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Century 21 China Real Estate Announces Completion Of Settlement Of Shareholder Petition
BEIJING, June 8, 2012 /PRNewswire-Asia-FirstCall/ -- IFM Investments Limited (NYSE: CTC) ("Century 21 China Real Estate" or the "Company"), a leading comprehensive real estate services provider and the exclusive franchisor for the CENTURY 21® brand in China, today announced that the Company and the Company's founders have completed the settlement with GL Asia Mauritius II Cayman Ltd. ("GLA") previously announced on April 30, 2011. The parties have jointly withdrawn or terminated all outstanding proceedings against the Company, including the petition to request an order to wind up the Company and other remedies filed by GLA on December 30, 2011, in the Grand Court of the Cayman Islands.
This settlement does not change the respective shareholding in the Company of the Company's founders and GLA. As of June 8, 2012, the latest practicable date, the Company's founders are the largest beneficial owners of the Company with approximately 40.5% of the Company's shares.
Amendment to Shareholder Rights Plan
The Company today also announced that its board of directors has adopted the first amendment to the Company's shareholder rights plan dated November 17, 2010 (the "Rights Plan"). The amendment, exempting certain circumstances from triggering the Rights Plan, was adopted on June 6, 2012 pursuant to the settlement among the Company, the Company's founders and GLA.
As of the date hereof, the rights under the Rights Plan have not been issued in response to any specific effort to acquire control of the Company. About Century 21 China Real EstateIFM Investments Limited ("Century 21 China Real Estate" or "CTC") is a leading comprehensive real estate services provider and the exclusive franchisor for the CENTURY 21® brand in China. CTC primarily focuses on China's fast-growing and highly fragmented secondary real estate market, providing company-owned brokerage services, franchise services, mortgage management services, primary services, commercial services and fund management services. CTC has experienced substantial growth since it commenced operations in 2000, and received numerous awards and recognition as franchisor and real estate services provider for its service quality and business achievements. Century 21 China Real Estate became a public company in January 2010 and its ADSs, each of which represents 45 ordinary shares of CTC, currently trade on the New York Stock Exchange under the symbol "CTC." For more information about CTC, please visit http://www.century21cn.com/english. For investor and media inquiries, please contact:
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Davis steps down from RLEF role
Posted 23 May 2013 - 10:10 AM
"My time with the RLEF has been extremely rewarding and I am very pleased to know that after my departure the systems we've implemented will continue to improve the standards of match official development among our member nations," he said. "European Rugby League has progressed to a point that is unrecognisable to when I joined the RLEF in 2007. I leave knowing that a large number of member nations are now qualified to internally deliver match officials development."
Click here to view the article
League Express is Britain's biggest selling Rugby League publication
On sale every Monday, in shops, on subscription and online for PC/MAC, iPad, iPhone, Android & Kindle Fire.
Follow us on Twitter @leagueexpress
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Posted 23 May 2013 - 11:57 AM
Best of luck Stevo.. not only a RLEF Officials Manager, but has also played a big part in the development of the game at many levels throughout London.
Newham Dockers - Champions 2013. Rugby League For East London. 100% Cockney Rugby League!
Twitter: @NewhamDockersRL - Get following!
Posted 23 May 2013 - 12:08 PM
A mind is like a parachute. It doesn't work if it isn't open. Frank Zappa (1940 - 1993)
Posted 23 May 2013 - 01:24 PM
Best of luck Stevo
I have to say when I saw this I started to panic, then re-read OP and clamed down.
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subset: commoncrawl


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Kilgore, TX — Kilgore police officials say they are looking into leads and believe a group of students are responsible for the early morning vandalism.
KPD offices say those responsible for the vandalism could face felony criminal mischief charges.
If you have any information contact the Kilgore Police Department at 903-983-1559.
Kilgore ISD officials say 39 out of 40 buses in their fleet had the stem valve removed causing flat tires.
Maintenance workers discovered the vandalism shortly after arriving to the bus barn located behind the high school at 3 a.m.
KISD officials say the bus barn is not equipped with video cameras, but is surrounded by barbed wire and the gates are locked at night.
The cost of vandalism is not known at this time.
KISD is not sure if students will have to make up this day or not.


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Colombian photojournalist escapes kidnappers
EDITOR'S NOTE: On January 29, 2015, reports in the Colombian press said Johanny Vargas issued a statement saying he had not been kidnapped and that his disappearance occurred under personal circumstances. CPJ is investigating.
Bogotá, January 23, 2015--The Committee to Protect Journalists condemns the abduction of Colombian news photographer Johanny Vargas and calls on authorities to bring the perpetrators to justice. Vargas, who had documented environmental damage caused by housing construction in the southern city of Popayán, was kidnapped Tuesday but escaped from his captors Thursday morning and is in good health, according to Vargas and news reports.


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- Tickets
NCAA Ticket ExchangeTM - Now Available! The official NCAA Ticket Exchange is the only guaranteed, NCAA-approved secondary ticket marketplace that allows fans to buy and sell NCAA Wrestling Championships tickets with other fans. Ticket holders who are unable to attend a session have a place to safely sell their tickets, and the buyer can be assured that the tickets are authentic and guaranteed.


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Supplements encourage unhealthy behaviour
Have you ever had that feeling, after an energetic gym session, or perhaps a long walk, that you've earned the right to a mountainous slice of cake, or to lounge lazily in front of the telly? Psychologists call these licensing effects and a new study has documented a similar phenomenon following the simple act of taking a vitamin pill. The researchers say the finding could help explain why the explosive rise in the consumption of dietary supplements (approximately half the US population take them, according to recent data) has not led to a commensurate improvement in public health.
Wen-Bin Chiou and his colleagues gave an inert pill to 82 participants recruited via posters in the Taiwanese city of Kaohsiung. Half the participants were told it was a placebo; the other half were told it was a vitamin pill...
For the full story visit the BPS Research Digest.
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Advertising technology and integrated strategic media services organization rVue Inc., a subsidiary of rVue Holdings Inc., recently announced the launch of the company's proprietary ad verification technology which provides real-time analytics across digital out-of-home networks.
The metadata initiative was developed to bring real-time accountability to advertisers' DOOH campaigns and enable buyers to review and optimize live campaigns across networks, according to rVue. Launched on the heels of the American Association of Advertising Agencies' (4As) and the Association of National Advertisers' (ANA) recent Embedded Metadata Manifesto initiative, rVue's ad tracking technology provides an industry solution which also conforms to IAB's VAST standards for reporting digital video advertising, the company said. For digital networks, rVue said its reporting beacon alleviates the time-consuming, manual process of data collection and delays in campaign performance reporting.
DOOH has grown to a multibillion dollar industry, which includes digital screens, tablets, kiosks and billboards in public locations where audiences dwell. According to industry research by Magna Global and PQ Media, DOOH is reportedly the third-fastest growing medium in the world behind the Internet and mobile, with double-digit growth projected to continue over the next few years.
The rapid growth of the industry is attracting advertisers due to its ability to target and engage consumers with rich digital media; however, buyers are finding it difficult to create media plans due to the fragmentation caused by fast industry growth, according to rVue.
Since exiting beta, the rVue ad-verification technology has been tracking multiple digital signage campaigns with acute accuracy, including a December 2011 campaign for a Fortune 150 brand, the company said.
"Advertisers are accustomed to accountability for the digital media they purchase and digital out-of-home is no exception," said rVue President and CEO Jason Kates. "... Real-time reporting for digital signage advertising campaigns provides the type of accountability advertisers receive from other digital media. By bringing digital out-of-home advertising and campaign analytics in line with the Interactive Advertising Bureau's standards, we increase current buyer confidence as well as invite new advertisers into place-based media who are looking for a greater degree of proof-of-performance reporting and accuracy."
The company said it verification technology embeds a beacon into video files which is compatible across all platforms and is accessible to most DOOH networks. The technology is built using a scalable cloud computing architecture and is designed to deliver daily reporting on over 100 million DOOH ad playback events each day to rVue's partners.
Read more about DOOH audience measurement.
Topics: Advertising , Audience Measurement , DOOH Advertising , Networks


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Advanced Search
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Leonar3Do, a developer of virtual reality software products, unveiled a new product line--dubbed Vimensio--targeted at the education market at this week's 2013 International CES show in Las Vegas. The new product line, slated for release in early 2013, is designed to allow educators and students to create and interact with 3D objects in virtual space.
Vimensio comes with a 3D application builder--Vimensio Edit--and interactive player, called Vimensio Play. Vimensio Edit enables users to produce 3D content and apps. The company plans to release two versions of the editor—one for beginners, with limited or no coding experience, and a Pro version for advanced designers. According to Leonar3Do, the Pro version will allow these developers to "build or modify 3DVR applications by modifying key functions of Vimensio (object handling, interaction, and moving) via a Python-based script interface." Vimensio works with 3D printers.
Vimensio Play, which enables users to present and work with 3D objects, also includes an online 3D app store. The company will encourage designers to share their apps through contests and a revenue-sharing model, to be introduced in the future.
Vimensio Edit will be available for $499 USD when the product is released this quarter. Vimensio Play will be available at no charge. Users will need Leonar3Do or HelloVR technology in order to use Vimensio.
Additional information about Vimensio is available online.
About the Author
Kanoe Namahoe is online editor for 1105 Media's Education Group. She can be reached at firstname.lastname@example.com.
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The BBA's Public Service blog, Beyond the Billable is your one stop shop for everything Public Service at the BBA. Check it out here.
How We Got Here. For the full post, click here.
In an attempt to integrate and utilize the many JD/LLM students in the Boston area who have an interest in International law, we have reached out to them to write articles on international law for our forthcoming Section newsletter. This will help further the Section's goal of bridging the gap between students and lawyers, increase the Section's exposure, and help students to get published. Once the student has written a first or second draft of the article, we are looking to partner that student up with a member of our Section - this is where you come in.
We are looking for lawyers to participate in this discrete initiative. It should not be too time-consuming, and would require you to edit an article that has already been written. We are not asking for lawyers to fact-check the article; we are looking for lawyers to bring a "practical" eye to marking up a document, as some law students might not have that skill-set. The student drafts are expected by the end of October, and we are hoping to finalize with a week to ten days after. Please let Darren Braham know if you're interested in helping: email@example.com.


