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Have you ever heard of Gotv Clear Error Codes ?
Gotv clear error codes is a term used to clear Gotv error codes both manually and automatically with your mobile phone or your desktop or laptop computer.
If you are using GoTV, you would have experienced error codes displaying on your TV screen, such as E16, E17, E32, E48-32 error codes etc
Those error codes are different and have ways of solving them, I can't go deeper into them now, if you are experiencing any of them right now… Read on.
How do I clear GOtv error codes?
GoTv error codes have different meanings according to GOTV but it is frustrating to see this errors on the TV screen even after subscribing.
How to clear GOTV error Codes
Method 1
- On the sim you used to register for GOTV, dial *288#
- On the next screen after dialing, select 1 for GOTV
- On the next screen select 1 again
- Enter IUC number (it is found under your GOTV Decoder)
- On the next screen input number near to clear error codes..
- It should be cleared if you follow the steps carefully…
Too long? Try the
- Go to your message box
- Send "RESET IUC NUMBER – 4688" (without the quotes), format – RESET 2021738348 to 4688
- Then you are good to go
I am not with the number I used to register for GOTV service or subscription
Then try this
3RD METHOD – How to clear GOTV Error Codes
This method requires internet connection
- Go to your browser
- Enter http://easy.gotvafrica.com/wap/ or http://gotvafrica.com – click easy self service
- Select Nigeria
- Click on Clear error codes
- Input your IUC number
- Input the security captcha on the screen
- Click the clear button
- Poof! Gotv error code is cleared.
The three methods to clear Gotv error code are tested…
Confused? Use the comment box
Another wonderful article AddedNew @ AddedNew.com, Always come for what's AddedNew.
Related Searches:-
- How to Clear Gotv Error Codes
- How to Clear Gotv E16 error code
- How to Clear Gotv E17 error code
- How to Clear Gotv E32 error code
- How to Clear Gotv E48-32 error code


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Thousands face losing jobs as Rok collapses
Rok filed for administration yesterday, putting thousands of jobs at risk just two months after the collapse of its rival building services group Connaught.
The property repair and maintenance group, which styled itself "the nation's local builder", called in administrators from PricewaterhouseCoopers.
The move comes months after it was embroiled in an embarrassing episode in which it revealed it had discovered a £6 million hole in its accounts. The Exeter-based company suspended Ashley Martin, its finance director, only to reverse the decision and apologise to the executive. Mr Martin then quit.
In a surprise announcement yesterday, the company said: "The board of Rok plc announces that it has resolved to put the company into administration and to make an application to the Financial Services Authority…

subset: commoncrawl


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I'd been hearing buzz for the smart, low-budget, limited release movie Moon by the time I got to see it in theaters last Saturday. My expectations were high, and I wondered if this film would do everything many people had said it would. Did I question my humanity? Did the movie ask what it means to be human?
Not as much as I assumed it would. But it did make me think, and is certainly a cerebral movie.
It's sometime in the future, and Earth has started using clean energy that comes in the form of Helium-3, an isotope I wasn't sure existed, but actually researched after the film. Turns out that it does, that it's valued highly for nuclear fusion research, that it's incredibly rare on earth, but that many people think it is on the Moon in abundance. Three points for accuracy. Anyway, an energy company is the sole supplier of Helium-3, and it gets all of it from a mining station on the dark side of the moon. The entire program is managed by one lonely occupant, who is under contract for three years before returning to earth, named Sam Bell, played by Sam Rockwell. With just two weeks remaining before returning home, Sam is starting to feel the weight of isolation of being away from Mother Earth for three long years. His only human contact is from pre-recorded messages from the company executives, and his wife and daughter. There's a satellite for live communication, but it's strangely broken and not set for repair any time soon. The job is taking its toll, Sam starts to have strange hallucinations and is on the cusp of insanity. Soon he finds himself interacting with somebody identical to him in every way, and also happens to go by the name Sam Bell.
He's (They're?) assisted at the base by GERTY, a high tech artificial intelligence system that speaks with voice of Kevin Spacey and shows emotion with the use of a select few smiley face emoticons on a small screen. What at first comes off as a creepy, HAL copy cat thankfully proves to be something much different.
I realized in October my freshman year of college, when I was still meeting new people in a place where I had known nobody coming in, that I had gone the longest duration of time without a hug. It was an odd, sad and sobering fact; something that had been daily routine for me, a natural human interaction, had suddenly vanished in my life. This movie takes that to the nth degree, viewing how somebody might act completely devoid of human contact. We then get what can only be described as a a 'mind-fuck' as we try to discern the circumstances surrounding this incident. Is he a clone, or has he completely snapped? The movie does a fantastic job of making it ambiguous and forcing you to think to discern what you think is true. In the end, I'm not sure it ever really tells you.
The cinematography on the film is perfect. It has a dated feel to it, where the CGI looks like it could be little models, but if it were little models it looks CGI. It gives it a rugged, realistic feel of what it would probably be like to drive a Humvee across the surface of the moon. Accompanied by a fantastic piano-heavy score by Clint Mansell, known best for his haunting music in Requiem for a Dream.
To say Sam Rockwell did a good job here is like saying Leonardo Da Vinci painted a pretty picture with the Mona Lisa. He has the whole spectrum of emotions to display, from despair to borderline madness, and it's his performance that gives the film much of its intrigue. Also considering the entire movie consists of him talking to himself, it's more than impressive to see what he's done.
If Moon is playing near you, go see it. It's the smart sci-fi film of the summer to balance out the bombastic fun that was Star Trek. My brain certainly churned more in this movie than in any other movie this summer, and it's certainly one of the best releases so far. The rare gem of a film that comes out during the season of popcorn fanfare.


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A Lakeview man accused of stealing a piece of Cubs memorobilia is facing charges.
According to the Chicago Tribune, Michael Vite ran onto Wrigley Field Sunday morning and grabbed some ivy.
He was reportedly caught on video surveillance entering the stadium through an area under construction along the west wall.
Vite was arrested by a police officer while trying to flee.
The 24-year-old is charged with criminal trespass to land.
Published at 4:35 PM CST on Jan 2, 2012 | Updated at 4:40 PM CST on Jan 2, 2012


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We couldn't be more excited for next weekend…because we're going to BottleRock! This is the festival's first year and we're thrilled to be a part of it. We'll be co-sponsoring a tent with model Michele Ouellet's Lorenza Wine, where we'll have wine tastings and some fun DIY projects like crystal bottle stoppers and decorating wine glasses. All of this on top of the amazing music that will be going on, and the incredible scenery of Napa valley!
Stay tuned next week for a post on what to pack, and what bands we're most excited to see! We'll also have live coverage on our Instagram and Twitter throughout the festival.
Who's going to BottleRock? Let us know, maybe we'll see you there!


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Question: I now have saved $50,000 and my husband has saved $40,000 in the bank. We pay mortgage each month about $1,000 and we still have more than $120,000 to pay for 29 years.
My question is: should we pay off our mortgage within the next year? If not, what should we do or invest with our savings? How will we plan for the future? We have no kids. I have a retirement plan, 403 with my employer but my husband has nothing. My husband plans to retire next year but I'll keep working. Thank you very much. Sue, Grand Fork, North Dakota
Answer: I think in the heart of every homeowner burns an intense desire to say goodbye to the bank for the last time and own a home free and clear. It's a wonderful moment, and if you have the money there's nothing wrong with paying off the mortgage to live debt free.
That said, I'd like to throw a couple of cautions against your getting rid of the mortgage right away. For one thing, you're putting most of your financial eggs in one basket -- a home. That's another way of saying that your financial health is now dependent on how one asset performs in coming years and, as we are witnessing right now, home prices can go down as well as up. I'd prefer that you build up a well-diversified portfolio in cash, stocks, bonds, commercial real estate, commodities, and international equities. Your home will then shrink as a percentage of your net worth. Once you've built up a well diversified portfolio, then by all means pay off the mortgage.
What else could you do with the money? I'm glad to see that you're participating in your retirement plan at work. One idea is to create a long-term portfolio in a taxable account. This way you can access the money without paying the 10% penalty you have to fork over if you tap into your retirement account under the age of 59 1/2. To keep your annual tax bill down, I'd recommend investing a chunk of the money into broad-based equity index funds. For example, with an equity index fund that mirrors the Standard & Poor's 500 or the Wilshire 5000 you'll pay very little capital gains tax every year since there isn't a professional money manager churning the portfolio every couple of weeks. You will, however, pay taxes on dividend payments and when a company is taken out of the index, say, through a merger or acquisition.
Another tax saving tip is to put the fixed income portion of this investment into I-bonds. Inflation is every saver's nightmare. Even if inflation averages a low annual rate of, say, 2% to 3% over the next quarter century, that's still enough to depreciate the value of a today dollar by nearly half. The I-bond is a savings bond, and it shares many of the same attractive investment features as its better-known cousin, the Series EE savings bond. There are no commission costs imposed when buying or selling I-bonds. I-bonds are sold at face value (up to $30,000 a year) and earn interest for 30 years, although you can sell after five years with no penalties. You don't pay any taxes on the investment until you cash in the bonds. There's no credit risk since the I-bonds are backed by the federal government. Its value adjusts to take into account changes in the Consumer Price Index, and you're also paid interest on your money. (You can get much more detail about I-bonds at www.savingsbonds.gov.)
A final point: It seems to me that you're at a major transition point with your husband retiring and you continuing to work. It's at times like this that a fee-only certified financial planner (CFP) can help you create a financial blueprint for the next stage of life. The planner would help you look at your whole portfolio, your stream of income and savings, and match your finances to your lifestyle goals. And you can run by the CFP the ideas I've suggested to see if they will really work for you. Good luck.


