text:
Year 4 Teacher
Job Category: Education
Salary/Rate: £100 - £170/day, we pay to scale!!
Location: South Birmingham Job Type: Contract
Start Date: September 2016
Contract Duration: Ongoing
The leadership team of the newly amalgamated Primary School is looking to appoint an enthusiastic and committed teacher to the school. The ideal candidate will be an experienced Key Stage 2 Teacher of any specialism who is able to plan, prepare and deliver stimulating, challenging and enjoying lessons that keep pupils engaged in classwork and progressing well in their academics. You will be teaching Year 4, so you will need to have recent experience.
Our client is looking for a Year 4 Teacher who is able to work until the end of the academic year July and someone who would like the opportunity of a permanent contract.
This is a large primary school with a vibrant and nurturing atmosphere where all young people are able to develop in their skills and grow as individuals. The senior leadership team and teachers are all privileged to teach the pupils at this school and their passion for education and determination to see all pupils succeed is at the heart of everything they do. The school delivers a broad and balanced curriculum and hold high expectations of traditional and exemplary standards of behaviour, effort and commitment to school life.
Are you a teacher who holds UK QTS? Did you know that with Lost & Found Recruitment is fully compliant with Agency Worker Regulations ensuring you receive equal treatment for pay and conditions after a 12 week qualifying period? Please ask for further details. Important Notice
It is Lost & Founds policy, as a matter of courtesy, to respond to all applications within three working days. However, because of the volume of applications, we are sometimes unable to respond to individual candidates. If we have not contacted you within three working days your application has been unsuccessful and your details have not been retained. Please apply for any other position that you may see in the future. Thank you.
Lost & Found Recruitment is a Recruitment Agency and is advertising this vacancy on behalf of one of its Clients.


text:
The fifth edition of the Transport Tracker looks at the challenges facing the global economy in the first half of 2015 and their impact on transport companies, and shows the latest developments in Shipping, Aviation and Express Logistics.
© 2017 KPMG International Cooperative ("KPMG International"), a Swiss entity. Member firms of the KPMG network of independent firms are affiliated with KPMG International. KPMG International provides no client services. No member firm has any authority to obligate or bind KPMG International or any other member firm vis-à-vis third parties, nor does KPMG International have any such authority to obligate or bind any member firm. All rights reserved.
KPMG has launched a state of the art digital platform that enhances your experience and provides improved access to our content and our people, whatever device you are on.

subset: commoncrawl


text:
Fact or Faked: Paranormal Files
Area 51/Cajun Apparition
Season 2 Episode 1
Bill's team goes to Louisiana to recreate a 2001 video taken at the historic Myrtles Plantation of an alleged ghost apparition. Ben's team goes to Nevada to recreate a UFO video allegedly sneaked out of Area 51.
- 43 min
- Premiered 10/22/2016


text:
Hot Links: Tent City's Birthday, Party Crews, and Taser Sales
Maricopa County Sheriff Joe Arpaio will celebrate the 16th anniversary of Tent City today. The planned fete includes a hot dog barbecue and an ice cream social for inmates. Arpaio says the 1,000 hot dogs and the ice cream were donated...Police arrested 48 people at a house party near 32nd avenue in Phoenix early Saturday. Police say those arrested were members of a "party crew," people who hop from house to house partying every night. Most of the arrests on Saturday were for underage drinking and liquor and curfew violations...The Arizona Game and Fish Department has lifted a hunting ban on lions in the Kofa National Wild Refuge near Yuma. The agency says mountain lions are not at risk, and is instead taking steps to protect bighorn sheep in the area...Queen Creek fire captain Eric Tinkham, 44, died in his sleep early Saturday at one of the town's fire stations. Tinkham had worked as a firefighter in Queen Creek for 24 years and is survived by his wife and three daughters...Scottsdale-based company Taser International Inc. hopes federal stimulus funds will stimulate sales of Tasers to more police departments. But many local agencies, including the Glendale Police Department and the Scottsdale Police Department, say they have no plans to increase their Taser purchases...A village in Lincolnshire, England suffered a power outage after a woman's thong got caught in overhead power cables. About 17 homes and businesses were without power until crews were able to free the entangled undergarment.
Tent City to celebrate anniversary
Police make 48 arrests at Phoenix party
Queen Creek fire captain found dead at station
Scottsdale's Taser hopes stimulus helps sales
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text:
As we celebrate the birth of Jesus Christ, it is also a time to reminisce of other great Christmasy things, like hallucinations on Christmas Eve. The Nutcracker is one of the craziest ideas of a Christmas tradition, but since the LDS Temple Hill Events sponsors a free Nutcracker, we figured it was a good time to test out this long running Christmas tradition.
Catherine joined us (of course, we can't do anything without our sister wife), and we spent a very memorable night watching ballerinas, toy soldiers, and rats dance around on stage.
I don't think we're going to keep this as a tradition. I'm not sure the ballet is really my favorite thing in the world, but it was fun to experience at least once. The temple also puts great lights around the grounds for everyone to enjoy. We loved going up and enjoying a night at the ballet for Christmas.


text:
1st: I tried to perform a search to see if this was covered already, but we all know how pathetic the search function is on this forum.
2nd: I tried to use Google but BMWMOA forum links were broken.
OK, so I don't use my car specific Garmin 40LM on my motorcycle, but with this unit having a built-in Lithium-Ion battery, the issue I had may apply to the Zumo and other similar models.
I started having short battery life and was about to order a new battery for this unit. But with it being about 18 months old and having worked in a defense lab with batteries and battery chargers, I knew the battery issue I was having was not the norm.
So I contacted Garmin; great service as usual by the way.
This is the response that I got and the instructions that I followed that worked. Note: leave the GPS connected for the duration recommended. It is a slow process.
Thank you for contacting Garmin International.
I am happy to assist you. It sounds like your device needs to be reset. Please turn the device on without any power connected to it. Once you do this please allow the device to completely drain of all battery. At this point please follow the instructions below. This should reset the battery so it will take a complete charge. Please advise.
Please plug your device into the computer and press and hold the power button for 8-10 seconds. The device may not power on immediately. Please allow the device to charge on the computer for a full 8-12 hours. Please be sure that the computer does not time out or have a screensaver come on. This can cut off power to the device and it will not charge. Once the charge has completed I recommend updating the operating system software. Please follow the link below for instructions. Please be sure to do all of the additional updates except for the voices you do not speak, the help files, and the vehicles. Please let us know if you have any further questions.