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Just curious.
Why brew your own beer when buying it from the store is so EASY? Just curious.
Number 1 cost. Number 2 control. Number 3 , ITS WAY MORE FUN!!
Quite honestly, all grain can be (and frequently is) as easy as partial mash. Granted, I only ever did one partial mash but since switching to all grain I've found the process to be as easy in most respects. The single biggest difference I have found in extract/partial mash and all grain is the initial investment, otherwise the process is easy for all of them. I am finding that I have a cleaner kitchen doing all grain because there's plenty of down-time in the process that I'm able to keep things organized and picked up, rather than when I did extract/partial I felt attached to the stove thus trying to fit in the remainder.
It's not about it being "easier"!! It is about enjoying the process of brewing. Brewing with extracts takes a large part of the brewing process out of your hands.
Check out my nanobrewery!http://www.facebook.com/#!/pages/Two...02323289804018
Extract beer causes shingles.


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when you read stuff like this, you gotta wonder how we consider ourselves the "beacon of light".... what an embarrassment... Munitions Dumping at Sea It is no secret that the U.S. military has used the ocean as trashcan for munitions in the past. Peter discussed at the Old DSN how federal lawmakers were pressing the US Army to reveal everything it knows about a massive international program to dump chemical weapons off homeland and foreign shores. "The Army now admits that it secretly dumped 64 million pounds of nerve and mustard agents into the sea, along with 400,000 chemical-filled bombs, land mines and rockets and more than 500 tons of radioactive waste - either tossed overboard or packed into the holds of scuttled vessels." Brian pointed me to the Daily Press's in depth coverage of this whole issue. Registration is free and only takes a minute or two and is extremely worthwhile. Included at the site are maps of disposal sites (downloadable as pdfs), stories, descriptions of items dumped including nerve and musturd gas, and rather depressing pictures some are below the fold (all from Daily Press).
Well, we're a beacon of light because of our free way of life. Furthermore, I'm not sure anyone else was much better back in 1958. The environment wasn't as big of a concern in that era, as it is now. The SS William Ralston filled with 301,000 mustard gas bombs and 1,500 1-ton canisters of Lewsite -- sinks in the Pacific off San Fransico in 1958 (Photo courtesy of the U.S. Army
I don't think "trashcan" quite covers the scope of what the Army did. That dolphin doesn't look like anything in MY trash can.
Not quite, he says it they way they say it in Upstate NY, not like that dumb ass texas hick in the WH says it.
tHE eND iS nEAR wE wILL aLL dIE mOTHER nATURE wILL sTART aLL oVER aGAIN mILLIONS oF yEARS fROM nOW tHERE wILL bE aNOTHER pRESS cOVERAGE :bricks:


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McFadden relishes top half finish
Scotland international striker James McFadden says Birmingham City's achievements in the Premier League this season should not be underestimated.Blues finished ninth under manager Alex McLeish in their first season after promotion from the Championship. "We'll enjoy the season we've had - we've put a lot of hard work in," McFadden told BBC WM. "Fifty points is a fantastic total and we'll go and enjoy the summer and come back fighting for pre-season." McFadden's season ended with a consolat.... read more
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FICTION: A North Dakota couple set out to bring their grandson home, and come face to face with evil.
Larry Watson
If there were ever any question about Larry Watson's reputation as one of the finest writers working today, there shouldn't be anymore. Not after "Let Him Go," Watson's ninth book of fiction, and his best.
Set in the autumn of 1951, "Let Him Go" is the timeless story of George and Margaret Blackledge, grandparents who set out to reclaim their grandson, who has moved from Dalton, N.D., to Montana with his mother and her suspect new husband, Donnie Weboy.
Donnie, in Margaret's mind, is a sorry replacement for her son James, whose recent and tragic death haunts her. It takes some arm-twisting, but Margaret convinces her reluctant husband to join her on the road, and before they leave Dalton behind, he makes a stop at their old ranch. Margaret, wise to his ways, says, "I've had enough loss, George." To which he replies, "You know that's what life is. Loss, fast or slow." It's a prescient sentiment, and the foreshadowing in their spoken words sets the novel in motion.
Readers familiar with Watson's stoic and laconic voice will surely recognize the timbre of "Let Him Go." The landscape is described in loving, gorgeous prose: "This is prairie, rolling gentle country where black seams of trees and brush stitch one grassy hill to another." And the subtle cleverness in both dialogue and in the emotional push and pull between characters is vintage Watson.
What distinguishes "Let Him Go" from Watson's previous novels is the relentless narrative energy. Without compromising any of his trademark style, Watson manages to tell a story that is riveting in its many twists, one that turns from sweetness to sorrow with an amazing economy.
When George and Margaret get to Montana and come face to face with Donnie and the rest of the Weboys, the pacing becomes downright explosive. The Weboys — especially the demonic matriarch, Blanche — are a villainous and territorial bunch of thugs intent on not giving up the grandson easily. What begins as a cordial request by Margaret to bring the boy back to North Dakota with his mother quickly becomes a mesmerizing and heart-stopping game of cat and mouse, one that builds expertly to a climax that is both horrific and inevitable.
Which is not to say that "Let Him Go" is simply a dark book, one that introduces evil characters merely for the sake of a driving story. There is redemption in these pages, too, and I don't think I've ever read a book that deals so convincingly with the staying power of memory, or of the power of familial love.
Peter Geye is the author of "Safe From the Sea" and "The Lighthouse Road." He lives in Minneapolis.


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Ehat a good beam for a gun green or red and wjy
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Romans 1:16 I am not ashamed of the gospel, because it is the power of God for the salvation of everyone who believes: first for the Jew, then for the Gentile.
No starting out first thread with gibberish. It's bad form.


text: NY exploring public-private pacts on road repairs By: Michael H. Samuels April 17, 2011 4 Comments The state is looking at private companies to help invest in infrastructure projects throughout the state. State Sen. Charles Fuschillo, R-Merrick, is planning on introducing legislation that will allow the state to partner with private businesses to facilitate and fund new capital projects, including road and bridge repairs and replacements. "New York's transportation infrastructure is at a ... Subscribe Login Email Address: Password: Already a paid subscriber but not registered for online access yet? For instructions on how to get premium web access, click here.Forgot your password? Print, Digital & Mobile1 Year$129 Digital & Mobile Only1 Month Intro Rate$2.99----------1 Year$99 Digital & Mobile Only1 Month Intro Rate$2.99----------1 Year$99 Print, Digital & Mobile1 Year$129 Print, Digital & Mobile1 Year$129----------Digital & Mobile Only1 Month Intro Rate$2.99----------1 Year$99 Share this:EmailPrintFacebookTwitterGoogle +1 Charles Fuschillo Dean Skelos DOT private firms road projects 3:41 pm Sun, April 17, 2011 Long Island Business News Michael H. Samuels


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Foursquare has developed a new version of its application that will include a new "explore" button in place of its current "tips" section.
The new button is designed to help Foursquare users discover new places in their surrounding areas.
Foursquare co-founder Dennis Crowley told ABC News that the new tool also will enable Foursquare to recommend a new restaurant based on users' check-in history, or based on what their friends like. A new search function, meanwhile, will let users type in keywords such as "margaritas outside" or "date night" and get suggestions for where to go.
The update is now available for iPhones and Android phones, and also will let businesses offer new types of incentives.
From ABC News:
The update brings three new types of specials, bringing the total to seven. "Swarm specials" will let places offer special deals if a ton of people — say, 50 or 500 — check in all at once. "Flash specials" are quick-hit deals — such as a free drink to the first 10 people to show up at a restaurant at a certain time. "Friends specials" are just that, deals you get when you bring a few extra guests to a venue.
Topics: Business Strategy and Profitability , Food & Beverage , Marketing / Branding / Promotion , Online / Mobile / Social


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Premier Urgent Care Monument
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Our family opened our Monument Urgent Care location in January 2008. Since opening day, our goal has been to provide the best in medical treatment and customer service at a fair price to our Monument and Tri-Lakes communities. We believe that having a medical doctor present at all times supported by a team of ER nurses/paramedics, licensed x-ray technologists, & medical assistants means our patients benefit most from that high-level of care. Over the years we have come to identify other individual medical providers with the same commitment to patient care who are the best in their specialties if a referral is necessary.
Premier also provides Level II worker's compensation and occupational medicine services to federal, state, and private employers. We are a Pinnacol SelectNet Preferred Provider and work closely with most work comp insurers as well. At Premier, we work closely with employers to meet the specific needs of a business and believe that strong communication is essential to a productive relationship.
Being a part of the Monument community is very important to us! Premier Urgent Care is a proud supporter of:
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Not all urgent care facilities are created equal. Know what level of medical professional is providing your care, and what that treatment will mean to your wallet. At Premier Urgent Care, we work hard to provide expert care and service - fast and fair!