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You have until Feb 1, 2018 to return or exchange items bought after Nov 1, 2017. All other standard return policy
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Product Highlights
The Teradek Cube-150 1 Channel HD-SDI Encoder is a camera-top HD video encoder, and is suitable for on-set video monitoring as well as full-scale film production. It features HD-SDI (serial digital interface) input, dual band WiFi (2.4/5.8 GHz), and an external USB port. In addition, the cube encoder features line-of-sight (waves traveling straight line) WiFi range up to 300' (91m).
A 30-day membership for a Premium Channel on Livestream allows users to utilize turnkey (ready for immediate use) solution for streaming live video to the internet. By entering Livestream details into encoders user interface, the users can start broadcasting on locating a broadband uplink.
Using a single Verizon 4G modem connected to the encoders USB port, it is possible to obtain upload/download speeds up to 8 Mbps for streaming to Livestream. In addition, this bandwidth can be used as a backup internet connection or to create a mobile wireless live streaming camera via 4G. Direct streaming to decoding devices such as a laptop or iPad is offered via the ad-hoc networking mode which eliminates the need for any other intermediary equipment.
Featuring a setup wizard, the Cube 150 HD-SDI Encoder web interface simplifies configuration. You can create a network, log on to Cube's web interface, complete the setup wizard, and start streaming video after connecting video to the Cube system. The Cube 150 HD-SDI Encoder features H.264 High Profile (level 4.1) video compression to offer high video quality at lower bit rates.
Downscaling to lower resolutions is possible via a built-in scaler that allows for output streaming resolutions of 1080p/PsF, 1080i, 720p, and 480i. Based on available IP bandwidth, you can choose target bit rates (HD - 2 to 4Mbps, SD – sub-1Mbps) and a video resolution as required. The Cube encoder offers end-to-end latency (time delay) of 1/8-1/2 second.
With a small size and compact design, the Cube 150 HD-SDI Encoder can be mounted to a rail system, cage, hotshoe, or camera baseplate. In addition, it can be used with camera systems such as Red One, Arri Alexa, and Panavision Genesis. The Cube encoder requires only 2.5W DC power.
Verizon 4G LTE Support for US Only.
Supports only Pantech UML290 USB 4G Model for Verizon, Requires Teradek Cube Firmware version 4.4.0 or higher.
|Supported Resolutions
1080p/PsF @ 23.98/24Hz
1080i @ 50/59.94/60Hz
720p @ 50/59.94/60Hz
480i @ 50/59.94/60Hz
Ethernet: 10/100BASE-T
Network Protocols: TCP/IP, UDP, HTTP, DHCP, NTP, SSL, IGMP
Transport Protocols: RTP, RTSP, RTP over HTTP, MPEG-TS, HTTP Live Streaming
Feature-rich WebUI for configuration and control
Point to Point (TCP or UDP), Multiple Unicast (UDP), or Multicast (UDP+IGMP)
USB 2.0: 1 Powered Port
WiFi: Internal 802.11n
Embedded Stereo
Audio Compression: AAC-LC, MPEG Layer 2
|Video Codec
Compression Algorithm: High Profile H.264 (L4.1)
Bit Rate: 250 Kbps to 10 Mbps
|Power Input
|Auxiliary Power Input: 9-24V DC
|Power Consumption
|802.3af Compliant
|Operating Temperature
|95% non condensing
|2.5 x 3.5 x 0.9" (64 x 89 x 23 mm)
|0.4lb (0.19 kg)
|Packaging Infoeyrqasquuevzvzcffaurduwbxurb
|Box Dimensions (LxWxH)
|6.2 x 6.2 x 6.3"


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If Nationals reliever Tyler Clippard has a sense of how his impending run through the arbitration process will unfold, he's keeping it close to the vest.
"I don't know, we'll see how it all plays out," the veteran right-hander said when asked if he expected to go to an arbitration hearing with the Nats.
Clippard has filed for arbitration and, since he wasn't able to reach an agreement with the Nationals, he and his representatives have exchanged proposed salary figures with the team.
The Nationals have submitted a proposed salary of $4.45 million, while Clippard has countered at $6.35 million, a difference of $1.9 million. Clippard made $4 million in 2013, when he went 6-3 with a 2.41 ERA and 0.59 WHIP in 72 games, the fourth straight season he's topped the 70-appearance plateau.
If the Nats and Clippard can't come to an agreement, the pitcher will have an arbitration hearing in February, and a three-person panel will decide on which salary he receives.
Asked if he was optimistic he could avoid a hearing, Clippard said, "Those discussions are all up to me, the Nationals, my agents and all that. Talking about it in the media does no good."
But he's at least paying close attention to what happens because it's the first time he's gone this far through what is sometimes a contentious process where teams take great care in pointing out every flaw in a player's game.
"It's part of the business and getting to this point in my career is something I've always wanted to do," Clippard said. "So in that aspect, it's enjoyable. But the business side of things - I like being a baseball player, I don't like being an agent or anything like that. So those type of things I leave up to them."
Earlier today, MASNsports.com's Dan Kolko got general manager Mike Rizzo's reaction to the impending arbitration hearings with Clippard and right-hander Doug Fister. Read Rizzo's comments here.


