text:
A middle-aged man has sacrificed living a full independent life to care for the mother he is devoted to.
The Rome of yesteryear casts shadows over Gianni Di Gregorio's charming debut, in which he also stars as a sixtysomething whose social life has been severely curtailed by his devotion to his 93 year-old mother. His dedication becomes positively heroic when he welcomes a trio of dotty dears to spend the midsummer holiday in their snug Trastevere apartment.
Making exquisite use of the musty rooms that are his mother's entire world, this semi-improvised saga is atmospherically photographed and exquisitely designed. But it's the gently farcical interaction and cutting dialogue of the non-professional cast that makes this so enticing.
The semi-improvised performances and gently nostalgic tone makes this endearing and captivating.

subset: commoncrawl


text:
This topic contains 1 reply, has 1 voice, and was last updated by James Keeling 8 years, 7 months ago.
SynapticJam on Toast – hhhmmm… tastes like chicken (aka PUVJK) #2 in crack hits (Special? Ain't I just)
S.H.I.T. Award Recipient and all-around uberjunkie!
When do we get a sales update? Is the FDO in Amazon top list?
You must be logged in to reply to this topic.


text:
Editors' Note: The "Download Now" link directs you to the iTunes App Store, where you must continue the download process. You must have iTunes installed in order to open the link, and you must have an active iTunes account to download the application. This download may not be available in some countries.
Sponsored Products
|Business Software
|Business Applications
Publisher's Description+
From Techpro Security Products: Access and control Techpro DVR cameras/IP Cameras directly from iPhone, iPad, iTouch. Watch live surveillance video streams - fast and simple.
read more +


text:
Minka Kelly Covers Self Magazine: "I Don't Want to Diet For the Rest of My Life."
A Healthy Bite: Minka Kelly
"Exercise is like meditation for me. And I'm giving myself that time . . . I cannot live without it now. When you have such a good workout, it clears everything up mentally, physically, and you just have a better day."
— Minka Kelly on the importance that exercise plays in her life. The actress is on the March cover of Self and shares with the magazine that working out with trainer Gunnar Peterson has been life changing. Minka also keeps things in perspective when it comes to food: she sticks to a healthy diet but also lets herself indulge. Deprivation diet is not in her vocabulary!


text:
Penn Hills 'birds and bees' program focuses strictly on nature
A pair of binoculars, a bird identification guide and a healthy respect for stinging insects will go a long way this weekend at the Penn Forest Natural Burial Park.
The "green" cemetery on Colorado Street will host a "birds and bees" talk on Saturday at 9 a.m., and while that phrase traditionally applies' to parents broaching the reproductive discussion with their children, on July 12 it will be solely focused on nature.
Chris Kubiak of the Audubon Society of Western Pennsylvania will lead participants on a bird walk, and Penn Forest's resident beekeeper Kristin Hauman will demonstrate the inner workings of a beehive.
For Kubiak, the Penn Forest grounds represent a wide variety of possibility when it comes to birdwatching.
"That's a place, particularly in an urban area like Penn Hills, that in a lot of ways is a good spot for finding birds," Kubiak said.
From resident species that remain in the area year-round — cardinals, chickadees, titmice and some of the woodpecker species — to migratory visitors such as the Baltimore oriole and scarlet tanager, the park's 32 acres should be home to an abundance of birds, Kubiak said.
"We'll likely see some of the summer migrants, along with resident species and probably a few types of local hawks," he said.
Data-collection website eBird.org, which allows users to tag geographical regions with bird counts, has entries with watchers spotting 52 species of bird in the nearby Dark Hollow woods, and 76 species along the Log House Trail in Monroeville's Boyce Park, just a few miles away.
Kubiak said western Pennsylvania is among the Audubon's top five chapters in the country for bird-watching.
"With all the habitat diversity, there are a lot of bird species here," he said.
Joining the local birds in flight are bees, which gather in two hives kept on the Penn Forest property.
Hauman, who will give a presentation on how the hive operates, works with Burgh Bees, a resource for local residents interested in forming an apiary, or bee colony.
"We teach all the classes in the area," said Burgh Bees President Steven Repasky. "We try to hook people up with experienced beekeepers to help them get a good start."
Repasky said he recommends beginners first sign up for a class.
"This isn't like going to get a dog from the animal shelter," he said. "It's such a steep learning curve, you really do need assistance. There's a lot of science involved and a lot of art in terms of the management."
Burgh Bees advises prospective apiarists to start with two colonies, to compare their progress. Between bees and equipment, the costs can run between $600 and $1,000, Repasky said.
The classes, which are typically offered in fall and early winter, are designed to help first-timers become more comfortable with the biology of honeybees.
"With the societal fear of stinging insects, opening up that hive and looking at about 30,000 bees can be intimidating," Repasky said.
The schedule for "Birds and Bees," will include Kubiak's bird walk at 9 a.m., Hauman's apiary presentation at 10:30 a.m., and a question-and-answer session on "green" burial with cemetery manager Pete McQuillin at 11 a.m.
For more information, call 412-927-0103 or visit Pennforestcemetery.com.
Patrick Varine is a staff writer for Trib Total Media. He can be reached at 412-320-7845 or email@example.com.