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Well, I run a dark theme and I love GO launcher, BUT.... I hated seeing that white menu pop up all the time.... so, I changed it!! That's the only changes but I think it makes a world of difference.... Instructions... Open GO launcher preferences and back up configuration... uninstall your current version download this.... http://bit.ly/GoLauncherEX239Dark find on your SD card select app select install.... Enjoy....
thanks for making this escnead. Just switched to go launcher, and this was much needed. Somewhat related:: u have any idea where the font color can be changed in the cap? I'm working on GOLauncher Calendar mod, and can't find the circled in green areas.
Is the Calendar built into the Launcher or is it one of the add ons? I don't use it... I will take a look at it tomorrow and see if I can find those colors...
Man you do truely awesome work. And you have given me a great reason to try out go launcher. Thank You!!
It's the addon widget - here's the market link I could post the apk orig and/or the mod so far -- lemme know
Looks good. The Go launcher devs should follow your lead and do this on the next update. I never cared for the white background either.
Wow, this is awesome! all I use is go lAuncher and I hated the white too. some themes get rid of it. But now its gone forever. DROIDX ApeXRC2.
I thought they might but I couldn't find any themes I liked that were free and why would I pay for a theme for a free app?? I just took care of it myself.. Sent from my SCH-I500 using DroidForums
awesome dude! How did you change the text color to white? If I want to update the new versions with this mod, which files do I copy to the new apk? Thank you
Usually, its best to edit the new xmls. I will be working on the update this weekend, and I will post it here when I get finished.... Glad you like it
Really nice Ecs! Another thing to help tide me over until my D3 can be rooted and I can really get my inversion on Sent from my Hitchhiker's Guide mark 2
Just a heads up, there is another update on the market now. I have a feeling they don't feel like slowing down right now. It may save your fingers just to wait for the next major update. Sent from my Hitchhiker's Guide mark 2


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quote:Liz Cheney announced Tuesday that she will challenge Wyoming Sen. Mike Enzi in a Republican primary next year. The daughter of former Vice President Dick Cheney made the announcement in a six-minute YouTube video. Most of her announcement was an attack on President Barack Obama, with only a veiled swipe at the the incumbent. "We can get our nation back on track," Cheney said. "Instead of cutting deals with the president's liberal allies, we should be opposing them every step of the way." "I'm running because I believe it is necessary for a new generation of leaders to step up to the plate," she added. "We can't continue business as usual in Washington." Enzi, 69, announced Tuesday that he will run for a fourth term. He said at the Capitol that Cheney did not give him a heads up and defiantly insisted that her move will not dissuade him. "She said that if I ran she wasn't going to run. That wasn't correct," he said. "I thought we were friends." National Republican Senatorial Committee Chairman Jerry Moran, a senator from Kansas, announced that the official party body will back Enzi. Cheney, 46, worked in the State Department and the Agency for International Development during the George W. Bush administration. But she moved her family to Jackson Hole last year and has traveled aggressively around the state laying the foundation for what everyone assumed would be a political bid.
quote:"She said that if I ran she wasn't going to run. That wasn't correct," he said. "I thought we were friends."
quote:Liz Cheney
quote: it would be an improvement to have a strong conservative woman in the senate...hope she wins.
quote:Plus she likes the vagina
quote: That's the other daughter.
quote:Another politicians child trying to belly up to the government feed trough? Shocking. These people are no better than welfare recipients.
quote:I get the feeling she's going to vote closer to McCain than Rand.
quote:Liz Cheney announces Senate bid
quote:Senate grid lock
quote:Then I do not want. She can keep her arse out of Washington. If she's another McCain, Graham, or a generic big govt RINO neocon, she should be told to frick off. We don't need anymore of those types in the party.
quote:She's running to the right of Mike Enzi, so I don't think she's what most would consider a RINO.


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So Western psychology has helped the Western mind to be very irresponsible. The Western revolts of the youth and other rebellions, destructive movements, they are really created by the Western psychology within these last fifty years. Freud is more responsible for this than Marx because he has said, "You are already complete by seven years of age. Your parents are responsible, you are not responsible. So whatsoever you are doing - if you are a criminal, if you are a murderer - you cannot do anything. You have to be. And now your dead parents cannot be changed. And those dead parents are also not responsible, because their parents.."
So, ultimately, Freud comes to the same conclusion that Christianity reached before: "Adam committed the sin and we are not responsible. He is responsible, Adam, the first father. And because of him everything is decided. Now we are born in sin and we will have to die in sin."
If you move with Freud you will reach the same conclusion: "If parents are responsible, then ultimately Adam and Eve are responsible. But how to change Adam and Eve now? That is impossible. So whatsoever is, is, continues."
This is not acceptance, this is defeatism. And human dignity is lost. If you cannot do anything, if you cannot transform yourself, you lose all human dignity. You have become just an automaton, a mechanical thing. You will run the course, because your parents have given you a winding, so you will run the course and then you will die. In the meantime, if you have the opportunity you will give a winding to other fellows, and they will continue.
This is most degrading. Man can change himself: that possibility is always with you. And this concept, this very concept that "I can change myself," starts the change. The revolution has taken place. The revolution has begun.
And, lastly, it has been asked, "Is it not necessary that the religious man should seek to bring about a radical change in the very pattern of society?"
He, the religious man, is the radical change in the pattern of society. He will not try to bring any radical change in the society. He is the radical change.


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Learn more about D-Link DSL-520B ADSL2+ Ethernet Modem:
The DSL-520B ADSL2+ modem router is an affordable high-performance ADSL device for home and the small office. With integrated ADSL2/2+ supporting up to 24Mbps download speed, firewall protection, Quality of Service (QoS) and 1-port switch, this router provides all the essentials that a home or small office needs to establish a secure and high-speed remote link to the outside world. Security features prevents unauthorized access to the home and office network from Internet intruders. The router provides firewall security using Stateful Packet Inspection (SPI) and Denial of Service (DoS) attack protection. SPI inspects the contents of all incoming packet headers before deciding what packets are allowed to pass through. Router access control is provided with packet filtering based on port and source/destination MAC/IP addresses. |


text: Even if MD5 is installed and working, programs like passwd won't use it by > default. Can I guess passwd is the place you're having the trouble? > > In login.defs make sure you set "MD_5_CRYPT_ENAB" to "yes" I was sure that I had fixed it (used sed), but when I checked the file, there was nothing(nothing at all) there. Now it's working.. -- Martin Holt Juliussen martin.juliussen at hamar.[remove to reply]online.no -- Unsubscribe: send email to listar at linuxfromscratch.org and put 'unsubscribe blfs-support' in the subject header of the message


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Each of these wines is an excellent representation of their varietal. The Rumeni Muškat is a very fun wine. A slight effervescent wine that is dry on the palate with just a touch of sweetness on the finish. Serve as an aperitif or at the end of the meal as a dessert wine; it is your choice, you can't go wrong either way. Serve this delicious un-oaked Chardonnay with the starter dishes, such as roasted vegetables, cured meats and salad. Now bring on the reds with a wonderful Cabernet Franc. We believe this is the best Cabernet Franc EVER!
Before there was wine in France, Spain and Germany, there was wine in the countries surrounding the Adriatic Sea; the Central part of Europe that shared cultures with the ancient Romans and Greeks. Since pre-roman times, this area has been the heart of wine making in Europe. These are the original Old World Wines that are hand-crafted in small batches by family-owned wineries using centuries old techniques to express the terroir (earth), the heritage and the history of this unique and ancient wine-making region.


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March 7th, 2014 at 2:39 pm
Thursday Final Ratings: 'The Big Bang Theory', 'Once Upon A Time in Wonderland', 'Parks and Recreation' & 'Elementary' Adjusted Up; 'The Millers' & 'Two and a Half Men' Adjusted Down
Thursday's final ratings.
[Read-More]March 7th, 2014 at 9:01 am
9 Percent Growth Week-to-Week for 'Parenthood' & 'Parks and Recreation'
"Parenthood" (1.2/4 in 18-49, 3.8 million viewers overall from 10-11 p.m. ET) grew week to week by 9% in 18-49 rating
[Read-More]March 7th, 2014 at 8:00 am
TV Ratings Thursday: 'Scandal' & 'Grey's Anatomy' Dip, 'The Big Bang Theory', 'The Vampire Diaries' & 'Parenthood' Up, 'American Idol' Hits Low
CBS was number one with adults 18-49 and with total viewers.
[Read-More]February 28th, 2014 at 1:55 pm
Thursday Final Ratings: 'The Big Bang Theory', 'Scandal', 'Grey's Anatomy', 'American Idol' & 'Parks & Recreation' Adjusted Up; 'The Millers' Adjusted Down
Thursday's final ratings.
[Read-More]February 28th, 2014 at 9:03 am
'Hollywood Game Night' Topped the Show's Final Thursday Original of Last Summer
With its first telecast in five weeks, "Parenthood" averaged a 1.1/3 in 18-49 and 3.6 million viewers overall
[Read-More]February 28th, 2014 at 8:36 am
TV Ratings Thursday: 'Community', 'Reign' & 'American Idol' Hit Lows + 'Scandal' & 'Grey's Anatomy' Return Up
CBS was number one with adults 18-49 and with total viewers.
[Read-More]February 14th, 2014 at 12:05 pm
Hollywood Game Night Party Game Hits Shelves February 15
Hitting shelves on February 15, the Hollywood Game Night Party Game will be available initially at Walmart
[Read-More]February 11th, 2014 at 11:01 am
NBC Dominates the Primetime Week of February 3-9 With Its Top Numbers Since Summer 2012
Feb. 6 Bonus Sochi Coverage Generates the Most-Watched Thursday for Any Network Since Early September