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Related terms for "aquaculture tank project ":
aquaculture companies
, aquaculture tank companies
Aquaculture Technology leads in consulting & planning, chemicals & hormons, diets & feeds, eggs & frys. Fish farming Systems and fish farming equipment. Consulting - Design - Planning for aquaculture and fish farms.
based in Kitzbuehel, AUSTRIA.
Asakua providing turnkey solutions for offshore fish farming projects. Offering solutions from bankable feasibility study to on site installations, monitoring and training as well. The main products of our company are fish farming cages, fish cage nets, mooring equipments for offshore farms, fish feed blowers, and fish tanks.
based in Bayrakli, Izmir, TURKEY.
Specialising in the development of Recirculation Aquaculture Systems for fish farming, all our systems have been tested on our own pilot commercial fish farm before making them available to our customers. Founded in 1999 and based on the Llyn Pennisular in North Wales, UK, we operate worldwide, supplying systems and advice in many different countries including, Australia, ...
based in UNITED KINGDOM.
AquaMaof Aquaculture Technologies, Ltd. designs and manufactures equipment for aquaculture sector, fish and food processing and construction of processing plants. The company has supplied many turn-key installations to major seafood companies in the world. The expertise and focus of AquaMaof Aquaculture Technologies, Ltd. is in its total commitment to giving fish growers ...
based in Rosh Ha'ayin, ISRAEL.
Scubla produces and distributes all over the world products and technologies for aquaculture, environmental monitoring, ecology and research. Scubla is a leading company, able in providing the latest technology and complete solutions for research facilities, fish hatcheries, recirculated aquaculture plants.
based in Remanzacco, ITALY.
AFE is a consulting and specialised advice in the field of aquaculture and micro algae and sells its own products dedicated to oxygenation basins: aerators basins aero range + AFE is the representative of the company Varicon Aqua Ltd specializes in microalgae: production systems BioFence ™ and Phyco-Pyxis ™, nutrients, algae pastes.
based in Challans, FRANCE.
AQUACULTUR is producing and selling aquaculture equipment worldwide. Fish farming equipment. Recirculating aquaculture systems. Fish feeders, aquaculture consulting, aquaculture water treatment, recirculation aquaculture systems (RAS), aquaculture engineering.
based in Nienburg, GERMANY.
Since 1984, long before it became popular to be `green`, Advanced Aquaculture Systems was designing and supplying recirculating systems. Advanced Aquaculture Systems continues to be at the forefront of innovation by offering our unique proprietary products and creative recirculating system designs. Advanced Aquaculture Systems is not a catalog house, but instead we select ...
based in Brandon, FLORIDA (USA).
Hesy Aquaculture B.V. is a private company, founded in 1984, With nearly 30 years of experience in the design and operation of intensive recirculation fish farms. HESY has developed systems in nearly 30 countries. The smallest system designed produces two ton /year, the biggest over 2000 ton/year. HESY offers training and education to all customers, which includes: ...
based in Kwintsheul, NETHERLANDS.
Aquaculture Equipment Limited was formed and managed by Ron Snedden, who worked in the aquaculture industry for over 25 years. As the company name implies, we specialise in the manufacture and supply of a wide range of equipment for fish farming, fisheries, fish keeping and all types of aquaculture. Although the company originally specialised in the production of fish ...
based in Middleton, UNITED KINGDOM.
Aquaculture Systems Technologies, LLC (AST) was founded in October 1995 to manufacture and market "Bead Filter Technologies" which were first developed at Louisiana State University's Civil and Environmental Aquatics Systems Laboratory in the early 1990s by Dr. Ron Malone. Today, AST's product line features a variety of different filtration products utilizing Bead Filter ...
based in New Orleans, LOUISIANA (USA).
Blue Ridge Aquaculture, Inc. is the world's largest producer of tilapia using indoor recirculating aquaculture systems (RAS). Each year, Blue Ridge Aquaculture produces 4 million pounds of tilapia, shipping between 10,000 and 20,000 pounds of live tilapia every day. These fish are raised without the use of antibiotics or hormones, and are free of mercury (undetectable ...
based in Ridgeway, VIRGINIA (USA).
Acui-T is specialized in the design and implementation of technical projects related to aquaculture equipment supply and water treatment in aquaculture.
based in Nantes - France, FRANCE.
RNT Tanks and Silos Ltd design, manufacture and supply a leading range of galvanised steel water tanks, silo storage systems and auger conveyor feed systems offering an all-round solution for a multitude of agricultural, horticultural, water management storage and material handling needs.
based in Bedfordshire, UNITED KINGDOM.
Fish graders, aquaculture filtration, recirculation aquaculture systems (RAS), fish egg incubators.
based in Larsgarden, NORWAY.
aquaFUTURE e.K. specialist in fish feeders, aquaculture consulting, fish cage systems, recirculation aquaculture systems (RAS), incubators, fish egg incubation boxes products.
based in Kreuztal, GERMANY.
Deep Blue Aqua was established in late 2007 by Brynn Simpson and Grant Brooker to fill the aquaculture technology supply gap in Southern Africa and assist in growing the burgeoning aquaculture industry in Africa. We have a highly competent team who are ready to assist with any aquaculture or related project.
based in Gordon Bay, SOUTH AFRICA.
Sterner AquaTech UK is one of the country`s leading providers of water treatment solutions to the aquaculture industry, commercial enterprise, and domestic installations. Our services range from supplying plant and parts, through to design and installation of major treatment projects. Fresh water aquaculture. Sea water aquaculture
based in Inverness, UNITED KINGDOM.
MaofMadan, a manufacturer and supplier of aquaculture products, offers a full range of products to meet all aquaculture farm and project needs. MaofMadan`s pledge is to serve the needs of all aquaculture applications in the most cost-efficient and economical way. We employ our deep understanding of both biological and technical aspects of aquaculture in order to come up ...
based in D.N. Menashe, ISRAEL.
Global Aquatics was a leader in aquaculture and fish farming technology for over 1/3 of the 20th century, now as aquaculture heads into the 21st. century. Over 40 years of refinement, our aquaculture and aquaponics systems have evolved into the world`s most efficient, state of the art, turn key production machines.
based in Aberdeen, MARYLAND (USA).
Need help finding the right suppliers? Try XPRT Sourcing. Let the XPRTs do the work for you
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Carole Hochman Midnight Love Me Sweet Sleepshirt 133351
This product was discontinued October, 2012 and is no longer available. Please consider one of the Customer-Recommended Replacements above, or try a new search.
Fall in love with the featherweight comfort of this sleepshirt. Features embroidered lace and pleating for an elegant look.
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By Carissa Wolf
WesCom News Service
BROOKINGS - The smells of chili and freshly baked honey corn bread wafted through the homey dining room on Chetco Avenue in Brookings.
Soon, some 20 hungry people lined up for a bowl of free chili, fruit salad and corn bread at His Banqueting Table. They all came to the newly opened soup kitchen to fill their stomachs. Many came to fill their souls.
andquot;People who don't have enough money to have a good meal, they can come here and have at least one a day,andquot; said Outreach Gospel Mission Executive Director Chuck Fidroeff. andquot;If they're hungry and cold, they have a place to go from 12 to 2. And they can hear the love of God.andquot;
The Outreach Gospel Mission opened the doors of His Banqueting Table for an inaugural feast this week, welcoming the hungry regardless of socio-economic class or religion.
andquot;Everyone is welcome,andquot; said Carrie Nelson, who lives at the shelter and volunteers in the kitchen.
Nelson spent Wednesday morning stirring an industrial-size pot of the chili concoction. She added chunks of meat to the hodgepodge of chili beans, vegetable soup, sun dried tomatoes and whatever else she had on hand. She couldn't follow a recipe. She had to make do with what ever donated canned food items she had on hand.
The name of the Mission's latest outreach service to the poor, homeless and hungry pays homage to a scripture, Song of Solomon and to the Bible song, andquot;His Banner Over Me is Love.andquot; In the popular church verses, the chorus proclaims, andquot;He calls us to His Banqueting Table. His banner over me is love.andquot;
To the men, women and children who cook and serve and eat at His Banqueting Table, the chili is as full of love and hope as it is of beans and sun-dried tomatoes.
As His Banqueting Table heralds a new beginning in the Brookings-Harbor social safety net, the volunteers and Mission shelter guests who staff the kitchen find new beginnings of their own.
Milissa Joe Good spends her days scraping paint from the peeling exterior walls of His Haven of Hope, cleans tables during His Banqueting Table hours and offers soup and bread to strangers who quickly become friends. Like many of the volunteers, she's quick to offer hugs. And she'll ask you to call her andquot;Missy.andquot;
Her days at His Banqueting Table stand in stark contrast to the last 20 years of her 36 year life.
andquot;I got high on my birthday which was March 7. I didn't want to do it anymore, so I moved here,andquot; the recent Crescent City transplant said.
andquot;Meth was my drug of choice,andquot; she continues, then proudly announces that she's been completely clean for 30 days. That's the longest her body has gone without a hit of something to give it a synthetic boost.
Methamphetamine and a little of everything else minus heroin pulsated through her veins since she was a teenager.
The meth use helped Good numb the pain of a gang rape she experienced at 16 years old. And in the years that followed, the meth raped Good of her family and her sense of self.
andquot;I had nightmares,andquot; Good said of the rape. Then she found meth or what was then known as andquot;crank.andquot;
andquot;And that was the end of the nightmares,andquot; Good said.
But new nightmares began.
Good married, had a family, held good jobs and even recently went to college. But she also stole, robbed and hustled the streets for cash to buy her next fix. And Good's addiction eventually robbed her children of a mother.
Good could have continued down that path if she hadn't gotten sick or found His Haven of Hope, she said.
andquot;This is the best I've ever felt. I want to give props to God,andquot; Good said.
In a little over a month, Good has put on weight, the sores on her neck have healed and a bright complexion has returned to her face.
But just months ago, Goods spent her days high and, like many meth users, she had compulsions to pick at her skin. At times she would hit a creative high and pour out verse after verse of lyrical poetry.
She also contracted methicillin-resistant staphylococcus aureus or MRSA, an antibiotic resistant staph infection. MRSA commonly infects hospital patients and healthcare workers but has recently been linked to methamphetamine use. The infection colonizes on the skin's surface, in the nasal passage, sputum or urine passages. It's easily passed between meth users who share straws to snort the powder and have a weakened immune systems or skin wounds. The infection left Good with an ulcer on her back.
andquot;I just got scared so I went to my pastor,andquot; she said.
Good's pastor referred her to His Haven of Hope where she found gospel-based recovery and other women hoping for a better future.
Good met 15 year-old Jacquie Nelson and her mom, Carrie. The two battled years of homelessness and addiction. Jacquie bounced around on friends' couches and lived with whomever would take her in. Sometimes it was a friend, sometimes it was an older guy. Most of Jacquie's living arrangements put her right in the middle of a drug scene.
andquot;I lived around it, so I thought, why not do it?andquot; Jacquie said.
His Haven of Hope helped mend the Nelson family and His Banqueting Table gave the pair a new purpose and a vision for the future.
andquot;I can see myself having a job and a car,andquot; Jacquie said.
She even found a way to dream like a teenager again. Jacquie jokes that she'd also like to be a model and a Dallas Cowboys cheerleader and a country singer. She also dreams that her best friend would stop using meth.
And Good found something she lost long ago: Herself.
andquot;People can look me in the eye and not wonder if I'm high or not. What's it called? Dignity.andquot;


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Roenicke thinks Greinke is still adjusting
Roenicke thinks Greinke is still adjusting
MILWAUKEE -- Brewers manager Ron Roenicke said he wouldn't have been surprised if Zack Greinke didn't have his best stuff in his first couple games after getting traded to the Angels. However, he didn't expect the 28-year-old right-hander to struggle quite like he has. In Greinke's first five games with the Angels, he has a 6.19 ERA to go with a 1-2 record and two no-decisions. His start on Sunday, when he gave up six earned runs on seven hits in six innings, marked the first time in his career he gave up four runs or more in four straight outings.
To Roenicke, he believes it's an issue of still getting adjusted to a new environment. "You take a guy like him, and he's real structured in everything he does," Roenicke said. "He got real comfortable here with how things were going, and now you put him with a whole new coaching staff, a whole new team and that's not easy to do. ... It's not easy to just be yourself and go through the same routine you usually do when your surroundings are so different." Roenicke said he doesn't expect Greinke's funk to last much longer, especially in Angel Stadium, which he considers a pitcher-friendly park. "I still think he's going to have some good starts for them," Roenicke said. "I think that last month, he's going to do well."


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This product was discontinued January, 2014 and is no longer available. Please consider one of the Customer-Recommended Replacements above, or try a new search.
This brief panty features smooth floral-print jacquard fabric and beautiful sheer lace for vintage romance. Made of 87% nylon and 13% spandex fabric.
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Everyone has their own individual learning styles. Everyone also has different hours during the day where focus and concentration are better. Or worse. Sometimes we can tell when those peak times are, but not necessarily with any real precision.
What if you could tell how well you perform in certain environments doing different activities at different times? What if you can gain insight into how your mind works by tracking your focus during any activity you choose. Well, the guessing is about to be over.
Now on Kickstarter is the Melon project. Melon is a headband and mobile app duo that tracks and helps you improve your focus in relation to your activity, your environment, your emotions, and any other behavior you want to track.
The Melon headband is a wireless brain-sensing device that uses EEG (electroencephalography) to measure your brain activity.
From this activity, the app runs algorithms detect your focus, and that data gives you personalized feedback on how to improve.
Input tags indicate activity, environment, behavior, and more. Wear the headband to monitor focus and learn what affects it.
Learn what helps and hurts your focus. Evaluate trends and gain insight at the end of sessions.
Visit the Melon Kickstarter page for more information, to read up on the science behind the Melon, and to set your pledge!


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American Academy of Pediatrics
Address:141 Northwest Point BoulevardElk Grove Village, IL 60007-1098
Internet address:http://www.aap.org/
Description of services provided:The American Academy of Pediatrics is a great resource for both parents and health professionals. Included on the site is a detailed policy statement on how best to manage sickle cell disease called "Health Supervision for Children with Sickle Cell Disease."
Sickle Cell Disease Association of America
Address:231 E. Baltimore St., Suite 800Baltimore, MD 21202
Internet address:http://www.sicklecelldisease.org/
Description of services provided:This site includes information on sickle cell disease, how to cope with its complications, who is affected, and how to join in efforts to research treatments. A chat room allows you to chat online with other people interested in sickle cell disease. Information on conferences, news releases, and a newsletter allow you to stay up-to-date on the latest research efforts.
Last reviewed October 2012 by Brian Randall
Please be aware that this information is provided to supplement the care
provided by your physician. It is neither intended nor implied to be a
substitute for professional medical advice. CALL YOUR HEALTHCARE PROVIDER
IMMEDIATELY IF YOU THINK YOU MAY HAVE A MEDICAL EMERGENCY. Always seek the
advice of your physician or other qualified health provider prior to
starting any new treatment or with any questions you may have regarding a
medical condition.
Copyright © 2010 EBSCO Publishing All rights reserved.
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Please use this identifier to cite or link to this item: http://hdl.handle.net/1783.1/4250
Image super-resolution using neighbor embedding over visual primitive manifolds
|Fan, Wei
|Issue Date
|Image super-resolution refers to the process by which a higher-resolution enhanced image is synthesized from one or more low-resolution images. It finds a number of real-world applications in computer vision and computer graphics. In this thesis, we propose a novel learning-based method for the problem of single-image super-resolution. Given a low-resolution image, its underlying higher-resolution details are synthesized based on a set of training images. In order to build a compact yet descriptive training set, we investigate the characteristic local primitive structures contained in large volumes of small image patches. Inspired by recent progress in manifold learning research, we take the assumption that these small primitive patches in the low-resolution and high-resolution images form manifolds with similar local geometry in the corresponding image feature spaces. This assumption leads to a super-resolution approach which reconstructs the feature vector corresponding to an image patch by its neighbors in the feature space. To speed up the process of searching for the nearest neighbors for any input image patch, we partition the training set into multiple clusters and also sample the training set probabilistically so that the data distribution is as uniform as possible. These two preprocessing techniques make the neighbor embedding algorithm more efficient as well as more effective. Experimental results show that our super-resolution method can synthesize higher-quality images compared with existing methods.
|Thesis (M.Phil.)--Hong Kong University of Science and Technology, 2007
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A MAN has undergone surgery after being slashed on the side of the face in an early-hours attack.
It happened at about 3.15am on Wednesday (August 6) in the Avenues area of Chester-le-Street, County Durham.
The 31-year-old victim was approached by a group of up to six in their late teens and early 20s.
Loading article content
After a brief confrontation the victim was slashed across the left side of his head, causing a deep cut to his ear and left cheek.
His attackers ran off from the scene.
A police spokesman said: "The victim is still in University Hospital North Durham having surgery on the left side of his face to the injuries."
Police in the town are also investigating two linked assaults outside Horners pub, Front Street, at 1.30am on Saturday (August 2).
A man in his early twenties was knocked to the ground and kicked and punched by another man.
The victim got up and walked away and has not been traced but police have arrested a man on suspicion of assault and bailed him pending further enquiries.
They need the victim to come forward to find out what injuries he suffered.
Seconds after the first incident a 27-year-old man who is 6ft 7in was knocked unconscious by a group of men and women who kicked and punched him on the ground before walking off towards Cone Terrace.
Anyone with information about any of the incidents should contact police on 101 or call Crimestoppers on 0800-555-111.


