage and securing the release of Shaker Aamer who was held by the US at the Guantanamo Bay detention camp in Cuba.
On answering his final question, Mr Cameron was greeted with a standing ovation from much of the chamber.
Speaking about how he'll be remembered, Sir Desmond Swayne said: "It's almost too soon to start talking of legacy because you don't know what's going to happen next.
"One of the things that might an enormous legacy is going to be the fact he gave us the referendum on the EU.
"But we won't know the nature of that legacy for 10 years or so."
David Cameron will continue in his role for MP for Witney. There has been suggestions he may even stand again at the next election.
Listen to Sir Desmond Swayne speaking to Premier's Antony Bushfield here:


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The complaint has been investigated and
resolved to the customer's satisfactionResolved Forex Turtle Trading — Scammed me out of $5000
resolved to the customer's satisfaction
I was called and told about an "expert" Forex trader, named Russell Sands. The caller said that if I invested $5000 he would guarantee that I would make at least 4x my investment back in 1 year!! The method would be automatic investing in the forex market. You wouldn't do anything; just sit back and let Russell do the work! Well it started off o.k. My $2000 grew to almost $6000 in just a few short months. But then it started going downhill fast. I watched it go down to to $239. In utter frustration I took what little was left, out of my account.
So in short this SCAM cost me almost $7000!!
I have in my files charts put out by this "expert" showing the record of his accomplishments all concocted I'm sure. I was even told at one point that if I had invested at least $10, 000 I would have been invested in more trading sessions. At some point I could see on a daily basis that I was losing money; when I called, I was told that the rules for the "small" guy had changed. Well LA DEE DAH! I feel as though I got Royally Screwed!!!

domain: economics


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Offense frozen out in pair of Northern Michigan University women’s basketball losses
MARQUETTE — The offense dried up over the weekend as the Northern Michigan University women’s basketball team suffered a pair of defeats at home as the Wildcats enter GLIAC play this week.
Minnesota-Duluth held NMU to under 40 points in a 50-37 victory, while St. Cloud State slipped past Northern 55-51.
The Wildcats open conference play on the road this week with games in Hammond, Indiana, at Purdue Northwest at 6 p.m. EST Thursday and in Kenosha, Wisconsin, at Wisconsin-Parkside at 2 p.m. EST Saturday.
NMU, which is now 4-3, returns home for another conference doubleheader weekend starting at 7:30 p.m. on Friday, Dec. 9, against Ferris State.
Here is a roundup of the weekend action:
Minnesota-Duluth 50, NMU 37
On Friday at the Berry Events Center, the Wildcats went into a deep freeze with their shooting, missing all 15 of their 3-point shots and only making 25 percent (11 of 44) overall from the field.
Even the free-throw shooting was iffy as they made 63 percent (15 of 24).
One bright spot was Northern’s second-leading scorer, Alyssa Nimz, who went 3 for 3 from the field and 4 of 4 on free throws for 10 points.
She trailed only Makaylee Kuhn’s 14 points for team scoring honors. While Kuhn also made all four of her free-throw tries, he managed just 5 of 14 from the field, including 0 of 5 on triples.
But Kuhn did rack up game highs of 10 rebounds and three blocked shots, while Nimz made seven rebounds and had no turnovers in 19 minutes court time.
No other NMU player scored more than four points.
Meanwhile, Brooke Olson led UMD (4-1) with 18 points even though, like Kuhn, she missed all five of her 3-point tries.
As a team, the Bulldogs made 37 percent (20 of 54) overall and 23 percent on 3s (5 of 22).
Northern stayed closer than it might’ve by outrebounding Duluth 42-28 and committing seven less fouls (19-12), though the Wildcats also had almost twice as many turnovers, 20-11, as the Bulldogs made 10 steals.
After NMU’s Kayla Tierney hit a jumper to tie the score 2-2 almost four minutes into the game, Duluth reeled off an 11-3 run to end the quarter with a 13-5 lead.
The Bulldogs’ lead quickly hit double digits in the second quarter and stayed there, ending with a 27-11 advantage at halftime.
And UMD took its biggest lead less than two minutes into the fourth quarter, 48-22, before the Wildcats ended the game on a 15-2 run. Nimz made three layups and two free throws in that stretch.
St. Cloud State 55, NMU 51
On Saturday at the BEC, the Wildcats shot better, but probably still below average, in a game that swang back and forth.
NMU was just 28 percent from the field (15 of 54), but did manage to hit three 3-pointers, but with 15 attempts it was just 20 percent success from long distance.
Their free-throw shooting markedly improved to 82 percent (18 of 22).
Kuhn led the Wildcats with 19 points as she made 11 of 14 free throws. While she was 2 of 4 on triples, her overall shooting was only 3 of 13.
But she did have a team-high seven rebounds to go with two steals and two assists.
Abi Fraaza added eight points for the Wildcats, shooting perfectly — 3 of 3 from the field and 2 of 2 on free throws. NMU’s Andrea Perez, Mackenzie Holzwart and Ana Rhude added six points apiece as Rhude and Fraaza each pulled down six rebounds.
Perez made a team-high three steals and Holzwart an NMU-best three assists.
Tori Peschel and Dru Henning paced the Huskies (3-2) with 13 points apiece as they combined to make 7 of 9 free throws.
St. Cloud led throughout the first quarter, moving its lead into double digits, 16-6, before Tierney hit a jumper with 2:31 left in the period to make it 16-8 after one.
Northern’s deficit swang between four and 10 points in the second quarter, Kuhn getting it down to 27-23 late with a free throw as it was 28-23 at halftime.
NMU took its first lead, 34-33, with 4:18 to go in the third quarter on a pair of free throws by Perez as the score was tied 37-37 entering the final period.
And while the Huskies spent most of the fourth quarter ahead, the Wildcats did take one final lead, 51-50, with 58 seconds remaining after a Fraaza layup.
But the home team wouldn’t score again as Henning made a pair of free throws with 45 seconds to go and a 3-pointer with 13 seconds left to provide St. Cloud’s winning margin.
Information compiled by Journal Sports Editor Steve Brownlee. His email address is firstname.lastname@example.org.