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Mute Monday: Tired Lucy
Fun Facts About Wine Country
The taste of wine varies from winery to winery – even those next door to one another may taste vastly different. The trees, whether Eucalyptus (there are seriously huge Eucalyptus trees all around) or Redwood or Black Oak, drop their leaves in autumn, allowing the different properties of the tree to soak into the soil. Eucalyptus is how a hint of mint shows up in wine.
Bat boxes & owl boxes & bluebird boxes are everywhere in Napa Valley. Bats eat insect pests. Owls catch rodents & snakes in the vineyards – grass is allowed to grow up over the roots of the vines during summer to keep the roots cooler. Bluebirds are very territorial songbirds & they don't eat berries! They help keep other songbirds out of the vineyard.
Roses & crepe myrtle plants are around every winery & vineyard, usually at the end of each row of vines. Insect pests that attack grapes will attack the flowers first – this way the vineyard master can deal with them before they ruin the grape crop.
Napa Valley doesn't allow pesticide use. Another reason I prefer Napa to Sonoma.
I miss my herb garden. Every winery uses herbs in their landscaping. Rosemary hedges, thyme creeping along the pathways… I MISS MY HERB GARDEN!
Vineyards Everywhere!
Cabernet Sauvignon
Sauvignon Blanc
Not-So-Mute Monday: California Olive Oil Only!
Only buy 100% California Olive Oil! Anything else is olive oil blended with less desirable oils. The FDA holds American companies to a higher standard.
Buy American!
Every small village in Israel has its own olive press, according to my #1 son who lived in Israel for a year. Each family brings their olives to the press & carts home their olive oil.
Final Day in Sonoma Valley
St. Francis Winery & Vineyards
St. Francis
Well known for their award winning restaurant (no reservations for us…Rats!), St. Francis Winery has an organic garden – veggies served in their restaurant, delicious, affordable wines & knowledgeable, fun servers behind the bar pouring wines. DH joined their wine club last year & we have not been disappointed.
The grounds are beautiful, the tasting room – love the ceiling!
We met a woman from Brentwood, TN who was vacationing with her friends from Michigan. We encouraged her to take them to Arrington Vineyards since they were going home with her after their California trip. (Tennessee grapes making excellent dry red wines!)
Kunde Family Estate
Five generations of the Kunde Family have been making wine on this estate for over 100 years. We enjoyed our tasting on the patio with a snack to serve as lunch.
After the cheese & meats they served a Port-style red with a sweet chocolate that smelled like a chocolate Easter bunny. Yum!
I joined this wine club.
Inman Family Wines
Our last stop was Inman, a very small winery & one of our favorites. Kathleen Inman, the wine maker served our tasting – try to get that at any other winery in Napa, Sonoma or middle Tennessee! She's just a normal person who loves her work, whose work is making excellent wine!
We shipped a few bottles home, but no wine club this time…yet.
Second Day Sonoma Wineries, part 2
Rodney Strong Vineyards
Rodney Strongis another wine you will find almost anywhere, but their reserve wines – what a lovely surprise!
The server behind the bar was friendly, engaging & wrote a lovely thank-you note to me, me having joined the Symmetry Wine Club. Why did I join this particular wine club – THE SHIPPING IS FREE! Wines only available at the winery & through the wine club are seriously excellent & make it worth joining!
J Winery
Sharing a parking lot with Rodney Strong, J Winery… oh my goodness, what to say… DH joined this wine club on our last visit. If you want to know what Sonoma Pinot Noir should taste like – open a bottle of J! Judy Jordan, daughter of the famous Jordan Winery family (we couldn't get a tasting reservation at this ORGANIC winery! Rats!) decided to go it on her own & has had great success producing delicious wines!
We chose the sparkling wine sampler with a tasting platter of cheeses. Perfect for early afternoon, having been tasting since 10am. A welcome respite & snack.
Martinelli Winery
The folks at Martinelli are so nice we had to stop in to say, "Hi!" The giant, ancient grapevine around the patio post greets all entering the tasting room.
We bought a bottle to give to our friends who couldn't join us on our trip.
The Martinelli's daughter lives in Brentwood, TN, just up the road a bit from us.
Another long day tasting wonderful wines. Time to join the afternoon rush-hour traffic heading to Whole Foods to grab something from their fresh food bar for dinner, then back to the hotel.
Giant Redwood Trees
This California Redwood Tree (Sequoia sempervirens) was in the picnic area of Chateau St. Jean Winery & Vineyard. The trunk was at least 6 feet across – I couldn't get my arms around it halfway.
Just so you know, I did not enhance the color of this photo! The sky was so clear, so blue, it took my breath away!
I am shocked at how tiny the pine cones from the giant redwood are.
You already know I'm a bird nerd & a nature freak, so to tell you I love the giant redwoods is not a surprise. The fact that I stopped at Ch. Montelena to speak to the redwood tree I had seen years before was a tipoff.
Giant Redwoods are remarkable to view, but one must remember, they are "giant" trees, just like St. Bernard's, Great Danes & Newfoundland's are "giant" dogs. They grow big because they are supposed to grow BIG. Saying that, I am wondering if I could grow one in middle Tennessee. It may not be moist enough, though we did get a ton of rain this past summer… I will do some checking, but if I can grow Myrtle, my Live Oak, why couldn't I grow a giant redwood?


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This is a great multicolor merino wool - fabulous pinks, purples greens and yellows blend to make a very spring like yarn -I understand the color name - I've seen some of these iris blends in the garden - they definitely have caught the spirit in this vibrant yarn color!
$9.20 or less + Free Shipping (Discounts will apply to Malabrigo when your order is $50 or more!) Content: 51% Silk, 49% Baby Merino wool Yardage: aprox 150 yards per 50 grams skein Gauge 5.5 Stitches per inch on a size 6 Needle Hand wash, Dry Flat Needles US 4 - 6 or 3.5 - 4mm Gauge 22.0 to 24.0 sts = 4 inches Please note: Malabrigo does not guarantee lot matches beyond bags of 10.We attempt to match lots on all orders. If we cannot, we visually match- we will contact you prior to shipping if we believe there is a problem.