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The EEOC and Jameson Memorial Hospital settled a federal lawsuit that was originally filed last February. The New Castle hospital will pay $50,000 to an African-American radiology technician who claimed the hospital denied him training opportunities because of his race.
The hospital must also revise its employee handbook and policies to clarify that denying training opportunities may be discriminatory.


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Which one of you VB dudes is this?
Results 1 to 8 of 8
Jul 25, 2014, 09:15 PM #1
Breaking the Stereo Type of GW Sharks
Jul 26, 2014, 01:34 AM #2
- Join Date
- May 2013
- Location
- Punching Latex Dummys in Barns
- Posts
- 4,712
- Images
- 4
that would be sick, i see what hes saying about the eye to eye thing. i mean they are big sharks they eat crap, fish people ..ect. if i was hungry and saw a deer i would shoot it and eat it.
Jul 26, 2014, 01:41 AM #3
Yeah the eye to eye thing is crazy when we're talking about a fish of that size, just impressive creatures all around
Jul 26, 2014, 03:47 AM #4
D@mnnn that dude is crazy, but in a good way. Mad kudos to him for his respect for sharks and just animals in general. I'd love to see a great white like that in person but def behind bars. Does this guy do caged dives or is he free ballin it?
Jul 26, 2014, 06:05 AM #5
Both caged and "un-caged" diving were being shown.
I have yet to see a shark while surfing. Back in 86, I was snorkeling off Key Largo (Pennekamp) with my
Dad. Before we went in the water, we were told about a 6 ft bull shark that frequented the area. They even had a name for him. Can't recall what it was - let's say Dave. I saw "Dave" after we had been out for a half hour or so. He swam right by my Dad and I. Dad never saw it. After the shark passed, I started to swim after it, and it quickly swam away.
During that same trip, I saw a 5 ft 'cuda while snorkling off Destin, FL. Dad was there too. Again, he didn't see it. Like Paulie, I was more concerned about those fish because of their sudden quickness and reputation for striking at a diver in reaction to seeing a shiny object, such as the sunlight reflecting off a metal watch.
I think I'd be more spooked seeing a large shark while out sitting on my board, feet danging in the water. Not sure which would be spookier, being in very clear water or murky water.
Jul 26, 2014, 11:25 AM #6
Good stuff guys, seen both shark and cuda while surfing, one day at the inlet the cudas were everywhere, just surfed and hoped for the best, nobody got bit, was just one of those days on high alert, there were so many pods of bait fish that every time a wave broke, you could see them all getting rolled up in the wave, talking hundreds of thousands of them, thought for sure one of was gonna get it
Jul 26, 2014, 01:36 PM #7
uh huh.
when that fish burps then squirts (the photographer)
we'll all say
r.i.p. steve irwin
Jul 26, 2014, 03:27 PM #8Senior Member
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- Mar 2014
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- wb
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whatever cranks your tractor dog


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Personal finance courses for aspiring entrepreneurs offered statewide
February 28, 2012Print
- Writer:
- Denise Horton
- Contact:
- Michael Rupured
Athens, Ga. - Before starting a business, it's essential that entrepreneurs have their personal finances in order, said University of Georgia consumer economics specialist Michael Rupured. He and other faculty in the UGA College of Family and Consumer Sciences will be offering workshops on personal finance throughout Georgia during the next several months.
"You may have the greatest idea in the world for a business, but if your personal finances are in disarray or if your credit scores are too high, it's almost impossible to get funding from banks or other lending agencies," Rupured said. "In addition, there's the reality that it can easily take six months to a year before a new business has any income. You have to be certain that you can continue to pay your home mortgage and buy groceries during that time."
Rupured will be joined by housing and consumer economics professor Joan Koonce and family and consumer sciences Cooperative Extension agents for the free workshops. During the four-hour sessions, participants will learn to set and achieve financial goals, plan their personal spending, learn how to work effectively with banks and other financial institutions, learn about credit, plan for the unexpected and take steps to protect their personal assets.
"These workshops are not designed to help entrepreneurs get their business started," Rupured said. "Instead, they're designed for people who understand that starting a new business is a tremendous undertaking and who understand the importance of having their personal finances in order before taking on the additional risks of business loans."
March workshops
• Valdosta - March 8
• Dublin - March 13
• Thompson - March 15
April workshops
• Athens - April 3
• Sandersville - April 12
• Bainbridge - April 17
• McDonough - April 26
May workshops
• Americus - May 15
• Griffin - May 29
June workshops
• Marietta - June 5
• Swainsboro - June 5
• Statesboro - June 12
• Fulton County - June 13
• Waycross - June 19
For more information on the workshops, see www.fcs.uga.edu/ext/econ/mm.php.
Filed under: Culture / Living, Business / Economy


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About This Activity
Run locally, think globally! Montessori Academy, Batavia, Illinois hosts this first annual event that spans two bridges and two continents. 100% of all registration fees go to important causes: locally, Tri-City Family Services; globally: Run for Congo Women.
Come run or walk our 5K, 10K or one of our three childrens'; races and support families in need in your community and across the globe. The 5K starts at 7:00 am, the 10K starts at 7:30 am, the Toddler Trot starts at 9:00 am, the Junior Jog starts at 9:15 am, and the Youth Mile starts at 9:45 am.
This race has a beautiful, scenic course that is on a bike path along the Fox River. The 10K crosses two bridges, so the racers will be on both sides of the river. The 5K is an out and back course that crosses the river and comes back. The Toddler Trot and Junior Jog will be held on the campus of the Montessori Academy while the Youth Mile will utilize the bike path along the river.
Race day parking and race starts will be at the VFW in Batavia, IL.
645 S. River Rd.
Batavia, IL 60510
Race Day registration will be CASH ONLY from 5:30 am to 6:30 am at the VFW (next door to Montessori Academy)
The 5K and 10K races will be chip timed with advanced chip technology -- the Innovative Timing Systems Jaguar timing system uses a disposable timing chip that is attached to the race bib.
We will also have tech race shirts for the first 200 to register. Please register by April 23, 2011 to be guaranteed a shirt.
We would like to let you know that packet pickup has been scheduled for Wednesday, May 4th and Thursday, May 5th.
We will also accept registrations at these locations for the race (cash only)
On Wednesday, May 4th, pick up/registration will be located at the New Balance Store in Geneva, Illinois from 4pm - 7pm.
1749 South Randall Rd
Suite E
Geneva, IL 60134
On Thursday, May 5th, pick up/registration will be located at Geneva Running Outfitters from 4pm - 7pm.
221 W. State Street
Geneva, IL 60134
Runners with baby joggers and stroller are not allowed in the race; bicycles, in-line skates, other wheeled vehicles (except wheelchairs) and dogs are likewise not allowed.
For information please contact Irene Crane at 630-853-1927 and firstname.lastname@example.com, or George Markham at 630-326-9000 and email@example.com
Organizer Info