text: Research 2012 Transition Management Survey Transition management is an industry that, while often thought of as staid, over the past year has been anything but. Published in PLANSPONSOR July 2012 Methodology Magazine Article Archive Well-known firms have recently experienced problems with their transition management services —some aberrations and some systemic and extremely serious. This survey indicates that all had an impact on how plan sponsors, even those that do not use transition managers, view the industry. Of the 119 U.S. plan sponsors that responded to the survey, none reported having complete trust in the industry as a whole, but roughly half admitted more transitions than the year before. Though numerous changes to the survey structure complicate comparisons to the results of previous years, the table above breaks down transitions conducted in 2011, offering a snapshot of the transition management industry as it stands today. Navigate Research BUYER'S GUIDE TRANSITION MANAGEMENT BUYER'S GUIDE PROVIDERSAbel Noser CorpBarclaysBNY MellonBrockhouse & Cooper Inc.CAPISCitiConvergEx Group LLCCredit SuisseGoldman Sachs International LtdJ.P. MorganNorthern TrustRussell InvestmentsState Street Bank and Trust Company Methodologyback to top Responses from 119 U.S. plan sponsors were accepted for the survey, over a period of three weeks ending May 22. PLANSPONSOR and aiCIO would like to extend a special thank you to all those who submitted responses for the survey, as well as those transition managers, plan sponsors and consultants who helped the editorial and survey teams construct the survey. For more information, contact Quinn Keeler at 203-595-3270 or firstname.lastname@example.com. Archive back to top TRANSITION MANAGEMENT SURVEY While there are not as many providers of portfolio transition management services as there are, say, defined contribution providers, this is an extremely competitive field and the stakes are high. The survey serves two purposes: first, to take the pulse of pensions and other institutional investors which use transition management; and second, to highlight all the providers in the field. 2014 Transition Management Survey2012 Transition Management Survey2010 Transition Management Survey2009 Transition Management Survey