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Rugby Union
Select a team
Six Nations
- England
- France
- Ireland
- Italy
- Scotland
- Wales
Aviva Premiership
- Bath
- Exeter
- Gloucester
- Harlequins
- Leicester
- London Irish
- London Welsh
- Newcastle
- Northampton
- Sale
- Saracens
- Wasps
The Rugby Championship
- Australia
- New Zealand
- South Africa
Guinness PRO12
- Cardiff Blues
- Connacht
- Dragons
- Edinburgh
- Glasgow
- Leinster
- Munster
- Ospreys
- Scarlets
- Treviso
- Ulster
- Zebre
Top 14
- Bayonne
- Bordeaux-Begles
- Brive
- Castres
- Clermont Auvergne
- Lyon
- Montpellier
- Racing Metro
- Stade Francais
- Toulon
- Toulouse
- Grenoble
- La Rochelle
- Oyonnax
Home team | Away Team | |
6 - 16 |
Match Links
- RBS Six Nations
- 9th Feb 2013
- Ground: Stade de France, Paris
France v Wales - Live
Full-Time: France 6-16 Wales
80 France trying to salvage something but the ball goes appropriately forward as the boos ring out. Wales end their losing run thanks to North's magic finish, but this was a truly dismal fixture. Next for France? A trip to Twickenham...
79 Last chance for France but they waste it through a tap penalty rather than the corner, knocking on outside Wales' 22. Excellent defence from the visitors who have earned their win late on.
76 Trinh-Duc, oddly out on the wing, tries to attack but with no fortune. Spectators pouring out of the ground as Wales have a scrum, counting down the clock.
PENALTY, WALES, HALFPENNY! 6-16 Wales regather possession from the restart and win the penalty! Halfpenny stepping up to win the match from a long way out, but he has the power in his boot. Up he steps and... it's over!
TRY, WALES NORTH! 6-13 Parra's box kick doesn't go far enough and Wales have an attacking lineout. North crashes up, chip over the top from Biggar and North is in! Try in the corner! Upstairs to the TMO to confirm... super finish.
70 Comedy moment from Huget as the ball goes forward off his head. Roberts kicks into touch and France will have to clear from their 22.
66 Wales win the penalty at the scrum - escaping from their half. The visitors now try to attack down the left, building the phases but lacking the quick ball required. Jamie Roberts isolated in midfield but clings on, before Bigger wastes possession with a terrible outside flick of the boot.
64 French attack continues to power forward - camped outside the Welsh 22. Spilled at the ruck and France have the scrum, which has been on top in the last few minutes.
62 Thunderous drive from France wins the penalty at the scrum and they move into Welsh territory.
60 More dross. Neither side able to make a mark and the crowd are showing their frustration.
58 Little structure for either side but Wales come again - a poor kick from Mike Phillips bringing the attack swiftly to an end.
56 Huge cheer as Morgan Parra enters the fray - France lineout results in a turnover by Ryan Jones and Wales have a good attacking scrum.
54 Driving maul leads to a blindside break for Picamoles, but the ball is lost and Wales counter - Halfpenny searching for space. Chip over the top from Ryan Jones of all people finds touch in the France 22.
PENALTY, FRANCE, MICHALAK! 6-6 Strong scrum from France and Michalak has the penalty, levelling the scores. Who will spark first?
49 Solid defence from Wales yields a scrum, Fofana wrapped up by Faletau. Both sides equally matched.
47 France attacking down the right, Fofana on an inside line trying to slide through the middle. Wales turn to batter away the attackers.
44 Break from Machaneaud from the restart and France are five metres out - back to Trinh Duc whose drop goal attempt is... wide! Poor option in the first place.
PENALTY, WALES, HALFPENNY! 3-6 Easy does it for Halfpenny and Wales take an early lead.
40 Francois Trinh-Duc for France leading to a reshuffle in the French backline - Mike Phillips not wasting any time from the restart as he charges forward. France recover the ball but Wales have the penalty, straight in front.
France 3-3 Wales
40 Carries from Tipuric and Phillips but Wales can't find a gap. Nothing comes off it, the boos ring out as both sides leave the field and that's a fair summary. Lots of possession for Wales but unable to use it, minimal opportunities created. Work to be done.
39 Penalty to Wales on the brink of half-time. Biggar kicks to touch and Wales will aim to salvage something before the interval.
37 Multiple phases for Wales but then Picamoles pounces at the ruck for the turnover. Plenty of territory and possession for the visitors but nothing coming.
35 Wales fail to use the space down the left-hand side, instead crashing the ball up with Faletau down the right.
33 Powerful maul well controlled by Szarzewski at the base, but Wales win the penalty, Gethin Jenkins turning the ball over at the breakdown. Biggar clears to halfway.
31 Advantage to France following the lineout - just out of Michalak's range who will target the corner instead.
28 Grubber through from Biggar causes problems for Huget who dabs the ball into touch. Welsh lineout in the France 22 - Wales set up a maul but Picamoles steals the ball. Fall clears upfield, Wales scampering but France turn over the ball. Kick through behind but Jonathan Davies clears up and Wales breathe again.
26 French defence holding firm as Wales attack - Biggar goes high but it's an easy take for Huget who skews his kick into the arms of Halfpenny. A troublesome bounce from the Welsh respondent kick leads to a 22 dropout for France.
24 Powerful scrum from Wales wins them the penalty, Biggar clearing to find touch in the France half.
22 Halfpenny charged down by Huget - Cuthbert regathers the ball but it slips and France attack in the Welsh 22. Smart choke tackle implemented by Ryan Jones and Wales have the scrum.
19 France create some handy possession in the Welsh half - powerful runs from Bastareaud and Suta. Wide to Huget who slips the tackle of Mike Phillips, Bastareaud on a jinking inside line. Huget well covered by Halfpenny but France press, a phenomenal turnover from Wales saving the day and Halfpenny clears.
PENALTY, WALES, HALFPENNY! 3-3 Easy for the full-back, Wales on the board.
16 Penalty to Wales from the restart - Biggar strokes it into the corner and Wales have a five metre lineout. Static maul so Wales go wide, Roberts then Biggar carrying up. France penalised and Wales will go for goal.
PENALTY, FRANCE, MICHALAK! 3-0 Penalty at the scrum to France and Michalak will have a shot at goal. No problem, France lead.
12 Knock-on from Dusautoir turns over possession and Wales clear from a Jonathan Davies grubber, thumped by Bastareaud as he kicked. Long lineout taken by Picamoles as France attack into the Welsh half.
10 Wales penalised for not releasing at the breakdown - Michalak clears into the Welsh half.
9 Atrocious state of the pitch not helping matters as multiple scrum resets cause more damage.
6 Wales enjoying plenty of early territory as Biggar goes for a drop goal. It falls short and Huget counters but to little effect. Scrum to Wales.
3 Excellent drive from Faletau puts Wales in the France, but the ball goes forward. Machaneaud clears from the scrum.
1 Early knock-on from Wales hands France a scrum outside their 22 - penalty to the hosts and Michalak clears to half-way. Pitch cutting up heavily.
Kick-Off Anthems sung and pyrotechnics set off - it's time to get underway in Paris. Dan Biggar to start us off.
Dry conditions so far in a freezing Paris. Soft hands required under a clear sky.
Team News Wales captain Sam Warburton is unable to take part in Paris due to injury, meaning the in-form Ospreys flanker Justin Tipuric comes in at openside with Ryan Jones on the blindside leading the side. Richard Hibbard is back at hooker whilst Lou Reed returns to the replacements bench.
Team News Injury to last weekend's captain Pascal Papÿ© sees Thierry Dusautoir re-gain the armband. Toulon's Jocelino Suta replaces Papÿ© at lock, with Mathieu Bastareaud returning in the centre to partner Maxime Mermoz.
welcome Hello and welcome to the Stade de France for Saturday's mammoth Six Nations clash between a wounded France and reigning Six Nations champions Wales.
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Time | Fixture |
Aviva Premiership | |
Friday 27th February | |
Aviva Premiership | |
19:45 | Northampton vs Harlequins |
Saturday 28th February | |
14:30 | London Welsh vs London Irish |
15:00 | Saracens vs Newcastle Falcons |
15:00 | Leicester Tigers vs Sale Sharks |
15:15 | Exeter vs Bath |
Sunday 1st March | |
13:00 | Wasps vs Gloucester |
Friday 6th March | |
19:45 | Bath vs Sale |
Saturday 7th March | |
15:00 | Exeter vs London Welsh |
15:00 | Harlequins vs London Irish |
15:15 | Gloucester vs Northampton |
Sunday 8th March | |
14:00 | Wasps vs Saracens |
15:00 | Newcastle vs Leicester |
Friday 27th March | |
19:45 | Northampton vs Wasps |
Saturday 28th March | |
15:00 | London Irish vs Newcastle |
15:00 | Leicester vs Exeter |
15:15 | Saracens vs Harlequins |
Sunday 29th March | |
14:00 | Sale vs Gloucester |
14:30 | London Welsh vs Bath |
Friday 10th April | |
19:45 | Newcastle vs Bath |
Saturday 11th April | |
15:00 | Harlequins vs Gloucester |
15:15 | Saracens vs Leicester |
Sunday 12th April | |
14:00 | Exeter vs Northampton |
14:30 | London Welsh vs Wasps |
15:00 | London Irish vs Sale |
Saturday 25th April | |
14:00 | Sale vs Harlequins |
Result |
Aviva Premiership |
Sunday 22nd February |
Aviva Premiership |
London Irish 6 - 12 Leicester Tigers |
Saturday 21st February |
Bath 13 - 21 Northampton |
Harlequins 21 - 32 Exeter |
Gloucester 48 - 10 London Welsh |
Sale Sharks 14 - 10 Saracens |
Friday 20th February |
Newcastle 23 - 23 Wasps |
Sunday 15th February |
Wasps 37 - 6 Harlequins |
London Welsh 12 - 52 Sale |
Saracens 34 - 24 Bath |
Saturday 14th February |
Exeter 46 - 17 Newcastle |
Friday 13th February |
Leicester 18 - 15 Gloucester |
Northampton 15 - 9 London Irish |
Sunday 11th January |
London Irish 28 - 26 Exeter |
Newcastle 38 - 7 London Welsh |
Saturday 10th January |
Harlequins 32 - 12 Leicester |
Bath 39 - 26 Wasps |
Sale Sharks 20 - 7 Northampton |
Friday 9th January |
Gloucester 24 - 23 Saracens |
Experts & Blogs
Phil Edwards
Read the thoughts and opinions of Phil Edwards with skysports.com


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Sir Cathcart D'Eath | E3 6a | ||
20m. 12m. No description yet. © ROCKFAX One of the better lines in the Valley with good rock and protection. Follow the steep leftwards-trending flake/crack, past a thread and peg, to flake-jugs at the base of a slanting groove. Launch up the groove which soon eases and a traverse left can be made to good small wires in a curving overlap. Climb to the lower off past a small knobbly ledge and flake above. FA. Stuart Cathcart, J.Dee 4.5.81 04/May/1981 Submit a new photo of this climb. | |||


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Showing 2 reviews for Metra MDF-8202-JBL-1
Customer Review Summary for Metra MDF-8202-JBL-1 | |||||||||||||||||||||||||||||||||||||||||
March 14, 2013 | Good for the Money |
2 of 4 people found the following review helpful:
Review by
desert rat from Blythe Ca
Good sounding easy to use, only down side I have noticed is that on sunny days its hard to see the navigation screen
July 22, 2012 | Nice sounding, Good looking upgrade w/ Quirky NAV/GPS feature. |
2 of 5 people found the following review helpful:
Review by
Oliver from Fremont, CA
Great replacement for those who have 2004 and up Toyota's with the OEM JBL package but want to upgrade to something with more up to date features like MP3, USB, etc and still use their existing amplified JBL speakers and subwoofer system.
Pros; Sounds great ! Fits perfectly in console. No cutting or snipping any existing vehicle wiring. 6.2 inch touchscreen Lots of extras like DVD, MP3/ MP4/ BT/ IPOD inputs...etc. Cons; NAVMATE software is quirky and setup is time consuming. So far it only worked accurately one time on a 40 mile round trip excursion.....the next day all setup data was lost and required reconfiguration. Although the system diagnostics reported 5 of 12 satellites in range the GPS would not track my movement. (the manual says 4 satellites in range is supposed to be sufficient for operation) The navigation software also runs off of an SD memory card so you cannot use the SD slot for anything else without forfeiting the Navigation feature. When exiting the Navigation software the touch screen locks up and so far the only way I've been able to recover is by removing and reinserting the SD card. No information regarding this unit or the NavMate software is available on the Metra website and Metra Tech Support is via phone mon-fri during standard business hours only. Attention DIY installers !! This unit is a direct fit but it requires alot more room than the OEM to allow for the wiring harness and JBL adapter so be sure to use wire ties carefully and stay clear of the HVAC cabling and all that other stuff behind your console.