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|May 16, 2005, 03:06 PM
Senior Member
Join Date: October 10, 2002
Location: Boston, MA
Posts: 1,242
Craig Spegel Grips?
I'm thinking of getting a pair for my new snubby 686. Does anyone have grips by Spegel? How do they hold up and do they make the recoil harsher? Thanks.
|May 16, 2005, 09:39 PM
Senior Member
Join Date: November 13, 2004
Location: PacWest
Posts: 455
Try'em, if you don't like'em you won't lose much on resale
Barring use to drive fencing staples I'd expect them to last as long as any - probably longer than guns will be legal in Massachussets . Recoil is pretty much a matter of physical laws - nice heavy grips will help - otherwise it's a matter of felt recoil and depending on how they fit your hands I'd expect felt recoil to be a little more severe with the boot grips I take it you are talking about.
If you get access to a time machine - see the MIT Timetraveler's convention this past weekend I'd go with Fuzzy Farant but you likely won't have that option.
For more comfort I'd get custom Herrett or Nill or another target/range oriented grip. Given that you say snubby I can't imagine a more or better fitting grip.
|May 18, 2005, 09:25 AM
Senior Member
Join Date: January 8, 1999
Location: Albuquerque, NM, USA
Posts: 2,541
Didn't Fuzzy's grips require grinding on the frontstraps of frames?
Anyway, I've never met anyone disappointed with Spegel's grips. Myself included.
|May 18, 2005, 10:48 AM
Senior Member
Join Date: December 14, 2000
Location: Upstate
Posts: 365
Spegels are more comfortable to me by a large margin than the Ahrends. Craig's Boot Plus is a little more controllable. The fit is fantastic, almost like custom stocks.
Yes, Erich.
Fuzzy's stocks (at least for the N frame) required grinding the front of the frame slightly, I had a set on a S&W 5" M27 in the early 70's. Great feel for a large frame revolver.
"We have seen the enemy and he is us", Pogo
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Discussion in 'General Pro Player Discussion' started by vkartikv, May 9, 2007.
Who has reached a GS final only once in their career and won it (besides Gomez 90 and Majoli 97)?
Gaudio, Costa, Johanson
Yannick Noah.
I think this is comprehensive among Open Era winners. Corrections welcome -- I did not spend more than half an hour on it.
Sue Barker (76 F)
Chris O'Neill (78 A)
Barbara Jordan (79 A)
Iva Majoli (97 F)
Anastasia Myskina (04 F)
Andres Gimeno (72 F)
Mark Edmonson (76 A)
Adriano Panatta (76 F)
Brian Teacher (80 A)
Yannick Noah (83 F)
Andres Gomez (90 F)
Thomas Muster (95 F)
Richard Krajicek (96 W)
Thomas Johansson (02 A)
Albert Costa (02 F)
Gaston Gaudio (04 F)
Ah yes, Adriano Panatta. The only guy to beat Borg at the French Open - Bjorn lost there in '73 and '76, and skipped the event in '77. Astounding!
What about Petr Korda's AO win. Was he in another Slam final?
Also, Kuznetsova 2004 USO
Think he was in the French final. Think he lost Courier.
Yes, in '92.
Kuznetsova lost last year's FO final.
she won 04 US open
She went to last years FO final and lost.
Didn't Pat Cash only get to one GS final?
Cash lost in 88 to Wilander at the Aus Open. Great match btw.
He also lost in 87 to Edberg at the AO, also in 5 sets.
Damm!, I thought this was question on Lendle who reached 19 slam finals...
5 setters in a Slam final are rare too. I remember Wimby 2001, some French Opens with Gaudio-Korea, Agassi-Medvedev but thats about it right nows. Anyone remember more?
Yeah there has been a drought recently. Agassi over Martin in the 99' USO comes to mind.
There was Sampras over Ivanisevic at Wimbledon the year before.
but it wasn't her only final i thought that waas the point of this thread only one grand slam final and won it too
Separate names with a comma.


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Had a chance to eat at this place a couple of days ago. Apparently, the menu is based on Dr. Weil's recipes and recommendations.
I had a "chopped" salad. The ingredients were fresh and simply prepared. I could tell when I ate it there was not a lot of salt, butter, oil, etc. More like what I would make at home. Hubby had the Ahi tuna sliders. These were absolutely fabulous. Delicious, possibly perfect, rare-cooked Ahi tuna with pungent wasabi sauce (mayo or something like it) on a small soft bun. The order came with two of these. I can't stop thinking about how good they were!
The wine list offered some interesting selections by the glass and many were priced very reasonably. I believe I had Tangent Pinot Gris for $6. The entire bill was $48 including tip for an entree salad, the sliders with a side of roasted sweet potatoes and two glasses of wine.
The patio decor was lovely, service prompt and with the mild weather, it was surprisingly relaxing for a mall setting. The only thing that I did not enjoy was that the tables are spaced very closely. We had to listen to a couple of grouchy people complain about God-knows-what the entire time.
I will be visiting again soon, and won't mind the 20 minute wait for a table :).


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I'm sure most of you know who I am but I thought it'd be fun to do this anyway. My name is Amy, I'm 21 years old. I live in the beautiful state of Utah. I've worked as a pet groomer for over 3 years. My 5th anniversy on Pet Talk was a few days ago.
I have 3 dogs. Nebo is the only one that's actually mine, and he's my spoiled baby. He's a 3 year old siberian husky. Reggie and Sydney are my parents dogs, Reggie is a 12 year old schnauzer and Sydney is a 3 year old schnauzer..and Syd is a girl, everyone thinks she's a boy. I also brought home a betta fish a few weeks ago, but he's still nameless.
I love photography, art (I draw pet portraits, link is in my signature, but I must apologize for the horrible non-updated website), and music (my favorite by far is U2!), and animals, of course....I'm a tad obsessed with siberian huskies.
*edit* For husky_mom my user name isn't that interesting, but it's because I love wolves and my last name starts with a Q. It's the name I've been using since we've had the internet and I just stick with it..
Here's two pictures of the dogs!


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Children bearing the brunt of the Syrian conflict, says UNICEFNEW YORK, 3 August 2012 - As violence engulfs many parts of Syria, UNICEF is alarmed that children continue to bear the greatest brunt of the tragedy.
UNICEF is working with its partners to deliver urgently needed assistance to hundreds of thousands of children and families who have fled their homes to escape the fighting and bearing the brunt of the conflict. Families sheltering in schools, mosques and other public buildings in Damascus and beyond are receiving hygiene kits, mattresses, blankets, as well as food and other items for their children. During the month of July, despite deteriorating security conditions and limited access in many areas, UNICEF and its partners were able to expand these and similar activities to reach around 94,000 people, the vast majority of them (around 90 per cent) children and adolescents.
UNICEF Executive Director Anthony Lake says the most urgent priority is to protect children and their families.
"As the eyes of the world focus on the mounting violence in Syria, we must not overlook the fact that while children are not responsible for this tragedy, they're paying a terrible price," said UNICEF Executive Director Anthony Lake. "Children are losing their lives, losing their homes, losing their parents and losing their schooling. UNICEF supports people of conscience everywhere calling on all parties to do everything in their power to protect the innocents."
The deteriorating situation is driving civilians to flee Syria to neighbouring countries in ever increasing numbers. To date, more than 130,000 Syrian refugees – around half of them children and adolescents -- have been registered in Jordan, Lebanon, Turkey and Iraq. UNICEF is joining with other humanitarian agencies to provide them with a range of essential services, including water, sanitation, education and psychosocial support.
For further information, please contact:
Newsline: Syrian Conflict
4 April 2014:
Statement by UNICEF Syria
15 March 2014:
Three years of Syria conflict have devastated lives of millions of children
12 March 2014:
European parliamentarians mark third year of Syrian conflict
11 March 2014:
Number of children suffering more than doubles in third year
24 February 2014:
Public urged to voice their outrage


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Recently there has been much discussion in the press about the Boy Scouts of America and the release of files on people deemed ineligible to be Scout volunteers. Unfortunately the issue has been sensationalized, largely by legal predators, dancing manically like savages discovering fire.
The reality is far different.
BSA as an organization, like the society it serves, has progressed in its recognition of and defense against issues of child abuse over the past century. Since its founding in 1910, the Boy Scouts have served more than 100 million youth. What may have been thought appropriate responses 50 or 100 years ago are unacceptable in the 21st century. This coupled with the unfortunate fact that some cases were not handled properly, even considering the mores of the time, has created concern about an organization that has given esteemed service to our nation.
A University of Virginia study cited in an Arizona Republic article on Oct. 23 found an incidence of abuse in Scouting programs of approximately two per 100,000. The same study found 70 cases per 100,000 in the general population. Although any statistic above zero is unacceptable, it should be noted that Scouts are 35 times safer at Scouting activities than the general youth population.
How has BSA achieved such an enviable record? Partly it has been through maintaining files on known and suspected predators such as those causing the current sensation. More importantly, however, it has been achieved through a comprehensive program of youth protection training and reporting.
All Boy Scout leaders are required to take youth protection training every two years. I just renewed my certification this month. The program covered topics such as inappropriate forms of contact, bullying and hazing prevention, privacy issues, cyber-bullying and adequate adult supervision. Parents are advised of youth protection guidelines on an annual basis, and policies are published in every youth and adult handbook and the Guide to Safe Scouting.
Additionally, Scouting's local partners, the churches and civic organizations that sponsor Scout units, are required to approve the application of each adult Scout volunteer.Comment on this story
For 102 years, the Boy Scouts of America has taught citizenship and character development to millions of boys. If you would like to see how successful BSA has been in this effort, I recommend you look at a study by Baylor University titled, "Eagle Scouts, Beyond the Merit Badge." It is available free online. Astronauts, Nobel scientists, world-class athletes, Pulitzer Prize-winning authors and even presidents have credited Scouting as being fundamental to their success in life. As importantly, millions of solid citizens and family men earning an honest living have had their character formed through scouting.
Any abuse is intolerable, but the Boy Scouts of America has an impressive record of proactively protecting the young people and families it serves.
John Keegan is the former Mayor of Peoria and currently serves as the volunteer BSA area president for the southwestern United States and Pacific basin. These comments reflect his personal opinions.