text:
Hello again friends, fans and supporters! Jack and I are so touched by the outpouring of virtual support for our team, and so hope we are able to continue on in the race so we can provide you with inside information behind the scenes of The Amazing Race 4!
We started off this leg of the race in Venice, Italy. At the start of the show, during our mandatory pit stop, you saw us all eating together and chatting. The pit stop is a great opportunity to sit back and take in the other teams' strengths and weaknesses and to plan whether or not we should try to ally ourselves with any of them. What CBS didnt show was that, after the meal, a few of us took a little trip over to St. Marks Square (the Piazza St. Marco, as its called in Venice. In fact, it is the only square called a Piazza; the others are called campo. Hey, some day that might come in handy in a heated game of Trivial Pursuit.)
St. Marks Square is undoubtedly the most photographed and famous plaza in Europe. Its the heart of the city and covered with tourists, sidewalk cafes and boutiques. Its also supposedly one of the most romantic spots in the world or at least thats what they infer in that ridiculous A Diamond Is Forever commercial. You know the one a loving couple strolling through the square and the man shouting I love this woman I love her, I love her, I love her. She gives the embarrassed look and tries to hush him. The she gets the three stone diamond anniversary ring, and whispers I love this man I love him, I love him, I love him. Sure glad we have no materialistic, gold-digging stereotyping going on there.
Now myself I think its lovely, but Im not so sure I think theres anything romantic about the famous pigeons which are an integral part of the setting. In fact, thousands of pigeons crapping all over the place in the relative vicinity of where I might choose to eat is pretty much just plain disgusting to me. But I digress again. We had a very pleasant evening with several of the teams, but unfortunately at this point, we still had mixed feelings about the sincerity of some of them and keep second-guessing our initial impressions of many. As our fun came to an end, we retreated to the hotel to rest up before our departure early the next morning still confused as ever, but excited for the next leg.
We departed early in the morning right on the tails of ReichenChip. Okay, maybe poor choice of words there (well, for me, anyway I'm sure Jack would have been more than pleased with the literal interpretation of that ), but we were right behind them. We learned that we needed to travel 300 miles to Vienna, Austria by train. Later on we heard of Millies asthma attack and were happy to hear it was short-lived. Scary stuff. We were already out of the station on the first train with Kelly and Jon, Tian and Jaree, ReichenChip, and David and Jeff. To be honest with you, I have a much harder time keeping David and Jeffs names straight than I did last year with Derek and Drew. All I know is one of them is a lot cuter (imo) the one with less facial hair (Jack says that one is Jeff). We were happy to stay far from the Tian and Jaree drama, as they dont seem to be a team that wed want to really even get to know better.
Upon arrival in Vienna, we walked the streets with the other teams and quickly found the Vienna sewers, only to realize that they were closed for the night. Oh well, what can you do? Jack and I followed suit with the other teams and found nearby hotels to get some rest. We awoke bright and early and found all the teams were together again for the trek through the underground. How fitting was this - a bunch of rats scurrying through the sewer with the local rodents.
Next we were to make our way to Schonbrunn Palace where our next clue was located by claiming a ticket to a fiacre, a horse-drawn carriage that would take us the 6 miles to the clue. Jack and I were the first team to arrive and claimed both the ticket and the carriage and left before anyone else even got there. Sounds like all we missed was a bunch of people who didnt read the directions accurately and then a physical race and fight between some of the teams. The more this leg continued, the more I thought we should stay far away from all the other teams.
Arriving at the Palace and detour, we immediately chose the Mozart option of lugging around the bass fiddle. A short while later, we saw Tian and Jaree and Monica and Sheree parading through the streets yelling girl power. Maybe they are thinking of creating their own Spice Girl band during their time in Sequesterville.
As we searched for Mozarts house, we got really hungry. So we stopped at a weinerschnitzel stand, one of Viennas best-known foods. Weinerschnitzel is a deep-fried breaded piece of pork or veal. Its delicious, but boy is it a guilty pleasure. Its important to keep your energy up though, so we scarfed that down as we continued to run around the city. Viennas hottest new girl band was able to find Mozarts house before us, and had the clue to the next destination in hand before we did. As we were leaving, we spotted Jon and Al and directed them to the house as well, thinking that of all the teams, we liked them the best by far. Perhaps they would be the team for us?
Arriving at the next clue after we opened it to find the roadblock challenge. The roadblock required one of us to take a 460-foot bungee jump off the Donauturm, the tallest tower-jump in Europe.
Oh Jack, I think youre going to have to do this one. Im not feeling so well from the weinerschnitzel we had.
Are you out of your mind, woman? Jack replied. It was all I could do to get through the rescue detour back in Italy! Theres no way in hell that I'm gonna do this one, too! You know how I feel about heights!
Okay, but if I lose the weinerschnitzel, youre giving me your shirt to wear. I figured that was as good as the negotiation was going to get at this point.
The jump was actually quite fun after I was on the ground again and the fear of dying was no longer an issue. Jack egging me on certainly helped, too. As I was walking out on the platform getting ready to jump, Jack screamed from the ground, "Hey, Jenn! If you get nervous, just imagine that some cute Navy SEAL is down here waiting to catch you in his open arms!" But there was no time to waste, as we had to travel 185 miles by train to Gmunden to the Seescholoss Orth as the last team to arrive would be philiminated.
On the ride, Jack and I discussed that we still had not come to any conclusions about forming any alliances with another team. Last year by Episode 4, Tracey and I were firmly entrenched in the brothers alliance so I knew that if we were going to do it, we should probably be thinking about it soon. But we dont want to make the wrong decision so I think were going to wait until the next leg. We arrived in 6th place this week right after Millie and Chuck and right before Jon and Al. Hopefully our current position will allow us to get to know both of those teams a little better.
Well, that's it for now! We'll catch you next week!
To contact the author of this article (BravoFan, aka Jenn), please send an e-mail to firstname.lastname@example.com
The Amazing Race Virtual Journal is for entertainment purposes only and is not an account of actual events.