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- 31Promote
Framed Art Print
Choose from a variety of frame styles, colors and sizes to compliment your favorite Society6 gallery, or fine art print - made ready to hang. Fine-crafted from solid woods, premium shatterproof acrylic protects the face of the art print, while an acid free dust cover on the back provides a custom finish. All framed art prints include wall hanging hardware.
islands of the whitsundays queensland australia
You should come on down, the ozzie dollars a bit high at the moment but you will have a great time. cheers :D
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- Home
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- Four Minute Mile Opaque Pink Colored
Original Recordings.
1. Coming Clean
2. Don't Hate Me
3. Fall Semester
4. Stay Gold, Ponyboy
5. Lowercase West Thomas
6. Washington Square Park
7. Last Place You Look
8. Better Half
9. No Love
10. Shorty
11. Michele With One "L"
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This recipe goes with Jerk Mackerel with Papaya Salad
Cooking Light JANUARY 2010
1. Cut Scotch bonnet pepper into quarters. Remove and discard seeds and membranes from 3 pepper quarters; leave seeds and membrane intact in remaining pepper quarter. Place pepper and remaining ingredients in a mini chopper, and process until a fine paste forms.
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"Rowden has wedded ethnomusicology and disability studies to offer a fresh approach to the study of African American popular music. The Songs of Blind Folk undermines many of the defining mythologies and tropes of blind musicians, including the perception that they are successful because they compensate for the loss of vision."
---Mark Anthony Neal, Duke University
"Illuminates how the enduring phenomenon of blind African American musicians emerged from brutal conditions, how these musicians were deployed in the burgeoning American iconography of race and 'freakdom,' and how they negotiated this hazardous cultural terrain . . . the book is timely, well-historicized, and rich in insight."
---Kari Winter, University at Buffalo
The Songs of Blind Folk explores the ways that the lives and careers of blind and visually impaired African American musicians and singers have mirrored the changes in America's image of African Americans and the social positioning and possibilities of the entire black community. The book offers a historically grounded consideration of African American performers and their audiences, and the ways that blindness, like blackness, has affected the way the music has been produced and received. Author Terry Rowden considers the controversial nineteenth-century prodigy Blind Tom Bethune; blues singers and songwriters such as Blind Lemon Jefferson, who achieved an unprecedented degree of visibility and acceptance in the 1920s and '30s; spiritual and gospel musicians such as the Blind Boys of Alabama; celebrated jazz and rhythm and blues artists Art Tatum, Rahsaan Roland Kirk, and Ray Charles; and finally, perhaps the best known of all blind performers, Stevie Wonder.
Terry Rowden is Assistant Professor of English at the City University of New York, Staten Island. He is coeditor of Transnational Cinema: The Film Reader.
Introduction 1
Chapter 1 Blind Tom and the Cultural Politics of Visibility 15
Chapter 2 Blind in Blue Blindness and Identity in the Blues Tradition 35
Chapter 3 The Souls of Blind Folk Blindness and Blind People in African American Spiritual and Gospel Music 65
Chapter 4 Blindness and the Rhetoric of "Genius" Art Tatum, Rahsaan Roland Kirk, and Ray Charles 85
Chapter 5 The Inner and Outer Visions of Stevie Wonder 105
Notes 123
Bibliography 143
Index 165