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Studies have been carried out previously to determine whether mesenchymal stem cells (MSC) influence the progression of pulmonary fibrosis. Here, we asked whether MSC (derived from mouse bone marrow and human umbilical cord blood) produce factors that mediate lung fibroblast (LF) growth and matrix production. MSC-conditioned media (CM) were found by ELISA to contain significant amounts of PDGF-AA and transforming growth factor-β1 (TGF-β1). Proliferation was increased in a concentration-dependent manner in LF cell lines and primary cells cultured in MSC-CM, but neither anti-PDGF antibodies nor PDGF receptor-specific antibodies affected proliferation, nor did a number of other antibodies to well-known mitogenic factors. However, proliferation was significantly inhibited by the Wnt signaling antagonist, secreted frizzled related protein-1 (sFRP-1). In addition, anti-Wnt1 and anti-Wnt2 antibodies attenuated MSC-CM-induced proliferation, and increased expression of Wnt7b was identified. As would be expected in cells activated by Wnt, nuclear β-catenin was increased. The amount of TGF-β1 in MSC-CM and its biological activity were revealed by activation at acidic pH. The stem cells synthesized and released TGF-β1 that increased α1-procollagen gene expression by LF target cells. Addition of anti-TGF-β to the MSC-CM blocked upregulation of collagen gene expression. These data demonstrate that MSC from mice and humans produce Wnt proteins and TGF-β1 that respectively stimulate LF proliferation and matrix production, two hallmarks of fibroproliferative lung disease. It will be essential to determine whether these factors can play a role in attempts to use MSC for therapeutic approaches.
- peptide growth factors
- pulmonary fibrosis
mesenchymal stem cells (MSC) have been derived from several sources including human umbilical cord blood (CBMSC) and mouse bone marrow (BMSC). These cells have broad potential and have been shown to differentiate into fibroblasts and myofibroblasts as well as bone, adipose, and epithelial cell types (7, 12, 30, 34). Initial genomic studies suggested that CBMSC and BMSC differ in expression of a large number of genes (29, 41). However, it is not understood whether these observed differences would translate to altered responses in models of stem cell therapy. MSC appear to influence the development of inflammation and injury in several model systems (28, 37). In the lung, introduction of MSC has ameliorated bleomycin injury (27, 33, 43) since a BMSC subpopulation expressing IL-1 receptor antagonist (IL-1RN) apparently provides protection from bleomycin-induced inflammation by inhibiting TNF-α and IL-1 production (27). In addition, BMSC were observed in the lungs of asbestos-exposed mice and correlated with decreased fibrosis (16). These data suggest that different MSC subpopulations can significantly modulate the onset of a fibrogenic response. Clearly, there is a great deal to be learned about the mechanisms through which MSC play a role in the development of lung disease and whether the cells could serve as potential therapeutic agents.
One approach to learning key aspects about the biology of MSC is to culture the cells and collect the conditioned medium (CM) to determine whether the cells release factors that control the proliferative and fibrogenic features of pulmonary target cells. We have been interested for some time in the biology of TNF-α, transforming growth factor-β1 (TGF-β1), and the PDGF isoforms that control growth and collagen production by lung fibroblasts (39, 42). Inasmuch as interstitial lung fibroblasts and myofibroblasts are the cells that synthesize collagen during the genesis of fibroproliferative lung disease, we are asking in the work presented here whether MSC express paracrine factors that regulate cell growth and collagen production. The results show that MSC derived from human umbilical cord blood (CB) or from the bone marrow of mice release Wnts 1, 2, and 7b, TGF-β1, and PDGF-AA into the culture medium. The CM induced a fibroblast cell line as well as primary lung fibroblasts to proliferate and express the gene that codes for α1-procollagen (COL1A1) in a concentration-dependent pattern. Our data demonstrate: 1) the Wnt signaling antagonist, secreted frizzled-related protein-1 (sFRP-1), inhibited the proliferative effects of the MSC-CM; 2) Wnt7b expression was measured along with nuclear β-catenin; and 3) anti-TGF-β1 antibody inhibited the induction of collagen gene expression in lung fibroblasts.
Culture of Primary MSC
BMSC were provided by the Tulane Center for Gene Therapy. Primary mouse BMSC were isolated from the femurs and tibia of male C57BL/6 mice as previously described (35). Cells were plated, and nonadherent cells were removed by washing with PBS. BMSC were further expanded in BMSC culture medium [Iscove's modified Dulbecco's medium (IMDM; Invitrogen, Carlsbad, CA) supplemented with 2 mM l-glutamine (Mediatech, Manassas, VA), 100 U/ml penicillin (Mediatech), 100 μg/ml streptomycin (Mediatech), 0.25 μg/ml amphotericin B (Sigma, St. Louis, MO), 10% FBS (Invitrogen), and 10% horse serum (HyClone, Logan, UT)]. Surface marker expression was determined using flow cytometry. BMSC were negative for CD11b and CD45 and positive for SCA-1, VCAM, and CD29 (5). The BMSC were used up to passage 8.
CBMSC from three different donors were provided by the Adult Stem Cell Core at the University of Vermont. Briefly, human CB was obtained from normal term deliveries, and Ficoll gradient centrifugation was used to isolate CB nucleated cells (38). Adherent cells were expanded in CBMSC culture medium [α-minimum essential medium (αMEM; Invitrogen) supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FBS]. CBMSC were assessed for the expression of MSC surface markers via flow cytometry (38). All experiments were conducted with CBMSC at passages 2-5. On receipt of all MSC, stemness was confirmed by inducing differentiation into osteocytes and adipocytes (data not shown). The CBMSC-CM data presented herein were reproduced independently with cells from each of the three donors.
Fibroblast Culture Conditions
Mouse Swiss 3T3 fibroblasts were purchased from American Type Culture Collection (ATCC, Manassas, VA) and cultured in DMEM (Mediatech) supplemented with l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FBS.
Human lung 16Lu fibroblasts were purchased from ATCC and cultured in MEM (Mediatech) supplemented with l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 10% FBS, nonessential amino acids (Mediatech), and sodium bicarbonate. Cells were kept subconfluent, and media was changed every 3–4 days. 3T3 and 16Lu fibroblasts were used up to passage 15.
Primary normal human lung fibroblasts (NHLF) were purchased from Lonza (Walkersville, MD) and cultured according to manufacturer's instructions in fibroblast basal medium (FBM) supplemented with hFGF-B, insulin, gentamicin, amphotericin B, and 2% FBS (media and supplements from Lonza). Fibroblasts were isolated from an 11-yr-old female and a 31-yr-old male. Experiments were conducted at passages 2-5. Data are representative of both donors and are not significantly different between the two.
Culturing Fibroblasts in MSC-CM
Subconfluent BMSC (Fig. 1A) and CBMSC (Fig. 1B) were trypsinized and plated in T-150 flasks at a concentration of 8 × 105 cells per flask. Cells were cultured in 20 ml of serum-free (SF) media for 48 h. Concentrated CM was filter-sterilized and stored at −20°C.
For all experiments, CM was thawed and concentrated with Centricon Plus-20 centrifugal concentration tubes (5,000 NMWL; Millipore, Bedford, MA) and resuspended at a fivefold concentration in SF DMEM, SF MEM, or SF FBM. The fivefold concentration corresponds to 2 × 105 MSC per milliliter. CM was serially diluted fivefold. Acid activation of TGF-β in CM was achieved by acidifying the concentrated CM with 1 N HCl followed by incubation at room temperature for 10 min. The CM was then neutralized with 1.2 N NaOH/0.5 M HEPES before resuspending fivefold in SF medium.
For the proliferation studies, fibroblasts were plated in 96-well plates at 1 × 103 cells/well in a 96-well plate for bromodeoxyuridine (BrdU) analysis or 2 × 104 cells/well in 12-well plates for cell counts. Cells were quiesced in SF media for 24 h before experimentation. Fibroblasts were cultured in serially diluted MSC-CM for 48 or 96 h. After 48 h, BrdU was added at a final concentration of 10 μM for 6 h. Proliferation was assessed using a cell proliferation ELISA commercial kit (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions. Cells were enumerated after 4 days with a hemocytometer. Viability was determined using trypan blue exclusion dye.
For the collagen studies, fibroblasts were plated at 2 × 104 cells/well in 12-well plates and quiesced in SF media for 48 h. Cells were treated with serially diluted MSC-CM for 48 h. Collagen expression was determined by real-time RT-PCR analysis of COL1A1 fibroblast mRNA expression.
Fibroblast Coculture Experiments
Target NHLF were plated at 1 × 104 cells/well on the bottom surface of six-well Transwell plates (0.3-μm pore size) and were cultured in serum-containing FBM for 24 h. Subsequently, MSC or NHLF cells were plated in the top well at 4 × 104 cells/well in serum-containing αMEM (CBMSC), IMDM (BMSC), or FBM (NHLF). Control wells contained αMEM only in the top wells. After 24 h, media in the top wells was replaced with SF αMEM (CBMSC) or SF IMDM (BMSC), and media in the bottom wells was replaced with SF FBM. After 72 h, media was aspirated from all wells, and the top wells were discarded. The target NHLF cells were trypsinized, resuspended in trypan blue, and counted using a hemocytometer.
Quantitative Real-Time RT-PCR
Total RNA was isolated and purified using an RNeasy Mini Kit (Qiagen). Two hundred nanograms of RNA were reverse-transcribed in 20-μl volumes using an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. One microliter of cDNA was amplified in 20-μl reactions containing 200 nM COL1A1, TGF-β1, or 36B4 primers in iQ SYBR Green Supermix (Bio-Rad). PCR was performed for 35 cycles at 95°C for 15 s and 60°C for 45 s using a MyiQ Real-Time Detection System (Bio-Rad). All samples were run in duplicate. PCR sequences were: COL1A1, forward, 5′-CTGGTAGCCGTGGTTTCCCT-3′, reverse, 5′-CCAGGGCTTCCAGTCAGACC-3′ (166-bp PCR product); 36B4, forward, 5′-TCTGGAGAAACTGCTGCCTCATA-3′, reverse, 5′-GCCAGCAACATGTCCCTGATC-3′ (90-bp PCR product); TGF-β1, forward, 5′-GGACACCAACTATTGCTTCAGCTCC-3′, reverse, 5′-AGGCTCCAAATGTAGGGGCAGGGCC-3′ (155-bp PCR product). Standard curves showed that PCR efficiency was 98–100% for the assays. Negative controls, such as cDNA reactions without RT or RNA, and PCR mixtures lacking cDNA were included to detect possible contamination. Melt curve analysis was conducted to confirm reaction specificity. Samples were quantitated by the relative standard curve method using standard curves made from serial dilutions of COL1A1, 36B4, and TGF-β1 plasmid standards. Expression of COL1A1 and TGF-β1 were normalized to that of 36B4 for each sample.
PCR Array
Two micrograms of total RNA from quiescent NHLF cells and CBMSC was converted to first-strand cDNA with a RT2 First Strand Kit (SABiosciences, Frederick, MD). Relative expression of an array of genes related to the human Wnt signaling pathway was determined using the NHLF and CBMSC cDNAs as templates in real-time PCR RT2 Profiler arrays (SABiosciences). A total of four arrays were analyzed, two for NHLF and two for CBMSC.
Measurement of Cytokine Concentration in the CM
The concentration of TNF-α, TGF-β1, PDGF-BB, and PDGF-AA in MSC-CM cultured in SF media for 48 h was quantified via ELISA (human and mouse TGF-β1, TNF-α, and PDGF-BB DuoSet and PDGF-AA Quantikine ELISA kits; R&D Systems, Minneapolis, MN). To quantify biologically active TGF-β1, the media was acid-activated according to the manufacturer's instructions and compared with untreated media.
Recombinant, human Dickkopf-1 (Dkk-1), sFRP-1, TGF-β1, PDGF, and recombinant, murine Dkk-1 and sFRP-2 were purchased from R&D Systems. Anti-mouse TGF-β, anti-mouse PDGF receptor-α (PDGFR-α), anti-mouse PDGFR-β, anti-human FGF-2, anti-human FGF-4, anti-human FGF-9, anti-human VEGF, and anti-human Wnt7b were all purchased from R&D Systems. Anti-Wnt1 (A-20), anti-Wnt2 (H-20), and anti-Wnt3 (N-15) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies and recombinant proteins were reconstituted according to the manufacturer's instructions.
Measurement of β-Catenin
NHLF were plated in triplicate at 1 × 105 cells/well in 12-well plates, quiesced in SF media for 48 h, and cultured in fivefold concentrated CBMSC- or BMSC-CM or 33 mM LiCl in SF media for 5 h as a positive control (9). Nuclear and cytoplasmic fractions were isolated according to the manufacturer's instructions (NE-PER Nuclear and Cytoplasmic Extraction Reagents; Pierce Biotechnology, Rockford, IL). Total protein concentrations were measured using the Bradford assay. Total β-catenin was quantified via ELISA according to the manufacturer's instructions (R&D Systems).
ANOVA was performed for all statistical analyses using a Tukey-Kramer t-test to perform multiple comparisons between all treatment groups. All values are expressed as means ± SE where n = 3 or 4. A P ≤ 0.05 was considered statistically significant.
MSC Produce TGF-β1 and PDGF-AA
Previously, we presented evidence both in vivo and in vitro that the peptides TNF-α (19), TGF-β1 (20), and PDGF-AA and PDGF-BB (21) play a role in the development of fibroproliferative lung disease. Here, we measured the production of these cytokines in medium conditioned by MSC. To determine the concentration of TNF-α, TGF-β1, PDGF-AA, and PDGF-BB in the supernatant of BMSC and CBMSC, MSC were plated at 80% confluence and cultured in SF media for 48 h. The CM was harvested and measured by ELISA. Gene expression of TGF-β1 was 2.5-fold higher in quiescent CBMSC than in quiescent NHLF as measured by RT-PCR (data not shown). Neither BMSC nor CBMSC produced TGF-β1 that could be detected before acid activation (data not shown). However, following acidification, CM from both MSC types contained similar concentrations of TGF-β1 (∼120 pg/ml; Fig. 2A). In contrast, PDGF-AA concentrations were significantly greater in BMSC-CM than CBMSC-CM. BMSC produced ∼90 pg/ml PDGF-AA, and CBMSC produced 10 pg/ml (Fig. 2B). PDGF-BB and TNF-α could not be detected by ELISA in the CM from either of the MSC populations (data not shown).
MSC-CM Induces Proliferation in Quiescent Lung Fibroblasts
Monolayer cultures.
Two primary hallmarks of pulmonary fibrosis are the proliferation of fibroblasts and the production of collagen. To determine whether the cytokines measured in the experiments reported above affect fibroblast proliferation, MSC-CM was concentrated, serially diluted, and applied to quiescent Swiss 3T3 fibroblasts and to NHLF for 48–96 h. Proliferation was assessed by BrdU incorporation and cell counting. CM from both BMSC and CBMSC induced proliferation at similar levels. Recombinant human PDGF-BB was included as the positive control for growth. Fivefold concentrated and unconcentrated CBMSC-CM increased BrdU incorporation 4- and 2-fold, respectively (Fig. 3A). Additional concentrations did not induce significant changes in BrdU incorporation. Serial dilutions of BMSC-CM produced similar proliferative effects (Fig. 3B). Swiss 3T3 fibroblasts were enumerated via trypan blue exclusion after 4 days of treatment with CBMSC- and BMSC-CM to confirm the BrdU incorporation data, and these counts showed that increased BrdU incorporation correlates with an increased number of cells (Fig. 3, C and D). To confirm that MSC-CM induces proliferation in primary fibroblasts as well as immortalized fibroblasts, primary cells from NHLF were purchased from a commercial source and treated with MSC-CM as in the previous experiments. CBMSC-CM concentrated 5-fold induced an ∼4.5-fold increase in proliferation of NHLF (Fig. 3E). In addition, NHLF proliferated at a similar rate as 3T3 fibroblasts following treatment with BMSC-CM (Fig. 3F). Together, these data suggest that CM from CBMSC and BMSC contain mitogenic factor(s) capable of inducing fibroblast proliferation at levels similar to 20 ng/ml recombinant human PDGF-BB. Acid activation of 5-fold concentrated BMSC-CM resulted in a significant decrease in NHLF proliferation compared with untreated BMSC-CM (Fig. 4).