text:
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LIGHTFOOT-L ArchivesArchiver > LIGHTFOOT > 2003-01 > 1041611835
Subject: [Lightfoot] Choctaw LIGHTFOOT family VA>GA>AL>MS>LA
Date: 3 Jan 2003 09:37:15 -0700
This is a Message Board Post that is gatewayed to this mailing list.
Surnames: LIGHTFOOT CHOCTAW/CHEROKEE
Classification: Query
Message Board URL:
Message Board Post:
Dear Missy and others,
It has been a couple of years since your postings regarding your search for Choctaw LIGHTFOOT. By this point in time you may be more aware that there are many others of us out here that are also searching for the same type of info. While we haven't resolved the whole situation yet, there may be missing pieces to be recovered by bringing more of us together in this search.
It would appear that there were one or more Choctaw family groups and Cherokee family groups or perhaps blended families of Choctaw and Cherokee and possibly other associated tribes of the name of LIGHTFOOT that received the name of LIGHTFOOT from marriages into the English descended LIGHTFOOT family group descending from (Reverend) Richard LIGHTFOOT of late 1500s Stoke Breurne, Northamptonshire, England, and his wife, Jane JONES, (daughter of (Captain) Roger JONES,) through their son, John (1) LIGHTFOOT, Sr., (Esquire,) a barrister, (Attorney at Law who studied at Oxford and Gray's Inn School of Law, (part of the Oxford University system,) of London, Middlesex, England, who married Elizabeth PHILLIPS, (daughter of Olive SAWYER and Francis PHILLIPS, (Exchequer to the Queen,) and through 2 of Elizabeth and John's several children who immigrated in abt. 1670 from England to Virginia who were Royal Navy sea (Captain) John LIGHTFOOT who began a family in Virginia before dying in!
Surinam in the West Indies in the Caribbean while his brother, Virginia militia (Colonel) Philip LIGHTFOOT stayed in Virginia and with wife, Alice CORBIN, (dau of Alice ELTONHEAD and Henry CORBIN who were shipowners,) and had children.
This particular group of LIGHTFOOT family and descendants were slave owners as well as the CORBINs and other connecting families including my HATTEN/HATTON family group and many others were also shipbuilders/shipowners and merchant mariners so it is highly likely that they were all or in part slave traders as well.
There was another John LIGHTFOOTE, (I believe descended from the Quaker LIGHTFOOT(e) group also with some out of London who also immigrated but earlier into Virginia.
There is one Cherokee group of LIGHTFOOT that the information begins about in the early to mid-1700s that cites their earliest ancestral grandfather as being a man who was named (Captain)/(Capt.)/(Cap.)/(Chief) John LIGHTFOOT who was the father of Nancy LIGHTFOOT and her sister Dorcas LIGHTFOOT who married into the DUNCAN, ADAIR and BENGE families. The BENGE family was from John "Trader/ Old Trader" BENGE who was possibly of Scottish or English origin and from Virginia where he had married Elizabeth LEWIS, daughter of William Terrell LEWIS, prominent Virginia family of Welsh descent. Elizabeth found out after the births of several children that John "Trader/Old Trader" BENGE had another Cherokee family happening at the same time with a Cherokee woman named WURTEH, born: Abt. 1750 in Tsalagi Town, (near/at Burnside, Kentucky which was a Chicamagua trading area,) who was the daughter of GREAT EAGLE, (descendants of MOYTOY.)
In addition, the BENGE family and connecting families also married into the family group of (Chief) John BOWLES, aka BOLD HUNTER, aka DUWALI/D'IWALI, born: Abt. 1750 in North Carolina, died: July 16, 1839 next to the current day small community of Redlands near Henderson, Rusk County, Texas and 20 miles east of current day Canton, Van Zandt County, Texas which is, I think, about 45 miles, give or take, from downtown Dallas, Texas, when he was killed on the 2nd day of fighting in the "BATTLE OF NECHES," which was the "LAST BATTLE OF THE TEXAS CHEROKEE," when as Chief of the Texas CHEROKEE, (Chief) John BOWLES, approximately 83 years old, led the approximate 800 tribal members, with approximately 600 of the group being women, children and elderly men, as they fought against the Texas Cavalry under the orders of Texas Republic President Mirabeau LAMAR who was taking back the apprx. 1.6 million acres of land given to the Texas CHEROKEE and the asssociated bands of Indians whic!
h had originally been orchestrated by former Texas Republic President Sam HOUSTON who also became a member of the Texas CHEROKEE tribe as well as marrying a CHEROKEE woman as his 2nd wife. LAMAR waited until HOUSTON was out of the state on business to take the opportunity to evict the Texas CHEROKEE from their land resulting in the "BATTLE OF NECHES."
It is not proven but I believe that this aforementioned (Chief) John BOWLES/DUWALI/BOLD HUNTER, born: Abt. 1750 in NC as John BOWLES, 1/2 Scottish, may be the same BOLD HUNTER that was my paternal 5th great grandfather. The aforementioned John BOWLES' father was said to have been a Scottish trader but I also find BOWLES from England immigrating in the 1600s, and 1/2 Cherokee from his mother, who later led his people out of the areas of NC and/or Georgia into Tennessee and across the south into Missouri where they were until the Great New Madrid Earthquake in 1811 then into Arkansas until 1819 when they moved into East Texas eventually settling in the aforementioned area between Canton, Van Zandt County and Henderson, Rusk County and I believe that before his known marriages when he was over 50 years old to the 3 wives recorded while in Texas and children that there were, most likely, other marriages and children in the eastern regions possibly North Carolina and/or Georgia!
. I believe it is highly likely that he is the same BOLD HUNTER, born: Abt. 1750 in North Carolina, died: in Texas, whom is known to be associated with the name of BOWLES and was the father of Sofia/Sophia HUNTER, possibly aka BOWLES and/or TOTER/TOATER who was the daughter of mother: BEAVER TOTER/TOATER whom is recorded in the CHEROKEE rolls of Georgia in 1835 and also mentioned in a book called "CHEROKEE PLANTERS OF GEORGIA," and is mentioned as a landowner so I think it highly likely that she was the daughter of a CHEROKEE Chief since it had only been about 3 years earlier that the first land deeds were given out by the U.S. Gov't. to CHEROKEE Chiefs. She was also the mother of sons, Jim TOTER, Light TOTER and Hog TOTER.
Sophia/Sofia HUNTER married Hugh PIERCE, (RS,) born: Abt. 1749 or in 1751 in Frederick County, Virginia, and Sophia died in 1859 in Henderson, Rusk County, Texas where she had moved to after her husband died with several of her sons and their families including George PIERCE and wife, Elizabeth McCRACKEN.
Sophia HUNTER and Hugh PIERCE, (RS,) were my paternal 4th great grandparents through their daughter, Elizabeth "Bettie" PIERCE, born: 1790 in 96th District, South Carolina, (formerly Spartanburg Dist. then 96th Dist. then Edgefield Dist. then County,) who married William MYRICK, born: Abt. 1790 in Barnwell County, SC, and they migrated into Mississippi Territory/Alabama settling in the area that became Jones County, Mississippi and Jasper County, Mississippi.
The PIERCE family traveling into Texas was with the KUYKENDALL and DeLOACH families that also married into the GOODWIN and MYRICK families and other connecting families including my connecting HERRINGTON, GRANTHAM, BARLOW, HATTEN/HATTON, LOTT families that married into the LIGHTFOOT family.
My maternal 3rd great grandmother, Frances Lucretia "Patsy" LOTT, born: Abt. 1802 in Alabama/MS Territory, married my maternal 3rd great grandfather, William HATTEN, born: January 01, 1800 in Tar River, Nash County, NC, (formerly Edgecomb County, NC,) who was the son of Lewis Lamkin HATTEN and wife, Sarah "Sally" UNDERWOOD, (who appears to have Indian descent possibly CHEROKEE.)
Frances Lucretia "Patsy" LOTT was the daughter of Absalom LOTT, born: Abt. 1758 in Edgecomb County, North Carolina,
(son of John (3) LOTT, (son of John (2) LOTT and wife, Elizabeth JOYNER, born: 1720 in Isle of Wight County, VA,) and Absalom's wife, Martha Jane Dorsey? LIGHTFOOT, born: Abt. 1763 in, perhaps, Virginia or in Antigua or onboard a ship to Antigua if, indeed, she is the daughter of the Philip LIGHTFOOT who was in Antigua from abt. 1763-1773. Martha Jane Dorsey? LIGHTFOOT's father is said to have been Philip LIGHTFOOT, born in early 1700s in Williamsburg, VA, of the English descended group from (Reverend) Richard LIGHTFOOT but not yet precisely sure which of the many Philip LIGHTFOOT men he was.
My aforementioned Frances Lucretia "Patsy" LIGHTFOOT, aka Mrs. William HATTEN, (the 1st wife,) died on or about May 01, 1840 during or immediately after the birth of her last son of 8 children as the family traveled from Mississippi through Ouachita Parish, Louisiana, (part of it is now Jackson Parish,) where, incidentally, a large contingent of CHOCTAW resided around that time under (Chief) TUSQUAHOMA, (I believe that is the correct name.) William HATTEN and his oldest sons fashioned a casket/coffin out of the family buckboard to bury Frances Lucretia "Patsy" in and she was buried near the road under/next to a tree on which, according to family legend, the words "CHOCTAW PRINCESS" were carved. Now, before the screaming starts, the CHOCTAW did not have a term "PRINCESS," however, it is possible that this was what was written precisely by her husband, for whatever reason in using the term, "PRINCESS," or it could be that this was the term given to whatever was actually writ!
ten by those latter descendants of hers as the information passed down through the family.
All of that said, Frances had a sister named Elizabeth LOTT who married a man named Thomas WATTS whose 'white family group' of WATTS is believed to somehow be related to the CHEROKEE family group of WATTS that married into the other CHEROKEE families mentioned above and Elizabeth LOTT's descendants also have the information in their group of descendants that she was part CHOCTAW.
The CHOCTAW descent 'legend' also exists in other of the LOTT branches from the marriage of Absalom LOTT and wife, Martha Jane Dorsey? LIGHTFOOT and appears to have entered with the mother of Martha most likely but we have no information, so far, on whom her mother was so that pretty much stops us at the moment. If Martha's father Philip was the Philip LIGHTFOOT that was in Antigua from Abt. 1763-1773 then his wife is thought to have been named Susannah ? and there was a daughter of theirs that was named Margaret Susannah LIGHTFOOT.
Martha Jane Dorsey? LIGHTFOOT, born: Abt. 1763, and Margaret Susannah LIGHTFOOT, born: Abt. 1765, both married in Abt. 1781 probably in Georgia.
And, of course, back to your original info that, perhaps, your CHOCTAW descended LIGHTFOOT family might have been those that inherited the name from plantation masters named LIGHTFOOT that could also have been the case. There, obviously, is not just one of LIGHTFOOT descent for all of the LIGHTFOOT individuals or for all of the LIGHTFOOT CHOCTAW and/or CHEROKEE individuals.
There is still much missing information and speculation here but, perhaps, there are some connections that someone will see that will lead us to new revelations.
Bellinda Myrick - Barnett
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text: This article about Chrysaor is a stub. You can help Mythology Wiki by expanding it. Chrysaor Chrysaor was the human brother of Pegasus. He was the son of Medusa and Poseidon and husband of Callihroe, daughter of Oceanus. He was the father of Geryon. Retrieved from "http://mythology.wikia.com/wiki/Chrysaor?oldid=28193" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Stubs Greek Mythology Demigods Greeks Offspring of Poseidon


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I agree with you. The Admins and moderators are way to strict and sensitive to this stupid code of conduct. They are also unfair.
I once created a thread.
I added "users are idiots, and are confused by functionality" mentality of Gnome is a disease. If you think your users are idiots, only idiots will use it. I don't use Gnome, because in striving to be simple, it has long since reached the point where it simply doesn't do what I need it to do.
And i was quoting wat linus says about gnome.
They really need to delete this stupid code of conduct


text:
So I have google wallet up and running on my S3 (ICS). Cards are loaded, NFC is on. When i went to my local CVS, it would not work. I took the phone out of the case as well just to see if there were any interference, but no dice.
Anyone else have positive results? Any way to test?


text:
Release Notes: Minor bugfixes and a new ooam_filter_ct method: hide_unknown_locations(), the only way to get rid of allocations done by code without debug info (like a stripped library).
Release Notes: The most important bugs fixed in this release are the removal of internal memory leaks, making list_allocations_on() work for custom debug devices, and making the old-ABI (pre-3.0 compiler) demangle routines thread-safe.
Release Notes: The debug output of one thread that interrupts so-called 'continued debug output' of another thread will now correctly cause "<unfinished>" and "<continued>" to be printed.
Release Notes: This release can filter the overview of allocated memory by the source file from which the memory was allocated, and by whether or not AllocTag was used. A minor bug related to the use of dlopen() has been fixed.
Release Notes: This release fixes a horrible bug that caused libcwd to crash on any call to realloc for a memory allocation without description.
Release Notes: This release adds support for g++ 3.1. A bug was fixed that caused compilation to fail on SuSE and Mandrake. Allocation administration is now kept in an STL map per thread which causes, for example, markers to work with threads too.
Release Notes: This release adds (non-threaded) support for Solaris 2.8.
Release Notes: This release fixes a deadlock which occurred during the initialization of threaded applications that use g++ version 2.96 or earlier. The library now comes in a thread-safe and a thread-unsafe version. It is now possible to filter the overview of allocated memory in various useful ways.
Release Notes: Minor bugs were fixed in the documentation. An API change was made: the macros related to configure-options have been renamed.
Release Notes: This release fixes a compiler error when passing a pthread_mutex_t* to debug_ct::set_ostream as locking facility. It also contains several bug fixes in the tutorial. A new tutorial was added on debugging threaded applications. A possible dead lock in object_file_ct::find_nearest_line() has been fixed. There are also several other minor bug fixes.