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Obamacare Countdown: Americans Still in the Dark about Health Insurance Exchanges
NEW YORK (MainStreet) ¿ Obamacare looms, but more Americans ¿ including those without health insurance ¿ have little idea how it works or what they should do. With health insurance exchanges set to open in days, a new Harris Poll reports 70% of those surveyed are not at all or not very familiar with them. Those without health insurance are even less familiar, with 73% not at all or not very familiar.
"As health insurance exchanges go live, consumers will have the opportunity to evaluate and select the health insurance plan and benefits that best fit their medical needs and wallet," says Debra Richman, Senior Vice President, Healthcare Strategy, and Co-Chair of Strategic Health Perspectives. "These findings suggest that unless knowledge and understanding of the exchanges begin to grow, particularly among those who aren't covered today, some of consumers may be slow to utilize them. Health insurers must first understand these new individual customers and work to close the huge consumer education gap."
Just 9% of the public report that they are extremely or very familiar with the term health insurance exchange, while another 21% are somewhat familiar. But one thing Americans can agree on: they don't want to pay the penalty for not having coverage. When asked what they would do if their employer did not provide insurance, over half (59%) of those with employer-provided coverage say that they would purchase insurance through the exchanges, as opposed to paying a penalty.
Nearly three-quarters of Americans believe the insurance exchanges will be difficult to understand and that health insurance purchased through an exchange will be expensive-- but still half believe that they will be able to find a health plan on an exchange that will meet their needs at a reasonable price.
"We see a fairly high percentage of Americans that actually believe the exchanges will enable them to find affordable health insurance that will meet their needs. This offers health plans a strong opportunity to engage consumers," Richman adds. "The key to a plan's success will be the ability to address consumers' exchange familiarity and also understand the important differences in consumer preferences and purchasing behaviors." While the Harris survey referenced the Obamacare insurance sources as "exchanges," the official government Affordable Care Act website (healthcare.gov) refers to them as "marketplaces." Beginning October 1, open enrollment will begin and consumers will be able to fill out an application for health insurance coverage through these marketplaces. Coverage commences on January 1, 2014. Americans without "minimum essential coverage" will still be responsible for paying any costs incurred for healthcare, as well as a penalty fee. The fee in 2014 is 1% of annual income or $95 per person for the year, whichever is higher. The fee increases every year. In 2016 it is 2.5% of income or $695 per person, whichever is higher. --Written by Hal M. Bundrick for MainStreetCheck Out Our Best Services for Investors
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Pseudofactin II is a recently identified biosurfactant secreted by Pseudomonas fluorescens BD5, the strain obtained from freshwater from the Arctic Archipelago of Svalbard. Pseudofactin II is a novel compound identified as cyclic lipopeptide with a palmitic acid connected to the terminal amino group of eighth amino acid in peptide moiety. The C-terminal carboxylic group of the last amino acid forms a lactone with the hydroxyl of Thr3.
Adhesion is the first stage of biofilm formation and the best moment for the action of antiadhesive and anti-biofilm compounds. Adsorption of biosurfactants to a surface e.g. glass, polystyrene, silicone modifies its hydrophobicity, interfering with the microbial adhesion and desorption processes. In this study the role and applications of pseudofactin II as a antiadhesive compound has been investigated from medicinal and therapeutic perspectives.
Pseudofactin II lowered the adhesion to three types of surfaces (glass, polystyrene and silicone) of bacterial strains of five species: Escherichia coli, Enterococcus faecalis, Enterococcus hirae, Staphylococcus epidermidis, Proteus mirabilis and two Candida albicans strains. Pretreatment of a polystyrene surface with 0.5 mg/ml pseudofactin II inhibited bacterial adhesion by 36-90% and that of C. albicans by 92-99%. The same concentration of pseudofactin II dislodged 26-70% of preexisting biofilms grown on previously untreated surfaces. Pseudofactin II also caused a marked inhibition of the initial adhesion of E. faecalis, E. coli, E. hirae and C. albicans strains to silicone urethral catheters. The highest concentration tested (0.5 mg/ml) caused a total growth inhibition of S. epidermidis, partial (18-37%) inhibition of other bacteria and 8-9% inhibition of C. albicans growth.
Pseudofactin II showed antiadhesive activity against several pathogenic microorganisms which are potential biofilm formers on catheters, implants and internal prostheses. Up to 99% prevention could be achieved by 0.5 mg/ml pseudofactin II. In addition, pseudofactin II dispersed preformed biofilms. Pseudofactin II can be used as a disinfectant or surface coating agent against microbial colonization of different surfaces, e.g. implants or urethral catheters.
Biosurfactant; Lipopeptide; Adhesion; Biofilm; Uropathogenic microorganismsBackground
Biofilms, which are formed by the majority of microorganisms in natural environments, are structures with low sensitivity to drugs [1]. Many laboratories are synthesizing or isolating new compounds preventing the formation of biofilms or causing their elimination [2,3]. Adhesion is the first stage of biofilm formation and the best moment for the action of antiadhesive and anti-biofilm compounds. Biosurfactants are promising compounds often showing antimicrobial and antiadhesive properties and sometimes penetrating and removing mature biofilms [4]. Microbial surfactants-amphiphilic, surface-active, secondary metabolites of bacteria or fungi ranging from low-molecular-mass glycolipids, sophorolipids, rhamnolipids and lipopeptides, to high-molecular-mass proteins, lipopolysaccharides and lipoproteins [5]-can interact with interfaces and inhibit the adhesion of microorganisms to different surfaces. They are an alternative to synthetic surface-active agents because of their low toxicity and biodegradability [6].
Another mechanism of biosurfactant action is the permeabilization of bacterial cells. The rhamnolipid secreted by Pseudomonas sp. S-17 permeabilized Gram-negative and Gram-positive cells, but a strong inhibition of growth was observed only in the case of Gram-positive bacteria [7]. Biofilm disruption was observed after the addition of rhamnolipids from Pseudomonas aeruginosa [8] and lipopeptide from Bacillus spp. [9]. A particular group of biosurfactants, lipopeptides, can act as antibiotics and also as antiviral [10] and antitumor agents [11]. Surfactin from Bacillus subtilis can interact with the plasma membranes of bacterial and fungal cells leading to their disruption [12]. The effects of biosurfactants on decreased microbial adhesion and detachment from different surfaces can be conveniently utilized in many fields, from medicine to various branches of industry, e.g., antimicrobial or antitumor activities [13,14] and their surface activity and antiadhesive properties can be suitable for preventing microbial colonization of implants or urethral catheters. Microbial surfactants from Lactobacillus fermentum and Lactobacillus acidophilus adsorbed on glass, reduced the number of adhering uropathogenic cells of Enterococcus faecalis by 77% [15]. A surfactant released by Streptococcus thermophilus has been used for fouling control of heat-exchanger plates in pasteurizers as it retards the colonization of other thermophilic strains of Streptococcus responsible for fouling [16].
Within the collection of 132 morphologically distinct bacteria isolated from water and ground provided from the wild environment around the University of Wroclaw scientific base in the Arctic Archipelago of Svalbard [17], Pseudomonas fluorescens BD5 was identified as strain which strongly reduces surface tension of its culture supernatant [18]. We purified and identified the chemical structure of two new P. fluorescens BD5 biosurfactants, pseudofactin I and II [19]. Both compounds are cyclic lipopeptides with a palmitic acid connected to the terminal amino group of an octapeptide. The C-terminal carboxylic group of the last amino acid (Val or Leu) forms a lactone with the hydroxyl of Thr3. The biosurfactant was found to be stable within the range from -20°C to 100°C, had the minimum surface tension (31.5 mN/m) and the critical micelle concentration (72 mg/L) [19]. Emulsification activity and stability of pseudofactin II was greater than that of the synthetic surfactants such as Tween 20 and Triton X-100.
The aim of this paper was to assess how the pseudofactin II influences the adhesion and biofilm formation of microorganisms such as Escherichia coli, E. faecalis, Enterococcus hirae, Staphylococcus epidermidis, Proteus mirabilis, Vibrio ordalii, Vibrio harveyi and Candida albicans found in gastrointestinal and urinary tract. Since the effects of a surfactant may differ depending on both the type of the microorganism and the type of surface it adheres to, we tested its action on the adherence of the above pathogenic microorganisms to three types of surfaces, polystyrene, glass (as standard laboratory surfaces for adhesion tests) and silicone (used in medical application such as urethral catheters).
Microorganisms and culture conditions
P. fluorescens BD5 strain was obtained from freshwater from the Arctic Archipelago of Svalbard [19] and maintained on the mineral salts medium MSM (7 g/L K2HPO4, 2 g/L KH2PO4, 1 g/L (NH4)2SO4, 0.5 g/L sodium citrate 2H2O, and 0.1 g/L MgSO4.7H2O) with 2% D-glucose.
The antimicrobial and antiadhesive properties of pseudofactin II were tested on several pathogenic strains that colonize animals gastrointestinal tract or medical devices. E. coli ATCC 25922, E. coli ATCC 10536, E. coli 17-2 (clinical isolate, Wroclaw Medical University), E. faecalis ATCC 29212, E. faecalis JA/3 (clinical isolate, Wroclaw Medical University), E. hirae ATCC 10541, S. epidermidis KCTC 1917 [20], P. mirabilis ATCC 21100 were grown at 37°C and V. harveyi ATCC 14126, V. ordalii KCCM 41669 were grown at 28°C in LB medium (10 g/L bacto-tryptone, 5 g/L bacto-yeast extract, 10 g/L NaCl). Two fungal strains, C. albicans ATCC 20231 and C. albicans SC5314 [21], were grown in a 6.7 g/L yeast nitrogen base (YNB, pH 5.5), broth (Difco Laboratories) containing 2% D-glucose for adhesion tests. To prevent filamentation of C. albicans, pre-culture was incubated at 28°C, while experiments with biofilms were performed at 37°C. RPMI-1640 medium (Cambrex, Verviers, Belgium) was used for Candida biofilms formation.
Isolation and purification of pseudofactin II
Pseudofactin II produced by P. fluorescens BD5 was obtained by extraction of cell free supernatant by ethyl acetate and evaporation of the extract under vacuum. The crude biosurfactant was separated by RP-HPLC in the same manner as reported earlier [19]. Purified pseudofactin II fraction was dried and stored at -20°C for further studies. Analytical RP-HPLC (data not shown) of purified pseudofactin II showed that its purity was > 99%.
Antimicrobial assays
The antimicrobial activity of isolated pseudofactin II was determined by the microdilution method in 96-well flat-bottomed plastic microplates (Sarstedt, Nümbrecht, Germany). Briefly, 50 μl volumes of sterile double strength LB (for bacterial) or YNB (for yeast) medium were dispensed into the wells of a 96-well microplate. Subsequently, 50 μl volumes of pseudofactin II (0.035 to 0.5 mg/ml) solution in phosphate-buffered saline (PBS) were added to the microplate wells and mixed with the medium. Negative and growth control wells did not contain biosurfactant. All wells (except for negative controls) were inoculated with 2 μl of overnight bacterial or yeast cultures (diluted to OD600 = 0.1) in LB or YNB medium respectively, and the microplates were incubated for 24 h at 37°C or 28°C for bacterial or yeast cultures, respectively. After 24 h of incubation, the optical density at 600 nm of each well was measured using an Asys UVM 340 (Biogenet) microplate reader. The growth inhibition percentages at different pseudofactin II concentrations for each microorganism were calculated as:
where ODT represents the optical density of the well with a given pseudofactin II concentration and ODC is the optical density of the control well (growth without pseudofactin II). Assays were carried out three times in three replicates.
Preadhesion treatment with pseudofactin II
Inhibition of microbial adhesion by pseudofactin II was tested in 96-well plates (Sarstedt, Nümbrecht, Germany). Briefly, the wells of a sterile 96-well flat-bottom plate were filled with 100 μl of 0.035-0.5 mg/ml pseudofactin II dissolved in PBS. The plates were incubated for 2 h at 37°C on a rotary shaker (MixMate, Eppendorf, Hamburg, Germany) at 300 rpm and subsequently washed twice with PBS. Negative control (blank) wells contained pseudofactin II at the highest concentration tested (0.5 mg/ml) while positive control wells contained PBS buffer only. The overnight cultures of microbial strains were centrifuged, washed twice with PBS (pH = 7.4) and re-suspended in PBS to an optical density OD600 = 1.0 for bacterial and OD600 = 0.6 for Candida strains. The highest adhesion without pseudofactin II were observed at these optical densities (data not shown). A 100 μl aliquot of a washed microbial suspension was added and incubated in the wells. After a 2 h incubation at 37°C in a rotary shaker (MixMate, Eppendorf, Hamburg, Germany) at 300 rpm nonadherent cells were removed by three washes with PBS. Then the plates were stained with 0.1% crystal-violet for 5 min and again washed three times with PBS. The adherent microorganisms were permeabilized and the dye was resolubilized with 150 μl of isopropanol-0.04 N HCl and 50 μl of 0.25% SDS per well. Crystal violet optical density readings of each well were taken at 590 nm on the Asys UVM 340 (Biogenet) microplate. Pseudofactin II did not affect the absorption of negative control (crystal violet in blank wells). The microbial adhesion inhibition was calculated as growth inhibition. Assays were carried out three times in three replicates.
Postadhesion treatment with pseudofactin II