NHLF were grown in coculture with NHLF, BMSC, or CBMSC so that the cells were in close proximity but were separated by a membrane (0.3-μm pore size). NHLF proliferation was significantly increased when cocultured with BMSC (Fig. 5) indicating that the BMSC are releasing a diffusible proliferative factor into the media.
sFRP-1 Inhibits the Proliferative Effect of MSC-CM
To determine the cytokine(s) responsible for regulating fibroblast proliferation, several blocking antibodies specific for known MSC mitogenic cytokines were added to the CM. The data in Fig. 1B show that MSC produced measurable levels of PDGF-AA, which is known to induce fibroblast proliferation (11). Anti-PDGFR-α and anti-PDGFR-β were added to the NHLF before treatment with MSC-CM. Growth rates of the NHLF treated with either BMSC- or CBMSC-CM were not significantly affected by the inhibition of the PDGFR (Fig. 6). In addition, a number of other proliferative cytokines such as FGF-2, -4, and -9 and VEGF reportedly are synthesized by BMSC and CBMSC (23, 34). Attempts were made to selectively block these factors by inhibitory antibodies, but each of these failed to suppress the mitogenic effects of the CM (data not shown).
Wnt proteins have been shown to be potent fibroblast mitogens (9, 44), and recent studies have demonstrated that MSC secrete several Wnt proteins (1, 8). To determine whether Wnt plays a role in the induction of proliferation by MSC-CM, several concentrations of recombinant human sFRP-1 and murine sFRP-2 were added to the CM before treating the NHLF. sFRP-1 inhibited NHLF proliferation induced by CBMSC- and BMSC-CM in a concentration-dependent manner (Fig. 7, A and B). However, sFRP-2 did not affect NHLF proliferation at any of the concentrations tested (Fig. 7, A and B). Stimulation of the Wnt signaling pathway has been shown to increase β-catenin concentrations (9). Nuclear and cytoplasmic β-catenin concentrations were measured via ELISA in NHLF following 5-h culture in CBMSC-CM, BMSC-CM, or LiCl (33 mM) as a positive control. Cytoplasmic β-catenin concentrations were not significantly changed in response to either CBMSC or BMSC (Fig. 7C), but both CBMSC- and BMSC-CM increased nuclear β-catenin concentrations similar to levels stimulated by LiCl (Fig. 7D).
Anti-Wnt1 and 2 Inhibit Fibroblast Proliferation, and Wnt7b Expression is Identified by PCR
MSC have been shown to produce a number of canonical Wnts such as Wnt1, 2, and 7b (1, 23). Anti-Wnt1 and 2 antibodies were added to CBMSC- and BMSC-CM before fibroblast treatment (Fig. 8, A and B). A nonspecific isotype control was added to the CM. The data indicate that both antibodies significantly attenuated fibroblast proliferation in a concentration-dependent fashion (Fig. 8, A and B). Together, these data suggest that MSC-CM contain a sufficient concentration of Wnt proteins to induce lung fibroblast proliferation. Expression of Wnt7b was determined by PCR array to be ∼50-fold higher in quiescent CBMSC than in quiescent NHLF; however, no difference in Wnt1 or Wnt2 expression was detected (data not shown).
MSC-CM Induces Collagen Gene Expression in Lung Fibroblasts
To determine the effect of the TGF-β1 produced by the MSC on collagen gene expression in fibroblasts, CM was concentrated, serially diluted, and applied to quiescent 16Lu and NHLF for 48 h. Gene expression was determined by analyzing COL1A1 levels measured by RT-PCR. When the TGF-β1 in the CM from BMSC or CBMSC remained latent, there was no effect on COL1A1 levels (Fig. 9, A and B). However, acid activation of the CM to remove the latency-associated peptide (24, 31) from TGF-β1 resulted in a significant dose-dependent increase in COL1A1 levels (Fig. 9, A and B). Both CBMSC- and BMSC-CM increased COL1A1 in 16Lu fibroblasts 5- to 7-fold when concentrated 5-fold (Fig. 9, A and B, respectively). In addition, unconcentrated CM from either source increased collagen gene expression approximately 3- to 4-fold. As conducted in the previous set of experiments, primary human fibroblasts were utilized to confirm the results seen with 16Lu fibroblasts. Both CBMSC- and BMSC-activated CM increased procollagen gene expression(s) in NHLF to similar levels as those found in the 16Lu cell line (Fig. 9, C and D).
Antibody Inhibition of TGF-β1 Blocks the Induction of COL1A1 in Lung Fibroblasts
The previous data show that activating TGF-β1 in the CM increases COL1A1 gene expression. To demonstrate that it was the TGF-β1 in the CM that induced collagen gene expression, anti-TGF-β was added to the CM before fibroblast treatment. The data indicate that specific blocking of TGF-β1 abrogates the enhanced collagen expression in fibroblasts treated with MSC-CM (Fig. 10, A and B). Together, these data suggest that the CM is able to induce matrix production in fibroblasts via a TGF-β1-dependent pathway.
The objective of our studies reported here was to determine whether a small set of well-characterized peptides are synthesized and released by human and mouse MSC and then to determine whether the factors mediate the growth and matrix production of lung fibroblasts as target cells. To determine the degree of biological variability that is likely to be present in both the stem cells and fibroblast targets, we compared CBMSC from 3 individual donors and BMSC from mice. The fibroblasts were derived from human and murine lungs. Our data demonstrate that CM from MSC derived from these two different sources clearly produced PDGF-AA, but apparently at a concentration insufficient to cause proliferation. On the other hand, latent TGF-β1 that could be readily activated was released at concentrations sufficient to induce collagen gene expression in lung fibroblasts. In addition, we present evidence that CBMSC- and BMSC-CM induce lung fibroblast proliferation via stimulation of the Wnt-β-catenin pathway. Human and mouse signaling proteins and their receptors are highly homologous as determined by amino acid sequence alignments; Wnts 1, 2, and 7b proteins are at least 96% identical, TGF-β1 proteins are 90% identical, and TGF-β and frizzled receptors are 95 and 93% identical, respectively.
Previous reports have demonstrated inhibitory effects of MSC on the growth of target cells such as T lymphocytes and cardiac fibroblasts (13, 18, 26). Conversely, MSC have been shown to induce proliferation and migration in keratinocytes and endothelial cells (3, 32). To our knowledge, the results reported here represent the first demonstration of the mitogenic potential of MSC-CM on lung fibroblast target cells in vitro. The earlier studies indicated above reported suppression of cardiac fibroblast proliferation and collagen production (18, 26), but these differ technically from our studies in several ways. Finally, on this topic, Li and colleagues (18) reported antiproliferative and antifibrotic effects when BMSC-CM was combined with a profibrotic protein such as angiotensin II. Thus conditions for collecting CM appear to vary greatly among these reported studies, and clearly future studies are required to characterize the role of culture conditions on the concentration of paracrine factors in media conditioned by MSC.
In our initial search for mitogenic cytokines, we found that both BMSC- and CBMSC-CM contain PDGF-AA and that BMSC produce ninefold more PDGF-AA than CBMSC. PDGF-AA is a well-established mitogen for mesenchymal cells, including lung fibroblasts, and is upregulated during fibrogenesis in animal models (21) and human lungs (25). However, we found that inhibiting the PDGF α- and β-receptors on lung fibroblasts did not reduce the ability of MSC-CM to enhance proliferation (Fig. 6) and antibodies to the AA and BB isoforms had no effect on cell growth (data not shown). The BB isoform was not detected, and our data suggested that either the concentrations of PDGF-AA in the CM were not sufficient to affect lung fibroblast growth or the AA isoform was not biologically active. Apparently, there is another, more potent proliferative factor in the media that is diffusible to the target cells in our coculture system (Fig. 5).
The Wnt family of proteins regulates a number of cell functions including proliferation, morphology, and migration. Several reports have observed an upregulation of Wnt-related proteins in fibrotic lungs (14). A recent study observed upregulation of Wnt1-inducible signaling proteins 1 and 7b in the lungs of bleomycin-exposed mice (17). Another study found that the Wnt downstream effector, β-catenin, was increased in the nucleus of spindle-shaped mesenchymal cells located in fibroblast foci in the lungs of 16 out of 20 patients diagnosed with idiopathic pulmonary fibrosis (IPF) or usual interstitial pulmonary fibrosis (4). Both BMSC and CBMSC express several Wnt genes such as Wnt1, 2, 4, 5a, 5b, 11, and 16 (1, 23). Our data demonstrate that the Wnt antagonist sFRP-1 significantly inhibits the mitogenic effect of both CBMSC- and BMSC-CM (Fig. 5). sFRP binds directly to Wnts and is hypothesized to inhibit the activation of frizzled/LRP receptors by the Wnt ligands (2). It is currently unknown how the different members of the sFRP family affect the proliferative capacity of the various Wnt proteins (2). In accordance with finding a Wnt ligand playing a role in fibroblast proliferation, we showed there was a clear upregulation of nuclear β-catenin in cells cultured in the MSC-CM (Fig. 7D). This suggests that the proliferative response is mediated through the canonical pathway as reported previously for Wnt proteins synthesized by MSC, i.e., Wnts 1, 2, and 7b signal through the canonical β-catenin pathway (36). Detection of increased expression of the Wnt7b gene in a PCR array suggests that this Wnt may contribute to the fibroblast proliferative response. Antibodies to other canonical Wnt proteins, i.e., Wnt1 and Wnt2, significantly attenuated fibroblast proliferation (Fig. 8, A and B), however, increased expression of these genes was not detected by the PCR array. Wnts 3 and 5 did not block cell proliferation in our system. The conservation of the Wnt pathway among many different cell populations (22) suggests that Wnt synthesized by MSC could affect other cell types in a similar manner. For example, studies have demonstrated that Wnt1 induces proliferation in endothelial cells and fibroblasts (10, 44). Future studies will determine whether Wnt synthesis by MSC and the biological activity of the Wnt-β-catenin pathway are altered as the stem cells differentiate and are exposed to other lung cell types in vitro and in vivo.
Expression of several cytokine genes by MSC has been demonstrated (3, 29, 41); however, to our knowledge, this is the first study to quantify the concentration of TGF-β1 and PDGF-AA produced by CBMSC. Our data confirm the findings of previous studies demonstrating TGF-β1 gene expression in MSC (3, 34). Interestingly, the concentration of TGF-β1 we report here in the CM from BMSC and CBMSC is similar to the levels produced by lung fibroblasts (39). In addition, all of the measurable TGF-β1 synthesized by MSC is present in the latent form as reported previously in lung fibroblasts (39). One of the many functions of TGF-β1 is to act as a potent stimulator of matrix production (40) and as an antagonist of cell proliferation (31). It has been well-established that upregulation of TGF-β1 in the lung is a hallmark of IPF (40), and TNF-α has been shown to regulate TGF-β1 expression in lung fibroblasts by activating the ERK-specific MAPK pathway (39). Our repeated attempts to identify TNF-α in the MSC-CM were unsuccessful, and this is consistent with other studies (15), suggesting that when the MSC synthesize TGF-β1, expression of this peptide is not influenced by TNF-α in an autocrine or paracrine fashion. Whether TNF-α from other lung or circulating cells would influence the expression of TGF-β1 by MSC is unknown at this time but could be an important parameter in an inflammatory process. As would be expected, activated TGF-β1 in the CM did block target cell proliferation (Fig. 4).
Activation of latent TGF-β1 is necessary for the ligand to bind to specific cognate receptors and upregulate matrix production in fibroblasts. Several studies have demonstrated that fibrotic lungs contain a number of factors that contribute to activating latent TGF-β1 (6, 24, 31). Consistent with this knowledge, we found that MSC-CM did not enhance collagen gene expression unless the TGF-β therein was activated at low pH. Activation of TGF-β1 in the CB and BMSC-CM upregulated collagen gene expression to levels similar to the TGF-β1-positive controls (Fig. 9). These data suggest that the TGF-β1 in the MSC-CM could act as a profibrotic paracrine factor, but this observation does not necessarily follow what has been seen in vivo. For example, studies demonstrated that amelioration of fibrosis followed bone marrow transplantation in mice exposed to asbestos, and these animals had decreased TGF-β1 in the bronchoalveolar lavage fluid (16). Ortiz and colleagues (27) found that a unique subpopulation of BMSC produce IL-1RN, which blocked TNF-α and IL-1 in bleomycin-treated lungs. These data suggest that MSC could inhibit the accumulation of TGF-β1 by inhibiting the production of TNF-α in alveolar macrophages (27). On the other hand, Xu and coworkers (43) demonstrated that BMSC expressing CXCR4+ contribute to the progression of fibrotic lesions. It is currently unknown whether the CBMSC and BMSC we used produce IL-1RN or are positive for CXCR4. Further investigations will determine how culture conditions as well as differentiation of MSC affects the production of TGF-β1 and its biological properties. At this juncture, our studies suggest that the TGF-β1 produced by MSC could induce matrix production following activation as discussed above.
In summary, our studies show that the MSC derived from mouse BM and human CB produce biologically active Wnts 1 and 2 as well as TGF-β1 (Figs. 8 and 10). Synthesis of these factors was not found to vary significantly between donors or species. Wnt ligand increased accumulation of nuclear β-catenin and induced cell proliferation. The TGF-β1 induced different populations of fibroblast target cells to upregulate the gene that codes for COL1A1. These two activities, fibroblast proliferation and extracellular matrix production, are two key features of lung fibrogenesis. Whether the MSC will influence lung cells in vivo as they have been shown to do here in vitro remains unclear. Certainly, little is known in regard to how exogenous treatment with cytokines or placing the stem cells in coculture conditions with different lung subpopulations would affect production of TGF-β1, Wnts, or other potent mediators. In addition, it will be critical to determine how differentiation into respiratory phenotypes such as myofibroblasts and epithelial cells affects synthesis of these fibroproliferative factors.
This work was supported by National Institutes of Health Research Grants ES-06766 and HL-60532.
- Copyright © 2009 American Physiological Society