text: Simoco DMR technology previewed ahead of February 2012 launch13 Dec 2011 05:44 PM Simoco gears up for DMR launch at IWCE 2012 with technology demonstrationsGlobal mobile radio specialist Simoco Group has successfully demonstrated its new DMR technology at Radio Comms Connect in Australia and the Federation of Communications Services (FCS) Business Radio event in the UK, ahead of its full international DMR product suite launch at IWCE in February 2012. Simoco sought to underline the interoperability of its technology and its commitment to interconnectivity policies that will drive open commercial markets by demonstrating its DMR mobile making and receiving calls with equipment from other manufacturers. Simoco is the third independent manufacturer of DMR base-stations and radio terminals to show working equipment, as the commercial viability, strength of standard and ecosystem for DMR products, applications and services continues to build demand. Ian Carr, chief executive officer of Simoco Group, said: '2012 is set to be a very exciting year for Simoco. DMR is recognised as a major radio standard for the future and we are committed to delivering a suite of products that provides the quality and performance Simoco is known for, together with the interoperability that gives customers greater choice and flexibility in their solutions. 'The success of our recent demonstrations, ahead of our upcoming launch at IWCE, underlines the quality of R&D and cutting edge products we continue to bring to markets around the world,' continued Carr. 'We are proud to be the third independent manufacturer to the market with a full end-to-end DMR solution, and with the imminent launch of this digital technology we will be bringing a new wave of open-standard, digital solutions to the market.' Simoco is also one of the key drivers behind the DMR Association's commitment to setting the standards for the future of DMR, helping ensure interconnectivity policies that drive open commercial markets. Simoco Group develops and manufactures PMR, DMR and TETRA products and services for markets including the emergency services, military, utilities and transport. It operates around the globe including the Americas, Australasia, Europe, the Middle East, the Far East and Africa.Wireless magazineLeave a CommentPlease write code to prove you're humanMore on... DMRPennine delivers Hytera radio solution at Etihad Stadium in ManchesterHytera Mobilfunk to provide DMR radio coverage for Metro Barcelona Birds Eye chooses Hytera IP-linked DMR system to aid pea harvestMore on... Critical CommunicationsFinland and Norway sign cross-border TETRA communication dealCC Europe 2017: Hytera showcasing new base station and handheld radioMore on... SimocoSimoco roll outs 80 site DMR Tier III network in CanadaSimoco launches Xd 700 Series of DMR portable and mobile radiosLatest in PMRPennine delivers Hytera radio solution at Etihad Stadium in ManchesterTCCA pinpoints key focus areas for LTE-based critical communicationsTait appoints Andrew Gill as managing director of EMEA region


text:
LAFAYETTE — Freshman starter Gunner Leger pounded the strike zone for seven strong innings, and the scorching Kyle Clement gave him an early cushion in Louisiana-Lafayette's 5-0 series-opening win against UT-Arlington.
"We pitched, we played defense and we got timely hitting," coach Tony Robichaux said. "It's hard to lose when you do all three."
It was the second shutout of the season for the Cajuns (26-16, 11-7), who have now won 11 of their last 13 games. The Mavericks (17-23, 8-11) have now lost 14 of their last 20.
Leger (5-2) was throwing darts from the onset. The book on the Mavericks was that they were an aggressive team, especially in the first few pitches of an at bat.
"They look for the fastball early in the count," Leger said. "We knew if we just came in and spotted up the fastball and mixed speeds, we'd be able to get them out."
Leger fired first-pitch strikes to 18 of the 26 batters he faced, and he lured the Mavericks into swinging at pitches they wouldn't cause much damage with.
The results were impressive. Leger retired the side in order in five of his first six innings of work, and he only allowed one base runner past second base in the outing.
Robichaux pulled Leger after he finished the seventh inning with 99 pitches, 69 of which came in for strikes.
"That's the name of the game, to throw strikes," Robichaux said. "The type of pitcher he is, he's got to be a strike thrower. He's not throwing 94 to 95 miles an hour. His pitchability's always been through the roof."
The Cajuns' offense made it a little easier for Leger to focus on throwing strikes by pushing four runs across the plate in the first three innings. Three of those runs were driven in by Clement, who continued his torrid play at the plate in recent weeks.
After Blake Trahan led off the bottom of the first inning with a bloop single, Clement pounded an opposite field double into the left field corner. Trahan wheeled around to score from first.
In that at bat, Mavericks pitcher Chad Nack (3-4) came inside several times before leaving a pitch out over the plate that Clement could barrel up. Clement armed himself with that knowledge the next time he came to the plate.
With Trahan again at first base, Nack tried coming back inside to Clement in the third inning. Clement made him pay for it by wrapping his fifth home run of the year around the right field foul pole, giving the Cajuns a 4-0 lead.
"When I'm feeling good I usually work the whole field," Clement said. "He tried to bust me (inside) the first at bat and then went away, so I had a feeling he was going to come back in the next at bat. … I was just guessing pitches."
Guessing pitches or not, Clement is on some kind of streak right now. After going homerless for his first 46 career games, he's got five long balls in his last nine games.
This week, Clement is hitting .583 with three doubles, a pair of home runs and six RBI with a 1.333 slugging percentage.
"There's an old saying: If you've got a broom handle in your hand and you think you can hit, you can hit," Robichaux said. "Right now, he's running high on confidence."
Nick Thurman added an RBI single in the fifth inning, scoring Stefan Trosclair, who extended his hitting streak to 11 games.
Junior Will Bacon finished what Leger started with two solid innings of relief to finish the shutout.
The Cajuns will go for the series win Saturday at 6 p.m. Freshman right-hander Evan Guillory (3-0, 4.22) will get the start for the Cajuns.


text: July 15, 2011 Apica Announces Webinar with Rackspace on Critical Need for Performance Monitoring and Load Testing of Cloud Apps • Attendees of Free Webinar to Learn How to Implement Simple Techniques to Prevent a Site from Becoming "the Next Bad Web Story"• Apica CEO Sven Hammar to Demonstrate How Utilization of Latest Technologies Results in Rapid Actionable Insight, Identifying Performance Issues Without Breaking the BudgetPalo Alto, Calif. – July 14, 2011 – Apica, a leading load testing and performance-monitoring provider for cloud and mobile applications, today announced a free webinar with Rackspace® Hosting, (NYSE: RAX), focused on the critical need for load testing and performance monitoring of cloud applications to prevent website failure.Webinar title: "No More Excuses!! Don't Become Another Bad Web Story" When: Tuesday, July 19, 2011 at 12:00pm PT/3:00pm ET Who: Sven Hammar, CEO of Apica with Adam Hansen, Marketing Coordinator at Rackspace The session will discuss how to not become another bad web story. As more businesses move to cloud computing, it's imperative these businesses test and monitor their cloud applications prior to deployment to ensure uptime and optimal performance levels. Apica CEO Sven Hammar will demonstrate how to work with selenium scripting, utilizing the latest tools and expertise that result in "Rapid Actionable Insight" and easy identification of performance issues without breaking the budget or schedules to improve overall performance monitoring strategy. Speaker Bio Sven Hammar has extensive business and technical experience in startups and public companies. Prior to founding Apica, Mr. Hammar was CEO and co-founder of Celo Communications. Before that, he was the VP Marketing at Gemplus (GEMP). Sven founded Apica in January 2005 in Sweden. As CEO, he has led the company from concept through development to becoming the premier provider of performance testing and monitoring solutions in Europe. In June 2011, Apica launched its U.S. headquarter offices. For more information and to register, go to: https://cc.readytalk.com/cc/schedule/display.do?udc=33hue7wfaysx Helpful Links • Apica • Rackspace Hosting • Apica Joins Rackspace Cloud Tools Program Announcement • Apica Cloud Performance blog Follow Apica on Twitter: http://twitter.com/#!/ApicaSystems About Apica Apica is a leading provider of load testing and performance monitoring solutions that test, monitor and optimize cloud and mobile applications. More than 250 customers, including Newsday, PricewaterhouseCoopers, eBay Sweden and Thomas Cook, use Apica for the highest possible performance standards globally and in the cloud. Apica's products are uniquely designed for cloud infrastructure and have performed some of the largest published load tests ever conducted. Apica WebExcellence Suite includes Apica LoadTest, Apica WebPerformance and Apica WebOverload, for testing applications for maximum capacity, real-time performance, improved load times and protection from peak load. Apica was founded in 2005 and is based both in Stockholm, Sweden and Palo Alto, Calif., with offices in the UK. To learn more about Apica, visit http://www.apicasystem.com. Media Contact: Danielle Salvato (for Apica) (650) 340-1982 Categories: Uncategorized


text:
Correction: Earlier versions of this article misstated where the business executives from New York were picked up by an UberBlack driver. The passengers were picked up in the District, and asked to go to Tysons Corner.
A group of taxi companies in Virginia is seeking to stop Uber and other ride services from operating in the state, the latest effort to rein in an increasingly popular transportation option that connects passengers with drivers through smartphones.
In a lawsuit filed Friday in Fairfax County Circuit Court, eight Virginia cab companies contend that the drivers who contract with Uber and similar services are ferrying passengers illegally because they lack taxi licenses.
The lawsuit is similar to one filed recently in Maryland, where five taxi companies are seeking to stop such services.
"We're playing by the rules, and the rules are in place to protect the public who ride in for-hire vehicles," said Spencer Kimball, owner of Northern Virginia Checker Cab, one of the Virginia plaintiffs. "It would be so easy for them to comply with the regulations, but they're choosing not to do so."
Uber, Lyft and other such companies have come under increasing fire after gaining a foothold in major cities. Critics complain of price gouging during emergencies and say the drivers, who work part time for ride services, are not always properly vetted.
This month, a group of New York business executives picked up in the District and headed to Tysons Corner were taken on a wild ride by an UberBlack driver who was questioned and then followed by a taxi inspector. The passengers were unharmed, but District police are investigating the incident.
With state and local officials pushing for licensing, the companies have lately sought a compromise.
Uber and Lyft recently applied for brokers' licenses in Virginia after receiving "cease and desist" letters from state officials — an approach that taxi companies say would still leave the drivers unlicensed.
"It's like a general contractor who was operating illegally decides to get a license but still allows his subcontractors to operate illegally," Kimball said.
Chelsea Wilson, a spokeswoman for Lyft, said the Virginia lawsuit is without merit.
"We are continuing to work with the [state Department of Motor Vehicles] and state leaders to come to a solution that preserves a future for ride-sharing in the state of Virginia," Wilson said.
Uber spokesman Taylor Bennett declined to comment on the lawsuit, but he said, "Uber will vigorously defend the rights of riders to enjoy competition and choice, and for drivers to build their own small business."