The 96-well flat-bottomed plates were incubated for 2 h on a rotary shaker (MixMate, Eppendorf, Hamburg, Germany) at 300 rpm with 100 μl of bacterial suspension (OD600 = 1.0) and Candida suspension (OD600 = 0.6) in PBS at 37°C. Unattached microbial cells were removed by washing the wells three times with PBS. Next, 100 μl of 0.035-0.5 mg/ml pseudofactin II was added to each well and incubated at 37°C for 2 h on a rotary shaker (MixMate, Eppendorf, Hamburg, Germany) at 300 rpm. Control wells contained only PBS. The plates were washed three times, adherent cells were fixed with 100 μl of 0.1% crystal violet for 5 min and again washed three times with PBS. The adherent microorganisms were permeabilized and the dye was resolubilized with 150 μl of isopropanol-0.04 N HCl and 50 μl of 0.25% SDS per well. The crystal violet optical density of each well was measured at 590 nm using the microplate reader. Assays were carried out three times in three replicates. The microbial adhesion dislodging percentages at different pseudofactin II concentrations for each microorganism were calculated as:
where ODT represents the optical density of the well with a given pseudofactin II concentration and ODC the optical density of the control well (without pseudofactin II). Assays were carried out three times in three replicates.
Confocal laser scanning microscopy
Confocal laser scanning microscopy (CLSM) was used for visualizing the formation of bacterial and Candida biofilms in the absence or presence of pseudofactin II (final concentration 0.25 mg/ml) in the culture medium. Bacterial and yeast biofilms were formed on Thermanox plastic coverslips (Nalgen Nunc International Co., Rochester, NY), glass microscopic coverslips (Menzel-Glaser, Germany) and segments of silicone urethral catheters (Unomedical, Denmark) placed in wells of 24-well plates (Nalgen Nunc International Co., Rochester, NY) containing LB medium for bacteria and RPMI-1640 medium for yeast. Inocula were prepared as follows: 24 h old overnight cultures were harvested and re-suspended at normalized dilutions (OD600 = 0.01). Five hundred microliters inocula were injected into the wells with the coverslips and incubated for 24 h at 37°C. After this time, the coverslips were washed with PBS for 15 min. Then, the bacterial biofilms were stained for 30 min at 37°C with 1 ml of 0.6% Live/Dead BacLight viability stain (Molecular Probes, Eugene, OR) dissolved in PBS, and PBS-containing concanavalin A-Alexa Fluor 488 (Molecular Probes, Eugene, OR) conjugate (0.025 mg/ml) for Candida biofilms. The stained biofilms were visualized by CLSM with an Olympus FluoView 500 (Olympus Optical Co. Ltd., Japan) microscope. The CLSM used an argon ion laser at 480-490 nm for excitation and a 500-635 nm band pass filter for emission. CLSM images were processed by Olympus FluoView 500 software. Assays were carried out two times. Representative images are presented on Figure 1.
Figure 1. Confocal scanning laser microscopy images of biofilm formation on polystyrene, glass microscopic coverslips and cut fragment of silicone urethral catheters by different bacterial strains: ((A, I, R) Escherichia coli ATCC 25922, (B, J, S) Enterococcus faecalis ATCC 29212, (C, K, T) Enterococcus hirae ATCC 10541, (D, L, U) Candida albicans SC5314) and biofilm inhibition after incubation with pseudofactin II (0.25 mg/ml) in the culture medium: (E, M, W) Escherichia coli ATCC 25922, (F, N, X) Enterococcus faecalis ATCC 29212, (G, O, Y) Enterococcus hirae ATCC 10541, (H, P, Z) Candida albicans mμSC5314). Scale bars: 50 μl.
Biofilm formation in urethral catheters
The uropathogenic strains E. coli, E. faecalis, E. hirae and C. albicans were used in these tests. Ten microliter volumes of overnight cultures of E. coli ATCC 25922, E. faecalis ATCC 29212, E. hirae ATCC 10541 were added into 1000 μl of fresh LB medium, and the same volume of C. albicans SC5314 was added into 1000 μl of fresh RPMI-1640 medium. To the medium was added 1000 μl pseudofactin II (final concentration 0.25 mg/ml) solution in LB medium (for bacterial) and RPMI-1640 medium for C. albicans and 4 cm long segments of sterile silicone urethral catheters (Unomedical, Denmark). The catheters were incubated at 37°C overnight. The cultures were removed and the catheters were washed with distilled water. After washing, 3000 μl of crystal violet (0.1%) was added to the catheters for 20 min. The stained biofilms were rinsed three times with distilled water and allowed to dry at room temperature for 15 min before examination. In a parallel experiment the catheters were pretreated with pseudofactin II by being placed in a tube with 2000 μl of 0.25 mg/ml pseudofactin II dissolved in PBS, incubated for 2 h at 37°C and subsequently washed twice with PBS. Then the experiment was carried out as in the case of adding pseudofactin II into the growth medium. Assays were carried out two times. Representative images are presented on Figure 2. This experiment was carried out under dynamic conditions using a peristaltic pump, where the flow of culture with or without pseudofactin II trough urethral catheters was 50 ml/h.
Figure 2. Pseudofactin II inhibits biofilm formation on silicone urethral catheters. The organisms were grown overnight at 37°C in a test-tube with sterile urethral catheters containing medium (A) with and without 0.25 mg/ml pseudofactin II and (B) where the urethral catheters was pre-incubated with biosurfactant at concentration 0.25 mg/ml as described in the text. Biofilms were visualized by staining with crystal violet.
Results and discussion
Antimicrobial activity of pseudofactin II
Lipopeptides have typical amphiphilic structure of a surfactant, where the hydrophobic moiety is a hydroxyl or α-alkyl-β-hydroxy fatty acid (e.g. 3-OH-C14, 3-OH-C15 and 3-OH-C10 fatty acids) and the hydrophilic moiety is a short chain or a cyclic peptide [22,23]. Instead, the hydrophobic moiety of pseudofactin II contains palmitic acid, which is a saturated fatty acid having no hydroxyl group. Rhodofactin, another lipopeptide with palmitic acid, has been described by Peng et al. [24]; however, contrary to pseudofactin II it has a short peptide chain which does not form lactone ring. Its antimicrobial activity has not yet been described.
The antimicrobial activity of pseudofactin II isolated from P. fluorescens BD5 was evaluated at concentrations from 0.035 to 0.5 mg/ml (Table 1). At 0.5 mg/ml the agent caused a total growth inhibition of S. epidermidis KCTC 1917 and considerable growth inhibition of P. mirabilis ATCC 21100 (37%), E. coli ATCC 10536 and E. coli 17-2 (32%), E. hirae ATCC 10541 (28%).
Table 1. Growth inhibition obtained with the pseudofactin II isolated from P.fluorescens BD5 at different concentrations (mg/ml). Values ± confidence interval, n = 9
In contrast to surfactin or iturin, produced by B. subtilis [25,26], lichenysin from Bacillus licheniformis [27] or polymyxin B and E from Bacillus polymyxa [28], pseudofactin II showed much weaker dose dependent antimicrobial activity against most strains tested in this work (Table 1). Only for two Vibrio strains pseudofactin II completely inhibited the growth in the lowest tested concentration, thus they were not used in further experiments (data not shown). This may be due to its unique chemical structure different from any currently known lipopeptides, which features a hydrophobic alkyl chain without a hydroxyl moiety attached to cyclic peptide.
Antiadhesive and cell-dislodging activity of pseudofactin II on polystyrene surfaces
Adhesion of pathogenic microorganisms to solid surfaces or to infection sites has been found to be inhibited by biosurfactants capable of modifying the physico-chemical properties of the surface to reduce adhesion and biofilm formation on a given biomaterial.
Pseudofactin II was found to possess antiadhesive activity against all tested microorganisms. The pretreatment of polystyrene surfaces with pseudofactin II significantly decreased the adhesion of all bacteria and yeast, and this antiadhesive effect was concentration-dependent (Table 2). The highest reduction of adhesion (80-99%) was observed for C. albicans SC 5314, C. albicans ATCC 20231, P. mirabilis ATCC 21100 and E. coli ATCC 10536. The dislodging effect of pseudofactin II on preformed biofilms on untreated surfaces was lower than the preventive effect of pretreatment and was in the range of 26-70% for 0.5 mg/ml pseudofactin II (Table 3).
Table 2. Microbial adhesion inhibition in the microtiter plate by purified pseudofactin II.
Table 3. Activity of cell dislodging in the microtiter plate by pseudofactin II.
The adhesion of pathogenic bacteria to polystyrene surfaces was inhibited by two lipopeptide biosurfactants produced by B. subtilis and B. licheniformis [9], and adhesion of Listeria monocytogenes to polystyrene microplates was reduced by 84% on pretreating the surface with surfactin (1 mg/ml), and by 82% when it was treated with purified rhamnolipid (7.5 mg/ml) [29]. Gudina et al. [30] characterized the anti-adhesive activity of biosurfactants against several microorganisms including Gram-positive and Gram-negative bacteria. This biosurfactant at concentration 25 mg/ml showed high anti-adhesive activity against Staphylococcus aureus (72.0%), S. epidermidis (62.1%), Streptococcus agalactiae (60.0%) and low anti-adhesive activity against P. aeruginosa (16.5%) and E. coli (11.5%).
Coating with pseudofactin II was effective above critical micelle concentration (0.072 mg/ml) [19]. Our results suggest that when the surface is covered by pseudofactin II micelles attached to polystyrene by van der Waals forces, the adhesion is inhibited more strongly than it is with monomers.
Pseudofactin II reduces biofilm formation on polystyrene, glass and silicone
Biofilms are defined as microorganisms attached to a diverse range of biotic and abiotic surfaces and proliferating on them. The human body and medical devices or implants including: urinary catheters, voice prostheses, orthopedic implants, ocular prostheses and contact lenses are exposed to adhesion and biofilm formation by many opportunistic microorganisms. Thus we have tested the influence of pseudofactin II on biofilm formation on different materials.
The activity of pseudofactin II against biofilm formation was visualized by confocal laser scanning microscopy (Figure 1). The biofilm growth of E. coli, E. faecalis, E. hirae and C. albicans on polystyrene, glass and silicone from urethral catheters is shown in Figures 1A-D, 1I-L and 1R-U, respectively. The biosurfactant inhibited biofilm formation at the concentration 0.25 mg/ml on polystyrene, glass and silicone surfaces (Figures 1E-H, 1M-P and 1W-Z). E. faecalis ATCC 29212 adhesion to all tested surfaces is less intensive than others strains (Figures 1B, J, S). In fact, the adhesion of this strain to 96 wells plate was between 2 to 4-fold weaker than others tested bacterial strains (data not shown). This effect may be due to small amount of adhesion proteins on E. faecalis ATCC 29212 strain. Pseudofactin in the tested concentration inhibited mixed biofilm formation (data not shown), similarly as for the single strain (Figure 1). In our experiments all the tested Gram-negative and Gram-positive bacteria showed decrease of adhesion. The results of the present study indicate that pseudofactin II have potential to be used for efficient removal and inhibition of biofilms for pathogenic microorganisms.
Rivardo et al. [9] demonstrated that biosurfactants obtained from Bacillus spp. were able to inhibit biofilm formation for two pathogenic strains E. coli at 97% and S. aureus at 90%, respectively. Irie et al. [31] demonstrated that rhamnolipids produced by P. aeruginosa were able to disperse biofilm for Bordetella bronchiseptica.
Pseudofactin II prevents biofilm formation in urethral catheters
To test biofilm formation on medical device, silicone urethral catheters, 4 cm segments of the catheters were incubated with E. coli ATCC 25922, E. faecalis ATCC 29212, E. hirae ATCC 10541 and C. albicans SC 5314. E. coli, E. faecalis and E. hirae formed biofilms mainly at the air-liquid interface, while the biofilm formed by C. albicans was dispersed along the whole growth surface (Figure 2). Even though the pseudofactin II present in the growth medium (Figure 2A), was at the concentration of 0.25 mg/ml which did not significantly affect the growth of the tested microbial cultures, biofilm formation was nearly completely prevented. The pretreatment of silicone urethral catheters with pseudofactin II prior to inoculation with medium was just as effective as including the biosurfactant in the growth medium (Figure 2B). We observed the similar effect in dynamic conditions for urethral catheters using a flow of 50 ml/h (data not shown).
Earlier reports noted an inhibition of biofilms formed by several microorganisms, e.g. Salmonella typhimurium, S. enterica, E. coli and P. mirabilis on vinyl urethral catheters by a surfactin produced by B. subtilis [32].
Our results show that pseudofactin II is promising compound for inhibition and disruption of biofilms and has potential applications in medicine.
The biosurfactant pseudofactin II, produced by P. fluorescens BD5 strain and purified by HPLC, showed antiadhesive activity against several pathogenic microorganisms, such as E. coli, E. faecalis, E. hirae, S. epidermidis, P. mirabilis and C. albicans, which are potential biofilm formers on catheters, implants and internal prostheses. Up to 99% prevention of C. albicans SC 5314 adhesion could be achieved by 0.5 mg/ml pseudofactin II. Confocal laser scanning microscopy confirmed the action of pseudofactin II as an inhibitor of biofilm formation. In addition, pseudofactin II dispersed preformed biofilms. Due to its surface tension properties and lack of hemolytic activity (data not shown), pseudofactin II can be used as a surface coating agent against microbial colonization of different surfaces, e.g. implants or urethral catheters.
Authors' contributions
TJ carried out experiments, ML participated in the design of the study, data analysis, coordination and helped to draft the manuscript, AK conceived the experiments and draft the manuscript. All authors read and approved the final manuscript.
This work was financial supported by grants from the Polish National Centre for Science N N302 640940, Polish National Centre for Research and Development KB/48/13639/IT1-B/U/08 and grant EU POIG.01.01.02-00-016/2008.
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A great agriturismo, superb location at the junction of Lazio, Toscana and Umbria, near a charming, undisturbed old italian village. Superb remodeling of an old building. Very kind hosts; possibility of having a very nice meal prepared with local products. Beautiful quiet settings and large swimming-pool.
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- Also Known As:
- Agriturismo Il Sentiero Italy/Acquapendente