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Flash Draw Created Feb 05, 2007 470 x 338px Unless noted Copyright 2007 The Master.
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this is so cool! very awesome ship! and i really like the way you did the planets! =)
very cool ship design. too bad the ink ran out.
yea... stars and more detail
9 for the ship ;)
cool ship also nice drawing too bad you ran out of ink some stars would've been helpful


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A contemporary family home with open-plan living, dining and kitchen area, 2 storage rooms, an indoor garage, a master bedroom with en-suite bathroom and dressing, 3 bedrooms and a bathroom. Upstairs the spacious hallway provides a study corner. Outside is a spacious garden with terrace.
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The Security and Exchange Commission (SEC) moved Friday to freeze a Switzerland-based account that was used in a case of suspected insider trading to collect more than $1.7 million ahead of this week's acquisition of H.J. Heinz Company.
The commission alleges that unknown traders took risky bets on Heinz' stock just before Berkshire Hathaway and 3G Capital agreed to acquire the company in a deal valued at $28 billion.
Upon public announcement of the acquisition, Heinz stock soared, rising 20 percent. Trading volume increased by more than 1,700 percent from the day before, the SEC noted.
SEC obtained an emergency order from Manhattan federal court to freeze assets in the Zurich-based account. The action ensures that any profits gained illegally cannot be siphoned out of the account while SEC investigates.
The commission's complaint had information about the deal when they purchased "out-of-the-money" Heinz call options on the day before this week's announcement. The account in question had not traded in Heinz securities in the previous six months.
General trading in Heinz options had been minimal before the deal went public, according to SEC.