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I own squat, Disney owns all. Bastards won't sell either. Sorry I took so long with the update, the story just wouldn't flow, and there is nothing worse than trying to force a story. / indicates Japanese.
Answers – Chapter 5
"Bonnie, is everything all right?"
Bonnie looked up from her food, a bit startled, as she looked across the table to Hirotaka. "Oh, I'm sorry. I just have something on my mind."
Hirotaka smiled lightly. "Is it the fight you had earlier today?"
Bonnie dropped her pizza slice back to the plate. "How did… I mean…"
Hirotaka just frowned slightly. "I am sorry if I intruded on a private subject. I had just heard through the… the grape vine… that you had had a fight. Is that what is on your mind?"
Bonnie sighed, picking up her slice once more. "Yeah, it is. Brick is… was my boyfriend, and I had just broken up with him. He didn't take it too well."
Hirotaka nodded as he took a bite of his own slice. "So Brick, he did something wrong?"
Bonnie looked startled, pausing mid-bite. "What… no, he didn't… I mean…" Bonnie put her slice back down, gathering her thoughts. "If you knew someone was going to do something, something that would hurt you, wouldn't it be better to stop it before it happened?"
Hirotaka paused himself. "That would depend on what was about to happen, and how sure you were that such a thing were to happen."
Bonnie slumped slightly in her seat. "I was pretty sure it was going to happen."
"Then the only other thing to consider would be how much you cared for the person, and how much you would do and deal with to be with that person. Is the pain he would cause you worth it, in the end?"
Looked away, sinking a bit lower into her seat. "Look, can we talk about something else?"
Hirotaka nodded. "As you wish."
Bonnie glanced down at her watch, her eyes widening slightly. "Oh, maybe we can get together later. I just realized I have to be somewhere right now. Later tonight?"
Hirotaka nodded, smiling lightly. "Of course. It would be my honour!"
He watched as the young woman quickly bolted from the table. He knew just what was so important to her, and he smiled. She is accepting her gift, even as she fears it. Sensei will be pleased.
A tap on his shoulder brought him from his thoughts, and he glanced up to see a dark skinned girl, smiling at him. The name escaped him for the moment, but he knew she was a friend to both Ron Stoppable and Kim Possible. "Hello."
"Hi, I'm Monique. I know you're new in town, and wondered if you'd like a guide for a bit. I can even get you a discount at Club Banana if you're interested in picking anything up?"
Hirotaka glanced towards where Bonnie had been, knowing she would be a while at least. Turning back, he smiled. "I would be honoured."
Sensei stood near the entrance, awaiting what he knew was inevitable from his talk with Ron Stoppable. It was true, he was the Chosen One of the prophecies, but for now, he lacked the confidence, both in himself and for what he might achieve. When he saw the young man approaching the gate, bag over his shoulder with a letter in hand, he knew that a further push was needed. He hated having used Yori in this manner, but when she was the first to volunteer, he could not deny her, her honour would have compelled her to question, and he was not ready for that.
When Ron called for her, he quickly realized that it was fate that had made the decision for him, and he smiled inwardly. It was one thing for Ron to shirk responsibility through doubt and fear, but he knew the young man could not leave a friend in danger.
"I am saddened to say that Yori is missing."
Ron's eyes widened slightly, surprise evident. "Missing? Missing how?"
"Since you refused the task of recovering the Lotus Blade, she seized the honour." Sensei motioned for Ron to follow him, and the pair quickly made their way down the trail. When they reached the small clearing, where it was obvious there had been a struggle, they paused. Sensei could see the warring emotions on the young man's face, and knew that he would do the right thing.
Bonnie paid for her final purchase, smiling as she left the store. She definitely felt better today, after pushing her talk with Brick to the back of her mind. Hirotaka had obliged her, going with her to shop, and she smiled at just how nice he had been. Most people followed her around for popularity, to boost their own image, but Hirotaka just wanted to be with her for her, and she liked that. It was too bad he would only be here a few more days. She was surprised though that she hadn't seen him coming though, this was normally the kind of thing she would have caught.
Smiling as she came out of the store, that smile faded when she saw Hirotaka talking with both Kim and Monique, and Bonnie suppressed an urge to tear their eyes out. She watched as he quickly bowed to them, and turned towards her. As he was leaving, she noticed Kim and Monique begin to fight, and smiled just a bit at that. Hirotaka's expression was one of resignation as he slowly approached her.
"What did they want?" The emphasis on they was not exactly hidden.
"They invited me to go with them to the Bravos tonight."
Bonnie looked shocked, a small fear welling up inside. "What did you tell them?"
Hirotaka looked almost hesitant before he responded. "I said it would be my honour to accompany them."
Bonnie's heart fell, and she looked away quickly. "Fine, go with them. Have a nice time."
Hirotaka tried to place a hand on her shoulder, but she brushed it off. "I'm sorry if I have…"
"Don't even bother. You just have a good time with little miss perfect and her friend over there. I'm going home."
Hirotaka watched her storm off, his pleas for her to stop falling on deaf ears. He could feel the guilt of what he had done, as he quickly realized why she had reacted the way that she had. She was scared, and she was lonely, he could see that in how her face fell as they had talked, and he had just left her. Now, she was hurting, and while his thoughts should have been on how he might accomplish his mission now, his only concerns were on how he might make things right with her.
Sensei frowned as he watched young Ron Stoppable heading past the gates, dressed all in black, Fukashima trailing slightly beside him. He knew all this had been anticipated but, even so, knowing and accepting were two different things.
"/Do you think he is ready? He is awfully young for such a heavy burden to be placed on him./"
Sensei frowned, glancing at the woman beside him. "/I do not know Nooni, I just don't know. The spirit is there, even if he does not yet realize it. Soon, the body will follow. This is but one of his early tests, and not even the prophecies are clear on just what will happen./"
Nooni nodded, glancing back to where the retreating students could just be barely made out. "/You do know that Fukashima is the betrayer, don't you/"
Sensei nodded glumly. "/I know. He has made his decision, just as we all have. There is nothing to be done about that now. All we can do is ensure the prophecy comes to fruition, preparing as best we can./"
Bonnie sat on her bed, knees tucked up to her chest, as she stared forlornly at the picture of Brick on her nightstand. She lost Brick, of that she was sure, especially after that tirade at school. Her parents never paid attention to her, and her sisters… well, they just weren't wanted in the first place. Of course, that didn't stop her from wanting their approval, even if she knew she'd never get it. Now, she had lost Hirotaka before she had had a chance to even really get to know him. She felt like crawling into a ball and staying there for a good long time.
A knock at her door startled her, and she pressed herself deeper into her pillows. "What do you want?"
"Bonnie, could I come in?" The sound of Hirotaka's voice was surprising at best, and she knew her mother must have let him in.
Bonnie jumped to her feet, straightening her blouse and wiping away any stray tears that might yet remain on her face. She checked herself quickly in the mirror, making sure she looked all right, before she cracked open the door. "What do you want?"
"Bonnie, I would like to apologize for my actions. I did not know that by accepting I would hurt you in this manner."
Bonnie mouth twitched slightly, but showed no other signs of emotion. "Didn't bother me one bit. What do I care if you hang out with Possible and her friend? Have fun."
Bonnie shut the door, but didn't move from there, wishing her stubborn pride hadn't taken over as he was apologizing. She beat her fist against her head, berating herself for being so stupid.
When she heard him speak again, she was almost overjoyed. "I did not take your feelings into account, and I am gravely sorry about that. I should have realized how much that might pain you. I have felt that myself, and for me to do that to you was unacceptable."
Bonnie slumped against the door, holding herself. "It's not your fault. I shouldn't have just taken off like that."
"It was still not my place to make you feel as you did. If you could forgive me, I would give anything to make it up to you. I value our friendship, more than anything."
Bonnie slowly slid to the floor. "Only friendship?"
She heard a pause, before he responded. "Your heart belongs to another at the moment, and there is nothing I could do to get between that. I know you still care for him, though you are afraid of the pain that might happen. But if you don't risk that pain, can you ever truly know love?"
"What if the pain might be too much?"
"You'll never know unless you try."
Bonnie slowly rose to her feet, opening the door slowly to see the faint smile on Hirotaka's face. She wiped a small tear from her eye, smiling at him. "I guess friends wouldn't be such a bad thing."
Hirotaka's smile broadened. "Friends are never a bad thing."
Sensei smiled at the almost festive mood at the school. With the return of the Lotus Blade, as well as Ron and Yori, the students were almost jubilant, and he allowed them this moment of peace. He walked among them as they talked and laughed of their victory, and the heroics of the two young students. There was also some talk of Fukashima's betrayal, but he knew that it would pass. He knew that the young man, who had allowed darkness into his heart and claim him, would return one day. He knew what would happen that day, and though he did not welcome it, he did understand his role to play. No one else knew that part of the prophecy though, not even Nooni, nor his successor, for they would try and stop him for sure.
Sensei took a moment for himself in his garden. Even with winter encroaching, the garden was still very fragrant, and he smiled. He had lived a good life, with much to be proud of. Many students had come through the school during his time, and many had gone on to do remarkable things. He would see his granddaughter grow to a beautiful woman before his time would come, and his adopted son become a man. He hoped he lived to see them share the love for each other that he could see when they looked at each other, though they did not know and understand it yet.
And he had started the Chosen One on his path. Though he did not know how that path would weave yet, he knew that he was started on the right one.
Sensei slowly made his way over to the gathered students, and smiled when Nooni came from the kitchen, bearing a tray of food. His smile broadened as he made out bite-sized portions of Ron's nacos, and even laughed, however slightly, when he heard the young man's yell of recognition.
The students lined up, and despite the festive mood, Nooni continued the tradition of "catching" their food. Sensei noted the look of determination on Ron's face, as he gripped his chopsticks in his hands. When his turn came, he swung wildly, and then again, as Nooni moved far to fast for him. When Ron paused, closing his eyes, Sensei smiled as he felt just a trace of the mystic power rise up within him, probably even unnoticeable to Ron. His eyes snapped open quickly, his chopsticks moving quickly, and just barely snagged one as Nooni moved. He could see the look of surprise on the young man's face.
Nooni bowed, smiling slightly. "Maybe nacos make Stoppable-san swift after all."
Sensei drifted away, returning to his room. It was beginning, and he feared, but seeing the young man, knowing what he might accomplish, he felt hope.
Bonnie smiled as she stepped out of the car, waiting as Hirotaka grabbed his bag from the back. He had had to return the bike to the rental place had she had volunteered to drop him off at the airport. When he had his stuff, he turned to her. "I will miss you Bonnie. It is good to have a friend, even if we must part this quickly."
Bonnie wrapped her arms around him, hugging him close. "I'm glad too. Now, you make sure you stay in touch, or even those kung fu moves you've got won't save you."
Hirotaka laughed, as they pulled away. They headed for the entrance to the terminal when Bonnie spotted Kim and Monique waiting by the entrance. "Oh, what are they doing here?"
"Perhaps they are here to greet their friend as he returns?"
Bonnie shook her head. "No, they're here to see you off. Probably still arguing over you."
The faintest trace of a smile crossed over Hirotaka's features. "Perhaps we can, as you say, kill two birds with one stone."
Bonnie looked over at him confused. "What do you mean?"
"A way that would force them to recognize the error of their ways, and to help patch up their friendship. A kiss."
Bonnie frowned. "How would kissing them help stop their fighting? That's just going to make matters worse."
Hirotaka's grin widened. "I meant that you should kiss me, when I give the signal. It would help alleviate their grievance, if they see that I am not choosing between them."
Now Bonnie was smiling. "As much as I don't really care for helping Kim right now, that's something I wouldn't mind."
Bonnie watched as he strolled over to them, placing his bag down behind the pillar. Slowly, she made her way over, watching them talk, and when he gave the signal, she ran over to him, jumping into his arms, her lips meeting hers. It was innocent to them, but to anyone watching, it looked impassioned. When she broke apart from him, she gave him a little wave. "Bye Hiro darling."
With a toss of her hair, she walked away, glancing over to share one last smile with Hirotaka as he was heading through the doors. In her hands she gripped the email he had given her, and she knew, though she might not see him again, that at least she had another friend out there.
Author's Notes – Well, this will be the last look back like this. I have way to many stories on tap right now to even try another episode prequel again. Though, there is always a chance in the future.