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By Harut Sassounian
Publisher, The California Courier
The Armenian government must take two important steps prior to opening the border with Turkey: 1) Forbid Turkish citizens from buying real estate in Armenia; and 2) Place tariffs on the import of products from Turkey.
Obviously, Turkish citizens do not have to cross the Armenian border to be able to buy real estate in Armenia. But, with the opening of the mutual land border, more Turkish citizens will be able to come to Armenia, thus increasing the flow of people and products from Turkey.
Allowing the citizens of a hostile country like Turkey to purchase real estate in Armenia is a serious national security threat, particularly if these properties are located near sensitive border areas.
There is a big contradiction between what the Constitution and laws of Armenia stipulate regarding the purchase of real estate by foreigners and what is actually practiced. Now that a Constitutional Committee has been set up to reform the existing Constitution, last amended in 2015, this is the right time to reconsider the existing provisions as to who can buy real estate in Armenia. There should be a ban on foreigners’ purchase of properties near Armenia’s border. In addition, citizens of Azerbaijan and Turkey should not be allowed to purchase any kind of property anywhere in Armenia.
This problem is particularly urgent because several years ago the Turkish government adopted a law that forbade the purchase of property in Turkey by citizens of four countries: Armenia, Cuba, North Korea and Syria. Citizens of another 35 countries are restricted to purchase property in Turkey based on the nature and location of the land. One would think that since the Turkish government has forbidden Armenian citizens from buying land in Turkey, Armenia should have reciprocated by banning the purchase of land in Armenia by Turkish citizens.
I wrote an article in 2012, informing Armenian officials of the Turkish law that banned the citizens of Armenia from buying land in Turkey and urged “the Armenian Parliament to consider adopting retaliatory measures against citizens of Turkey interested in purchasing Armenian properties.” Regrettably, my suggestion was ignored.
The 1995 Constitution prohibited foreigners from purchasing land in Armenia. However, this was contradicted by the Armenian government’s subsequent report to the World Trade Organization: “foreigners have the right to own real estate properties built on Armenian land.” The report also stated that “the [Armenian] legislation grants the Government the power to limit and prohibit foreign investment for national security concerns.”
In line with the Constitution of 1995, the subsequent Armenian constitutions of 2005 and 2015 also stated that “Foreign citizens and stateless persons shall not enjoy ownership right over land, except for cases provided for by law.”
If foreigners are not allowed to purchase land or real estate in Armenia, then how were they able to buy them? In 2019 alone, foreigners, contrary to the Armenian Constitution, purchased 186 apartments, 72 houses, two factories, nine public properties and even 121 plots of land. How was this possible?
Much more concerning is that citizens of the enemy states of Azerbaijan and Turkey have been buying properties in Armenia without any objection. According to the figures released last week by the Armenian government’s cadastre or official registry of real estate, from 2010 to 2021 citizens of Azerbaijan purchased six properties in Armenia, which included five apartments and one public property. During the same period, citizens of Turkey bought 71 pieces of real estate, including 55 apartments, five houses, one garage, seven public properties and three plots of land in Armenia.
I assume that many of the Turkish citizens who purchased real estate in Armenia are of Armenian origin. I suggest that the Armenian government make an exception for those who are of Armenian origin, if and when the purchase of real estate by Turkish citizens is banned.
Finally, turning to the import of products from Turkey and other countries, the Armenian government must impose tariffs to protect the viability of domestic production. Since Turkey has a very large population, it is able to produce items much cheaper due to mass scale. Armenian producers, unable to compete with them, will go out of business. Already the Armenian market is flooded with Turkish products. After opening the border, Turkish products will no longer have to go through the expense of importing them via Georgia, which means that they will be even cheaper creating a bigger problem for domestic producers. Making matters worse, the collapsing value of the Turkish Lira has made the prices of imported products from Turkey cheaper.
Before several sectors of Armenia’s economy are completely devastated, the Armenian government must place tariffs on imported Turkish products to protect Armenia’s vulnerable producers.

domain: law