text:
Holiday Shoppers Beware
Legislative column by Assemblyman Brian M. Kolb (R,C,I-Canandaigua)
December 12, 2008
The holiday season is once again upon us, which means it's that time of year to spend with loved ones. It is also a time to go holiday shopping – often the most stressful part of the season. In addition to the worries of acquiring the perfect gift for everyone on your list, there are also people out there who take advantage of this time of year to attempt to part you from your money through fraudulent scams or out-and-out theft, leaving you with an empty wallet. It is impetrative that everyone is cautious of such con artists. With a few simple steps, it is easy to ensure that your hard-earned money does not end up in the wallet of these holiday "Grinches."
- Limit the amount of cash you keep on hand.
- Keep wallets and money in your front pockets or an inside coat pocket to reduce the chance of being pickpocketed.
- If you suspect your credit card was lost or stolen, call your issuer immediately.
- Be cautious of "all sales are final" purchases.
- If you are forced to shop at night, bring a friend or family member.
- Always save your receipts.


text:
Beatdown 12/02/11
Beatdown 12/02/11 - Mixed Martial Arts (MMA) Event on December 2, 2011 in Saginaw, Michigan USA
Donofro MMA & The Impact Fight League in Association with The Dow Event Center presents: "BeatDOWn". Another installment of the best Mixed Martial Arts in the mid-west. Join us on Friday, December 2, 2011 at 7 pm as Donofrio MMA and The Impact Fight League bring the most talented combatants from Michigan and beyond to engage in the clash of "BeatDOWn"


text:
What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
I'm unable to import forms from FormStackAsked by daberistic on June 16, 2015 at 04:04 PM
I have tried a few times with different forms, none of them can be imported into Jotform using your Import Form function. Please help!
Another example:
Unfortunately, we don't have an import tool that would allow you to automatically import forms from FormStack.
What I can suggest is to check our templates section and our theme store where you can find ready-to-use forms that you can use as a startup point. In case you don't want to recreate the forms from scratch.