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RevoStock Ramadan Ident 934519
After Effects Project \ Commercials \ Intros \ Religious \ Holidays \ Opening Sequences \ Credits
After Effects Project - Ramadan Ident 934519 by wongjowolondo @ RevoStock
After Effects Project \ Commercials \ Intros \ Religious \ Holidays \ Opening Sequences \ Credits
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A short and simple project, perfect for TV ident, greeting, opener etc. Included: 3 text, 1 logo. No plugins required! CS5.5 and CS6 versions included. Flares are pre-rendered.
This product is an Adobe After Effects project file. It requires Adobe After Effects version CS5.5 or higher as well as any plug-ins listed fellow: Required After Effects Plugins: No Plug-Ins required.
Keywords: ramadan, islam, moslem, idul fitri, eid mubarak, ramazan, ramadan kareem.


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Eckartsau Vacation Packages
Vienna, the national capital, is located 20 miles (32 kilometers) to the west of Eckartsau, while Graz is 100 miles (161 kilometers) to the southwest.
See firsthand why Lower Austria is so universally-loved by tourists, by hitting the road and exploring the wonderful man-made and natural attractions surrounding Eckartsau. Two examples of places in the vicinity of Eckartsau are Donnerskirchen, 19 miles (31 kilometers) to the north, and Leopoldsdorf, 19 miles (31 kilometers) to the east.
While you are in Eckartsau, experience Neusiedl am See. Approximately 14 miles (23 kilometers) away, this regional city is where you'll find Frauenkirchen Basilika, Podersdorf Beach and Neusiedler See National Park. Schloss Halbturn and Pannonisches Heimatmuseum are also good spots for a visit before returning to Eckartsau.
On sunny days, why not take the opportunity to investigate Lower Austria's gardens and open-air museums?. This region is well known for its many old towns; these are often evocative testaments to the area's colorful past.
Hop into your snow gear and go snow shoeing or cross-country skiing. These winter sports are popular with locals and tourists alike. Have a break from traveling and spend a relaxing afternoon boating or swimming.
You'll be reluctant to bid "Tschüss" (farewell) to this unique area.


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Recent studies have demonstrated that augmentation of lymphangiogenesis and tissue engineering hold promise as a treatment for lymphedema. The purpose of this study was to determine whether adipose-derived stem cells (ASCs) can be used in lymphatic tissue-engineering by altering the balance between pro- and anti-lymphangiogenic cytokines.
ASCs were harvested and cultured in media with or without recombinant VEGF-C for 48 h. ASCs were then implanted in mice using Matrigel plugs. Additional groups of animals were implanted with ASCs transfected with a dominant-negative TGF-β1 receptor-II adenovirus with or without VEGF-C stimulation, since TGF-β1 has been shown to have potent antilymphangiogenic effects. Lymphangiogenesis, lymphatic differentiation and cellular proliferation were assessed.
Stimulation of ASCs with VEGF-C in vitro significantly increased expression of VEGF-A, VEGF-C and Prox-1. ASCs stimulated with VEGF-C prior to implantation induced a significant (threefold increase) lymphangiogenic response as compared with control groups (unstimulated ASCs or empty Matrigel plugs; p < 0.01). This effect was significantly potentiated when TGF-β1 signaling was inhibited using the dominant-negative TGF-β1 receptor-II virus (4.5-fold increase; p < 0.01). Stimulation of ASCs with VEGF-C resulted in a marked increase in the number of donor ASCs (twofold; p < 0.01) and increased the number of proliferating cells (sevenfold; p < 0.01) surrounding the Matrigel. ASCs stimulated with VEGF-C expressed podoplanin, a lymphangiogenic cell marker, whereas unstimulated cells did not.
Short-term stimulation of ASCs with VEGF-C results in increased expression of VEGF-A, VEGF-C and Prox-1 in vitro and is associated with a marked increase lymphangiogenic response after in vivo implantation. This lymphangiogenic response is significantly potentiated by blocking TGF-β1 function. Furthermore, stimulation of ASCs with VEGF-C markedly increases cellular proliferation and cellular survival after in vivo implantation and stimulated cells express podoplanin, a lymphangiogenic cell marker.
Secondary lymphedema is a dreaded complication of cancer treatment resulting from injury to the lymphatic system [1]. Patients who suffer from lymphedema have severe, life-long and often progressive arm or leg swelling resulting in frequent infections, secondary malignancies, functional issues, such as heaviness, stiffness and pain, and decreased quality of life [2]. Unfortunately, lymphedema is very common and affects a large number of cancer survivors. It may seem surprising, for example, that lymphedema is more common than chemotherapy-induced cardiomyopathy or secondary tumors resulting from chemotherapy or radiation [1,3,4]. In fact, it is estimated that 20-40% of breast cancer patients who undergo axillary lymph node dissection go on to develop lymphedema [1,5]. Lymphedema is also a common complication of other solid tumors, with one recent meta-analysis reporting an overall rate of 16% in a variety of cancers [6].
Current treatment for lymphedema is palliative and designed to mechanically drain interstitial fluid from the affected extremity, and then prevent its re-accumulation by wrapping the limb in tight compressive garments. Recent experimental animal studies, however, have shown promise in curing lymphedema by transferring lymph nodes or lymphangiogenic cytokines to the area of injury [7-9]. Clinical application of these studies has been hampered, however, by the fact that harvesting even a few lymph nodes from another area can potentially cause lymphedema in the donor extremity [10]. In addition, delivery of lymphangiogenic cytokines alone has had limited success in some circumstances [11,12].
Our group and others have recently shown that lymphangiogenesis is a complex process and it is dependent not only on molecules that promote lymphangiogenesis, but also cytokines that prevent lymphangiogenesis [13-15]. This balance of pro- and anti-lymphangiogenic forces is critical and may be responsible for circumstances in which delivery of lymphangiogenic cytokines, such as VEGF-C, fail to induce lymphatic repair [11,12]. For example, we have shown that lymphatic fluid stasis during wound repair induces the expression of TGF-β1, an anti-lymphangiogenic growth factor that directly decreases lymphatic endothelial cell (LEC) proliferation, tubule formation and migration [13,15]. Increased TGF-β1 expression in this setting prevents lymphatic repair and results in markedly diminished lymphatic function despite high levels of VEGF-C expression. As a result, inhibition of TGF-β1 promotes lymphatic repair resulting in improved lymphatic function and restoration of homeostasis [14]. Collectively, these findings suggest that the regulation of lymphangiogenesis, similar to angiogenesis, is a complex and well-orchestrated balance between pro- and anti-lymphangiogenic mechanisms.
Tissue engineering may hold promise in the treatment of lymphedema, since this approach may obviate the need for harvesting lymphatic tissues from another region of the body, thereby removing the potential for lymphedema at the donor site. In this way, lymphatic vessels can be engineered in vitro or in vivo and used to bypass damaged lymphatic channels. In support of this concept, we have recently shown that acellular matrices can support lymphatic regeneration and that transferred lymphatic structures can be used to bypass damaged lymphatics [16,17]. Delivery of mesenchymal stem cells (MSCs) in these settings may be even more effective since recent studies have shown that these cells can improve angiogenesis and wound repair [18]. Adipose-derived stem cells (ASCs) hold particular promise since these cells are readily available with minimal donor site morbidity and share significant phenotypic similarity with other MSCs by retaining the capacity to differentiate into a number of adult cell types [18-20].
The purpose of the current study was to determine if ASCs can be used in lymphatic tissue engineering by altering the balance between pro- and anti-lymphangiogenic cytokines. We demonstrate that short-term stimulation of ASCs with VEGF-C or inhibition of TGF-β1 potently induces lymphangiogenesis. Concomitant stimulation with VEGF-C and blockade of TGF-β1 was even more effective. Furthermore, we show that exposure of ASCs to VEGF-C induces the expression of podoplanin, a lymphatic cell marker in transferred cells and significantly increases cellular proliferation of transferred cells.
Three-week-old green fluorescent protein (GFP) transgenic mice (C57BL/6-g[CAG-EGFP]1Osb/J; Jackson Laboratories, Bar Harbor, ME, USA) were euthanized via carbon dioxide asphyxiation. ASCs were isolated and cultured using a modification of our previously published methods [21]. Briefly, inguinal fat pads were excised and finely minced. Tissues were digested with 0.1% type II collagenase (Sigma, St Louis, MO, USA) dissolved in RPMI with 10 mM Hepes incubated in a shaking water bath for 45 min at 37 °C. The collagenase was then inactivated by adding an equal volume of media with 10% fetal calf serum. The digested fat was centrifuged at 350 g for 10 min. The supernatant was discarded and the pellet resuspended and filtered through a 100-μm cell strainer to remove undigested tissue fragments. Cells were resuspended and cultured in Mesencult media (Stemcell technologies, Vancouver, BC, Canada) supplemented with penicillin and streptomycin, in a humidified 5% CO2 incubator at 37°C. Nonadherent cells were discarded after 48 h and media was changed every 2-3 days. Only early passage cells (<5 passages) were used. All animal experiments were approved by the Institutional Animal Care and Use Committee at the Memorial Sloan-Kettering Cancer Center.
ASCs were characterized for expression of known MSC markers (Sca1, CD29, CD73 and CD105) and absence of hematopoietic cell markers (CD34, CD45 and CD31) using our previously described flow cytometry techniques [21,22]. Briefly, cells were trypsinized, washed and 1 × 106 cells were incubated with fluorescent-tagged primary antibodies (from eBioscience; catalogue numbers as follows: Sca1 17-5981–81, CD29 12-0291-81, CD73 12-0731-81, CD105 12-1051-81, CD34 12-0341-81, CD45 15-045-81 [San Diego, CA, USA]; and CD31 from Biolegend [San Diego, CA, USA]) for 30 min at 4°C. Cells were then washed and assayed using the FACSCalibur flow-cytometer (BD Biosciences, San Jose, CA, USA) and data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).
In order to analyze the pluripotential differentiation capacity of harvested ASCs, we induced bone, fat and cartilage differentiation using our previously published techniques [22,23]. Briefly, bone differentiation was induced by exposing cells to MesenCult® stem cell media supplemented with 5 mM β-glycerophosphate, 10−8 M dexamethasone and 0.28 mM ascorbic acid (all from Sigma-Aldrich, St Louis, MO, USA) for 14 days. Von Kossa staining was then performed as previously described to stain mineralized bone nodules [22,23]. For fat differentiation, cells were initially cultured in MesenCult stem cell media supplemented with 10−6 M dexamethasone (Sigma), 1 mM insulin (Sigma) and 0.25 mM 3-isobutyl-1-methyl-xanthine for 4 days and then maintained in MesenCult supplemented with just insulin (1 mM) for 10 days. Cells were then stained with 0.18% Oil Red O (Sigma) to stain fat droplets. Finally, to promote chondrogenic differentiation, cells were cultured in serum-free μ-Minimal Essential Medium (Gibco BRL, Gaithersburg, MD, USA) with 10 ng/ml TGF-β3 and 0.2 mM ascorbic acid (Sigma) for 14 days. Cartilage cells were stained with Alcian blue (Sigma) dissolved in glacial acetic acid for 15 min.
In order to block TGF-β signaling, we transfected isolated ASCs with an adenovirus-expressing a soluble, defective type 2 TGF-β receptor (DN-RII) lacking the intracellular signaling domain of the endogenous receptor as described in detail in our previous publications [14,24]. Briefly, the transgene in this construct is expressed under the control of the constitutively active cytomegalovirus, and overexpression and secretion of the defective receptor by transfected cells competes with the endogenous TGF-β receptor, thereby acting in a dominant-negative manner to suppress TGF-β function. We have previously shown that secretion of the transgene by transfected cells results in regional blockade of TGF-β activity, and improves lymphangiogenesis by increasing LEC migration, proliferation and tubule formation [14]. An adenovirus with the identical genetic background expressing the bacterial B-Galactosidase gene (B-Gal) was used as a control for adenoviral transfection.
Adenoviruses were grown in 293 cells, purified and quantified using our previously published methods [25]. ASCs (1×106) were transfected with DN-RII or B-Gal viruses at a multiplicity of infection of 100 plaque forming units per cell. Cells were harvested 48 h later for evaluation and in vivo experimentation as outlined below.
We used Matrigel as a carrier to determine whether ASCs can be used in lymphatic tissue engineering and whether changing the balance of pro- and anti-lymphangiogenic cytokines affects this process. The use of Matrigel in this context was based on the fact that this material has been previously used successfully for lymphangiogenesis assays and also because Matrigel contains basement membrane proteins, thereby preventing fibrous tissue ingrowth and re-absorption of ASCs [15,26]. We divided the animals (n = 6-8 per group) into five groups and used adult female (8-12 weeks old) C57B6 mice (Jackson Labs). These mice have the same genetic background as the mice from which we harvested ASCs but do not express GFP, thereby enabling us to track implanted cells. Groups 1 and 2 served as controls and were subdermally implanted with 300 μl of growth factor-depleted Matrigel (BD Biosciences) containing no cells or unstimulated ASCs (5 × 105 cells). Group 3 animals also served as controls and were implanted with ASCs transfected with the B-Gal virus. Animals in groups 4-6 were experimental and were implanted with Matrigel plugs containing ASCs transfected with the DN-RII virus, ASCs cultured for 48 h prior to implantation in media supplemented with recombinant human VEGF-C (100 ng/ml; R&D Microsystems, Minneapolis, MN, USA), or ASCs transfected with DN-RII virus and 1 day following transfection cultured for 48 h prior to implantation in media supplemented with recombinant VEGF-C. Each group had six to eight animals and each animal underwent placement of two Matrigel plugs (one on either side of the upper, dorsal back). We specifically chose not to add VEGF-C to the Matrigel plugs as this intervention is known to promote lymphangiogenesis independent of ASCs [27].
Matrigel plugs were harvested 2 weeks after implantation, fixed in 4% paraformaldehyde at room temperature overnight, embedded in paraffin and sectioned (5 μm). To ensure identical specimen harvest and fixation, plugs were harvested using a 5-mm punch to include the overlying skin, thus enabling us to use capillary lymphatics in the dermis as our positive control for immunohistochemistry. LECs were localized using antibodies directed against lymphatic-specific markers, podoplanin (syrian hamster monoclonal; Ab11936; Abcam, Cambridge, MA, USA) and LYVE-1 (AF413; mouse monoclonal; R&D Systems). To localize blood vessels and microvascular endothelial cells, sections were stained with antibodies against von Willebrand Factor ([vWF] rabbit polyclonal; Ab6994; Abcam). GFP-positive cells were identified with mouse monoclonal anti-GFP antibodies (Abcam). We chose to use immunofluorescence staining of GFP rather than endogenous florescence based on our preliminary studies demonstrating insufficient tissue section quality with frozen sections. Finally, proliferating cells were identified using mouse monoclonal antibodies against PCNA (Ab2426; Abcam, Cambridge, MA, USA).
Immunofluorescent secondary antibodies used were Alexa Fluor 594 and 647 (Molecular Probes®, Invitrogen, Carlsbad, CA, USA) and counterstaining was performed using Prolong mounting medium with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen). For immunohistochemical staining, secondary antibody from the Vectastain® ABC Kit (Vector Laboratories, Burlingame, CA, USA) was used and developed using diaminobenzidine with counterstaining performed using hematoxylin (Dako). Negative-control sections were incubated with secondary antibody only. Images were obtained using bright-field microscopy (Leica TCS, Buffalo Grove, IL, USA) for immunohistochemistry and confocal microscopy (Leica) for immunofluorescence. Cell and vessel counts were performed in three to five high power fields per section (n = 8-10 per group) by two blinded reviewers.
ASCs were cultured for 2 days in recombinant human VEGF-C (100 ng/ml; R&D Microsystems) or media alone followed by harvest and isolation of RNA using the QIAGEN Allprep RNA/DNA/Protein Isolation kit (Valencia, CA, USA) according to the manufacturer's instructions. Reverse transcription was performed using TaqMan® Reverse Transcription reagents (Applied Biosystems, Foster City, CA, USA) followed by semiquantitative reverse transcriptase-PCR (RT-PCR) using TaqMan® Universal Mastermix (Applied Biosystems) and LightCycler® thermocycler (Roche Diagnostics, Indianapolis, IN, USA). Expression of Prox-1 (primer Mm00435969_ m1; Applied Biosystems), LYVE-1 (primer Mm00475056; Applied Biosystems), VEGFR3 (primer Mm0043337_m1; Applied Biosystems), VEGF-A (primer Mm00437308m1; Applied Biosystems) and VEGF-C (Mm01202432_m1; Applied Biosystems) was normalized to 18S (primer Mm03928990_g1; Applied Biosystems) measured concurrently. Experiments were performed in triplicate.
Statistical analysis was performed using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA, USA). Multigroup comparison was analyzed by one-way ANOVA with post-hoc Tukey-Kramer test. Mean values and standard deviations presented with p < 0.05 are considered significant unless otherwise noted.
Adipose-derived stem cells harvested from GFP transgenic animals strongly expressed GFP both in situ and in culture (Figure 1A & B). In addition, these cells maintained the potential to differentiate along the bone, fat and cartilage paths when exposed to appropriate environmental stimuli (Figure 1C-e). Flow cytometry analysis demonstrated that, although ASCs were comprised of a mixed cell population, the majority of cells expressed typical MSC markers (Sca1 [56% positive], CD29 [98.4% positive], CD105 [32.4% positive], CD73 [11.7% positive]) and lacked cell surface expression of hematopoietic stem cell markers (CD45, CD31, CD34; Figure 1F-L ). The LEC marker VEGFR3 was positive in 3.1% of ASCs (Figure 1M). As we have previously shown, transfection of ASCs in vitrowith the DN-RII or B-Gal virus was highly efficient (>75% of cells) and resulted in high-level transgene expression (not shown) [25,28].
We sought to determine whether unstimulated ASCs or ASCs stimulated briefly in culture with VEGF-C can modulate lymphangiogenesis in vivo. In addition, we aimed to determine whether the balance of pro- and anti-lymphangiogenic stimuli can alter this response. First, we characterized phenotypic responses of ASCs after 48 h of culture with VEGF-C in vitro prior to implantation within Matrigel. PCR analysis revealed significant upregulation of Prox-1 (1.2-fold; p < 0.05), VEGF-C (1.4-fold; p < 0.001) and VEGF-A (1.13-fold; p < 0.01, Figure 2A) expression but no significant changes in LYVE-1 or VEGFR3 expression, suggesting that VEGF-C exposure introduced early but incomplete differentiation toward the LEC lineage. In support of this finding, flow cytometry analysis of cells before and after VEGF-C stimulation similarly revealed no changes in VEGFR3 expression (Figure 2B) but demonstrated a reduction in the expression of stem cell markers initially highly expressed by ASCs (46% reduction in Sca1 expression; 12.8% reduction in CD29; Figure 2B).
Localization of the lymphatic specific marker podoplanin demonstrated specific staining for capillary lymphatics in the dermis (Figure 3A). Cell counts of podoplanin-positive cells surrounding the Matrigel plug demonstrated no statistical differences between acellular Matrigel plugs, Matrigel plugs containing ASCs alone or Matrigel plugs containing ASCs transfected with B-Gal adenovirus (Figure 3B-e). By contrast, constructs containing ASCs transfected with DN-RII virus demonstrated a 4.5-fold increase in the number of podoplanin-positive cells/high-powered field (HPF) surrounding the Matrigel plug (p < 0.001; Figure 3B & F). Culturing ASCs in media containing VEGF-C for 48 h prior to implantation also significantly increased the number of podoplanin positive cells/HPF (p < 0.001; Figure 3B & g). Interestingly, ASCs transfected with the DN-RII virus and subsequently cultured for 48 h with VEGF-C prior to implantation resulted in an even more potent lymphangiogenic response than either the DN-RII virus alone or pretreatment with VEGF-C alone (p < 0.001; Figure 3B & H). Augmentation of lymphangiogenesis in the region surrounding the Matrigel plug was secondary to expansion of host cells rather than direct differentiation of donor ASCs since we did not observe GFP-positive cells in the regenerated lymphatic tissues (not shown).
Examination of LYVE-1, another lymphatic specific marker, demonstrated similar patterns as those noted for podoplanin (Figure 4) . Once again, we noted specific staining of capillary lymphatics in the dermis (Figure 4A). In addition, similar to podoplanin staining, we noted no differences in the number of LYVE-1-positive cells/HPF when comparing Matrigel without cells, ASCs alone or ASCs transfected with the B-Gal adenovirus (Figure 4B-e). Analysis of constructs containing ASCs transfected with DN-RII or after pre-treatment with VEGF-C prior to implantation demonstrated a significant increase in the number of LYVE-1-positive cells/HPF (1.8-2-fold, respectively; p < 0.001; Figure 4B & F-H). Further, similar to our findings with podoplanin staining, we noted a significant increase in the number of LYVE-1-positive cells/HPF when concomitant blockade of TGF-β1 and pretreatment with VEGF-C was performed (p < 0.001 compared with DN-RII or VEGF-C alone). Taken together, these findings suggest that even a short-term exposure of ASCs to lymphangiogenic stimuli (VEGF-C) can induce lymphangiogenesis in the host tissues after transplantation. In addition, the balance of pro- and anti-lymphangiogenic cytokines is important since inhibition of antilymphangiogenic mechanisms in combination with short-term stimulation with prolymphangiogenic cytokines is even more effective than either therapy alone. Finally, our findings suggest that ASCs delivered without either of these stimuli do not inherently promote lymphangiogenesis.
Mesenchymal cells and ASCs have been shown to differentiate into endothelial cells with appropriate environmental stimuli [19,29]. In addition, MSCs have been shown to express lymphatic markers after stimulation in vitro [30]. Therefore, in order to determine whether ASCs can express LEC markers in vivo, we analyzed ASCs within the Matrigel construct for expression of podoplanin and LYVE-1. We identified donor ASCs and their potential for expression of LEC markers by co-localizing GFP and podoplanin or LYVE-1. We found very few podoplanin-positive cells in unstimulated ASCs (received no VEGF-C prior to implantation [Figure 5A, F & g]). Transfection of ASCs with DN-RII resulted in a 92% increase in the number of GFP-positive/ podoplanin-positive cells per HPF; however, this change was not statistically significant (Figure 5A & H). By contrast, stimulation of ASCs with VEGF-C prior to implantation in Matrigel resulted in a significant increase in the number of double-positive cells (97% increase as compared with ASCs alone; p < 0.001; Figure 5A-D, i & J). Blockade of TGF-β in conjunction with pre-implantation exposure to VEGF-C further increased the number of GFP-positive/podo-/podoplanin-positive cells; however, this difference was not statistically significant when compared with VEGF-C stimulation alone.
We found that some of the podoplanin-positive cells in the groups transfected with DN-RII or stimulated with VEGF-C had assumed a tubular, vessel-like structure in the Matrigel (Figure 6A & B). The number of these tubular structures was significantly increased by DN-RII treatment but even more so when cells were stimulated with VEGF-C prior to implantation (p < 0.001). By contrast to our podoplanin-positive cell counts, we found that combination of VEGF-C stimulation and DN-RII transfection was additive with significantly more podoplanin-positive vessels as compared with VEGF-C stimulation alone (p < 0.01). Interestingly, we found that ASCs did not express LYVE-1, another lymphatic cell marker, even when cells were stimulated with VEGF-C, in any of the experimental groups evaluated (not shown).
Analysis of GFP staining in the Matrigel plug demonstrated that constructs containing ASCs stimulated prior to implantation with VEGF-C had significantly more donor ASCs (GFP-positive cells) as compared with other groups (Figure 7A-F; p < 0.001). In fact, Matrigels implanted with VEGF-C-treated ASCs contained twofold more GFP-positive cells as compared with ASCs transferred without VEGF-C treatment (p < 0.001). TGF-β1 blockade also modestly increased the number of GFP-positive cells; however, this increase was not statistically significant (Figure 7A & D). Similarly, concomitant TGF-β1 blockade and preimplantation exposure to VEGF-C did not result in increased numbers of GFP+ cells as compared with VEGF-C stimulation alone. As expected, we found no GFP-positive cells in acellular Matrigel constructs (not shown).
In order to determine how VEGF-C increased ASC numbers, we co-localized GFP with PCNA; Figure 8A-D. We have previously shown that this technique can be used to identify proliferating cells.[15] This analysis demonstrated a sevenfold increase in the number of proliferating ASCs when cells were stimulated with VEGF-C prior to implantation (GFP-positive/PCNA-positive; Figure 8A; p < 0.001). We also found a modest increase (twofold) in proliferating ASCs after TGF-β1 blockade (p < 0.01). However, similar to our GFP counts, we found no significant increase in the number of proliferating ASCs when TGF-β1 blockade was combined with VEGF-C stimulation. Taken together, these findings suggest that stimulation of ASCs with VEGF-C or blockade of TGF-β1 increase ASC proliferation in vivo, however, these effects, unlike the changes in lymphangiogenesis, are not additive.
In order to determine whether blockade of TGF-β1 in conjunction with VEGF-C stimulation also has a synergistic effect on angiogenesis, we evaluated the expression of vWF in the regions surrounding the Matrigel plugs in our various groups (Figure 9). Similar to our findings with lymphangiogenesis, we found very few vWF-positive cells or blood vessels in animals treated with acellular Matrigel, Matrigel-containing unstimulated ASCs, or Matrigel plugs with B-Gal transfected ASCs (Figure 9A-D). By contrast, we found a significant (fivefold) increase in the number of vWF-positive cells in the regions surrounding Matrigel plugs containing ASCs stimulated with VEGF-C prior to implantation (p < 0.001; Figure 9A & F). In contrast to our lymphangiogenesis findings, however, we saw no significant effect of TGF-β1 blockade on vWF expression either when this treatment was performed in isolation or in combination with VEGF-C stimulation (Figure 9e & g). These findings suggest that TGF-β1-induced inhibition of lymphangiogenesis is independent of effects on microvascular endothelial cells.
In this study we found that short-term (48 h) stimulation of ASCs with VEGF-C in culture can induce a potent lymphangiogenic response when the cells are subsequently implanted in vivo. Our findings are supported by and add to those of Hwang et al., who recently demonstrated that delivery of ASCs in hydrogels containing VEGF-C significantly increases lymphangiogenesis when implanted in a mouse hindlimb model [31]. One notable difference between our study and those reported by Hwang et al. is that we stimulated ASCs with VEGF-C prior to implantation in vivo (i.e., the Matrigel plug contained no additional VEGF-C). This difference is important and suggests that VEGF-C stimulated ASCs have an altered phenotype and can independently promote lymphangiogenesis. This hypothesis is supported by our data demonstrating that VEGF-C stimulation for 48 h leads to significant upregulation of Prox-1, VEGF-C and VEGF-A mRNA expression by ASCs. Furthermore, in response to VEGF-C stimulation, ASC stem cell marker expression, including Sca1 and CD29, was reduced, suggesting these cells begin to differentiate in response to VEGF-C. This finding is further supported by a recent study by Conrad and colleagues demonstrating that stimulation of peripheral blood MSCs with VEGF-C induces the expression of LEC markers [30]. Stimulation of stem cells with VEGF-C prior to implantation has important clinical implications since this approach avoids delivery of large amounts of VEGF-C, thereby potentially avoiding regeneration of hyperplastic but dysfunctional lymphatics as has been previously reported [11,12]. In addition, delivery of high concentrations of VEGF-C in cancer patients (the population at highest risk for developing secondary lymphedema in developed countries) may have the unwanted effect of stimulating cancer recurrence or metastasis, as this cytokine is a central regulator of tumor growth and spread in a wide variety of solid tumors [32-34].
An additional finding in our study that adds to the existing literature on stem cells and lymphangiogenesis is the observation that blockade of TGF-β1, an antilymphangiogenic cytokine, can also induce lymphangiogenesis and that concomitant blockade of TGF-β1 significantly potentiates the effects of VEGF-C. This finding is important because it may provide an alternative approach to circumstances in which augmentation of VEGF-C expression alone is insufficient to promote lymphatic regeneration. For example, using the mouse-tail model Rutkowski et al. have shown that lymphatic stasis increases VEGF-C expression, but that this response actually exacerbates edema due to formation of poorly functional, hyperplastic lymphatic vessels [12]. Similarly, Goldman et al. reported that overexpression of VEGF-C in this model failed to increase LEC migration or function despite regeneration of hyperplastic lymphatics [11]. Using a similar model, our group has shown that blockade of TGF-β1 markedly accelerates lymphatic regeneration in this scenario, and that this molecule has direct antilymphangiogenic effects in wound repair [14,15]. Thus, the balance between pro- and antilymphangiogenic factors may be a critical regulator of lymphatic regeneration and efforts directed at optimizing these forces are likely to be an important step in clinical application.
In the current study, we found that unstimulated ASCs did not induce lymphangiogenesis when implanted in vivo. This finding contrasts with to previous studies by Hwang et al. and Conrad et al. [30,31]. For example, Hwang found that ASCs alone, delivered via a hydrogel, led to an increased number of lymphatic vessels in a hindlimb lymphedema model in which the lymphatic vessels were ligated after a circumferential incision of the thigh. Similarly, Conrad et al. showed that intradermal injections of MSCs in a mouse-tail skin excision model were associated with improved lymphangiogenesis and lymphatic function in this model. One possible explanation for the differences noted between these reports and our current study is that the former experiments were confounded by the presence of a wound. Therefore, delivery of ASCs to wounds may have simply led to enhanced overall wound healing secondary to delivery of ASCs or MSCs, subsequently leading to enhanced lymphatic regeneration rather than direct stimulation of lymphangiogenesis by ASCs. This is supported by previous studies demonstrating improved wound repair after local or systemic delivery of ASCs [35-37]. It is also possible that differences in delivery (i.e., hydrogel or direct intradermal injection) had an effect on lymphangiogenesis. These concepts are interesting and require additional investigation.
We found that exposure of ASCs to VEGF-C resulted in expression of podoplanin in vivo. This is supported by the findings of Conrad et al. who showed that in vitro exposure of peripheral blood-derived MSCs to LEC conditioned media or VEGF-C resulted in upregulation of podoplanin expression [30]. In addition, we found that the number of podoplanin-positive vessels in Matrigel constructs containing ASCs stimulated with VEGF-C was potentiated by inhibition of TGF-β1, suggesting the combination of VEGF-C and blockade of antilymphangiogenic signals is more effective in inducing lymphatic differentiation. This is supported by the findings of Oka et al. and our group demonstrating that inhibition of TGF-β increases expression of LEC markers, including LYVE-1 and Prox-1, by isolated human dermal LECs [15,38].
Interestingly, we found that stimulation of ASCs with VEGF-C or inhibition of TGF-β1 did not result in expression of LYVE-1. This finding is similar to those of Lee et al., who found that stimulation of bone marrow derived mononuclear cells with VEGF-C in vitro results in only temporary expression of Prox-1 and LYVE-1; expression is present for the first 4 days in culture but is completely lost on culture day 7 or longer time periods [39]. By contrast, podoplanin expression is maintained even 10 days after initiation of VEGF-C stimulation. Similarly, Conrad et al. reported lymphatic differentiation of MSCs in response to in vitro stimulation with VEGF-C resulting in podoplanin expression; however, expression of Prox-1 or LYVE-1 was not analyzed [30]. Thus, it is possible that lymphatic differentiation of MSCs in response to VEGF-C is limited and requires additional stimuli to acquire the complete phenotype of mature LECs; however, this hypothesis warrants additional investigation.
Only one recent study has suggested that MSCs could differentiate into LECs, incorporating into newly formed lymphatics and expressing LYVE-1 after in vivo transfer into a wound [31]. However, in this study the authors used co-localization without z-stacking to evaluate coexpression of MSC markers and LYVE-1 making it difficult to conclude definitively that in vivo co-localization was due to expression in a single cell with a single nucleus rather than simply adjacent cells. In fact, it is possible that ASCs may contribute to supporting structures around collecting lymphatics since these vessels are known to have pericytes [9]. This hypothesis is supported by the findings of Sasaki et al., who showed that MSCs contribute to wound healing by differentiating into a number of skin cell types including endothelial cells and pericytes [37]. Similarly, Zannettino et al. have shown that ASCs can also differentiate into pericytes [40].
In the current study, we found that VEGF-C pre-treatment significantly increased ASC proliferation after implantation in vivo. In fact, exposure of ASCs to VEGF-C doubled the number of donor cells present in the Matrigel plug after 2 weeks and was associated with a sevenfold increase in the number of proliferating ASCs in vivo. These observations are supported by and add to those of Hwang et al. who demonstrated that stimulation of ASCs with VEGF-C increased cellular proliferation in vitro [31]. It is also possible that VEGF-C increased ASC numbers in our study by other mechanisms distinct from increased cellular proliferation alone. For example, VEGF-C treatment may inhibit programmed cell death or senescence as supported by the findings of Wang and colleagues on the effects of VEGF-C on LECs [41]. In addition, this hypothesis is supported by the findings of Pons and colleagues, who demonstrated that VEGF-A increases survival of bone marrow-derived MSCs in vitro and in vivo by inhibiting cellular senescence and decreasing expression of proapoptotic genes [42].
Finally, we found that VEGF-C pretreatment of ASCs significantly increased angiogenesis surrounding Matrigel plugs after in vivo implantation. This finding suggests that stimulated ASCs produce cytokines that promote both angiogenesis and lymphangiogenesis. Interestingly, we found that concomitant inhibition of TGF-β1, by contrast to lymphangiogenesis, had no effect on angiogenesis, suggesting that the antilymphangio genic effects of TGF-β1 are specific rather than global changes in cytokine expression patterns.
In conclusion, we have shown that short-term stimulation of ASCs with VEGF-C results in a marked increase lymphangiogenic response after in vivo implantation and that blocking TGF-β1 function potentiates this response. Furthermore, exposure of ASCs to VEGF-C markedly increases cellular proliferation and cellular survival after in vivo implantation and stimulated cells express the lymphangiogenic cell marker podoplanin.
Transfer of ASCs is a technique that holds promise for the reconstruction and regeneration of tissues, given the differentiation potential of these cell populations. Furthermore, the availability of the cells and ease in attaining them with minimal morbidity to the donor makes ASCs an attractive candidate for tissue reconstruction. This study, and others, have recently suggested that these cells have the potential to differentiate in response to lymphangiogenic signals, including VEGF-C, and promote lymphangiogenesis through direct or indirect mechanisms. Given these findings, the therapeutic potential of ASCs in the treatment and/or prevention of lymphedema is strong; however, future investigation is required to characterize the role of these cells in promoting lymphatic repair.
Adipose-derived stem cells are responsive to VEGF-C stimulation & can promote lymphangiogenesis
Modulation of TGF-β1 function can alter lymphangiogenic response by stimulated ASCs
Financial & competing interests disclosure
This manuscript was funded in part by a Plastic Surgery Education Foundation Grants to Tomer Avraham and BJ Mehrara. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Ethical conduct
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.
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PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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At a local antique and collectibles event at the weekend I bought two review programmes from the Folies Bergere in Paris in the 1930s and I am utterly enchanted by the deco glory of it all and can easily see myself being seduced into collecting more of them. However, I was surprised, among all the bouncing breasts and beautiful bling to see more than a shadow of well-oiled beefcake. And is it possible... to look at the final image below, that I may have stumbled on the inspiration for the famous Jabba the Hut and Princess Leia scene in Star Wars?
Tuesday, July 31, 2012
The Men of the Folies Bergere
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How the Elite Will Sell Global Collapse To The Masses
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Listening to vid now
"I love the man that can smile in trouble, that can gather strength from distress, and grow brave by reflection. 'Tis the business of little minds to shrink; but he whose heart is firm, and whose conscience approves his conduct, will pursue his principles unto death."
Thomas Paine, The American Crisis, No. 1, December 19, 1776
"Those who expect to reap the blessings of freedom, must, like men, undergo the fatigues of supporting it."
Thomas Paine, The American Crisis, No. 4, September 11, 1777
"I believe that banking institutions are more dangerous to our liberties than standing armies. Already they have raised up a monied aristocracy that has set the government at defiance. The issuing power (of money) should be taken away from the banks and restored to the people to whom it properly belongs." -- Thomas Jefferson, U.S. President.
"A majority of the people of the United States have lived all of their lives under emergency rule. For 40 years, [79 years now in 2012] freedoms and governmental procedures guaranteed by the Constitution have in varying degrees been abridged by laws brought into force by statutes of national emergency."
|No Dhimmi (OP)
I am the infidel your imam warned you about
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Are you ready?
Background of my avatar: [link to en.wikipedia.org]
The religion of submission cannot survive an open and honest discussion of its convoluted and foolish scriptures, its sexually perverted terrorist prophet, or its deceitful and demented god. When Western leaders become unified and resolute in their hostility to Islam's violent and ungodly beginnings, Muslims will flee the religion because they will be horrified by its endorsement of terrorism, mass murder, slave trading, plunder, kidnapping, and rape.
If Muslims & their apologists really want to cure "Islamophobia," here is an easy way. Focus their indignation on Muslims committing violent acts in the name of Islam, not on non-Muslims reporting on those acts.
[link to www.cspipublishing.com]
[link to www.faithfreedom.org]
[link to shoebat.com]
[link to thereligionofpeace.com]
[link to www.bibleprobe.com]
[link to www.fatherzakaria.net]
[link to www.answering-islam.org]
Ecclesiastes 10:2 "The heart of the wise inclines to the right, but the heart of the fool to the left."
We are all born ignorant, but it takes a lot of work to keep looking the other way to remain stupid.
Islam is not a race - the racism rant is lame, used by the intellectually lazy to derail the debate.
Democrats do and always have loved poor people. . . they continue to make so many of them.
"Evil is." And being a useful idiot enabling it is no excuse.
Thread: You're Part of the War with Islam - Which side are you on?
Furthermore, I consider that islam must be destroyed.


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|Sebastiaan van Erk <sebs...@sebster.com>
|Re: Another Comet 6.0.10 question
|Wed, 04 Apr 2007 15:38:22 GMT
Ok, so I get a -1 on a read. Now what do I do? If I just return from the event handler I immediately get another read :-(. If I close the event I can't do output anymore. Can't it just send me ERROR/TIMEOUT events, just like what would happen if no READS arrive at all? In that case I can do async output (like in the Messaging application example). The CoyoteInputStream I called the read() on should know it reached the end of stream when I called read(), so why does it cause another READ event immediately even though it should know there is no more data? Shouldn't it behave in the same way that it would normally behave after handling a read event and there being no more data, i.e., do ERROR/TIMOUT events until the event is closed or new data arrives (which in this case can't happen)? Regards, Sebastiaan Rémy Maucherat wrote: > On 4/4/07, Sebastiaan van Erk <email@example.com> wrote: >> Hi, >> >> The pages on Comet say the following about the read event: >> >> EventType.READ: This indicates that input data is available, and that >> one read can be made without blocking. The available and ready methods >> of the InputStream or Reader may be used to determine if there is a risk >> of blocking: the servlet should read while data is reported available, >> and can make one additional read without blocking. When encountering a >> read error or an EOF, the servlet MUST report it by either returning >> false or throwing an exception such as an IOException. This will cause >> the error event to be invoked, and the connection will be closed. It is >> not allowed to attempt reading data from the request object outside of >> the execution of this method. >> >> However, I just got a READ event where available() returns 0 (because >> the next byte to read would be -1 indicating the end of the stream). >> >> Is there any way to test for the end of the stream without calling >> read() ? > > Ok, that javadoc is a little bit out of date (ex: returning false). > However, it is still valid: if you get a read event, you're supposed > to do at least one read, and it could return -1 (if it got that from > the client). If it fails to read data because the client disconnected, > you'll get an IOException. > > Rémy > > --------------------------------------------------------------------- > To start a new topic, e-mail: firstname.lastname@example.com > To unsubscribe, e-mail: email@example.com > For additional commands, e-mail: firstname.lastname@example.com > --------------------------------------------------------------------- To start a new topic, e-mail: email@example.com To unsubscribe, e-mail: firstname.lastname@example.com For additional commands, e-mail: email@example.com
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Let's make a tribute to Steve Jobs
This image above is what got me interested in making art & code with computers. I was in 7th grade in the early 90's and lucky enough to have a few Macs in our school library. I started playing with HyperCard, creating interactive stories that you could click through. Choose your own adventure style :) ... I was hooked. I spent many long afternoons after school in the library, not wanting to go home. Soon I figured out how to make web pages, went to art school, started connecting with other like-minded creatives on the Internet, started Threadless, etc.
Steve has changed so many of our lives. If your life was impacted by him, please share your story here. Or make something in tribute and post it up.