text:
Formed under the sea more than 100 million years ago, emerged as a result of large tectonic movements, the massive limestone Ankarana suffered for 3 million years unrelenting erosion. This mammoth task on its surface gives the strange appearance of sharp ridges called "Tsingy" (walk on tiptoe in Malagasy).
The Tsingy of Ankarana are formed by an extensive network of underground rivers that have created real natural cathedrals where, amidst stalactites and stalagmites, live colonies of bats.
This massive unusual not just a simple mineral world as the flora and fauna that thrive there are among the most attractive in a country which is considered one of the most important spots of biodiversity in the world. Amphibians and reptiles, birds and lemurs are numerous and easy to observe.
Natural lakes and points of view to bear with the Mozambique Channel in the foreground, this incomparable massive jagged surely represent one of the most lasting memories that you can return to Madagascar.
Subscribe to our newsletter :


text:
MADISON, N.J., March 21 /PRNewswire-FirstCall/ -- Wyeth Pharmaceuticals, the pharmaceutical division of Wyeth , announced today that three vaccine scientists, Drs. Ron Eby, Velupillai Puvanesarajah and Dace Madore, have been awarded the prestigious Discoverers Award from the Pharmaceutical Research and Manufacturers of America (PhRMA) for the development of Prevnar(R), Pneumococcal 7-valent Conjugate Vaccine (Diphtheria CRM197 Protein), the first conjugate vaccine to help prevent invasive pneumococcal disease caused by Streptococcus pneumoniae in infants and children up to the age of two. The scientists accepted the award at PhRMA's Annual Meeting in Washington, D.C.
Each year, PhRMA presents the Discoverers Award to scientists whose research and development efforts have greatly benefited humankind and whose dedication and interest in improving the quality of life exemplify the best in the research-based pharmaceutical industry. Drs. Eby, Puvanesarajah and Madore were selected this year for their pioneering work in the discovery and development of Prevnar(R).
"The Discoverers Award is one of the most prestigious awards within the pharmaceutical industry. Prevnar(R), a dramatic example of the power of prevention that can be achieved through vaccination, is certainly worthy of this honor," says Robert R. Ruffolo, Jr., Ph.D., President, Wyeth Research and Senior Vice President, Wyeth. "While it is recognized that many scientists contributed ideas and skills to the Prevnar(R) program, the efforts of these three scientists stood out and significantly impacted the successful development of Prevnar(R)."
Streptococcus pneumoniae, also known as pneumococcus, is responsible for more than one million deaths worldwide in children under the age of five, especially in developing countries. In the U.S., prior to the introduction of Prevnar(R), pneumococcus was a leading cause of childhood meningitis, bacteremia, pneumonia and acute otitis media. In addition, the most prevalent strains were becoming antibiotic resistant.
Since the introduction of Prevnar(R) into routine use in infants in 2000, more than 65 million doses have been distributed in the U.S., and the public health impact has been dramatic. One year after vaccine licensure, core surveillance sites of the Centers for Disease Control and Prevention (CDC) noted a 78 percent reduction in cases of invasive pneumococcal disease caused by vaccine serotypes in children younger than two years. In 1998, the rate of invasive pneumococcal disease in children younger than two years of age was estimated to be 188 cases per 100,000 population. Data from 2002 indicate that rates of invasive disease have declined 70 percent to 80 percent in this age group.
The scientists being honored developed a vaccine that is actually seven different vaccines combined to prevent the majority of invasive pneumococcal disease in children. Each component is made using a novel, patented conjugation technology that allows the vaccine to work in infants as young as two months of age. It is the only vaccine of its kind in the world. In 2003 these scientists were honored with the Heroes of Chemistry award from the American Chemical Society, and Prevnar(R) has also received the prestigious Prix Galien award in the UK, Switzerland and France.
About Prevnar(R)
Prevnar(R) is indicated for active immunization of infants and toddlers against invasive disease caused by S. pneumoniae due to the capsular serotypes included in the vaccine. The routine schedule is 2, 4, 6, and 12 to 15 months of age.
Risks are associated with all vaccines, including Prevnar(R). Hypersensitivity to any vaccine component, including diphtheria toxoid, is a contraindication to its use. Prevnar(R) does not provide 100 percent protection of children vaccinated. Please see Prescribing Information found at http://www.prevnar.com/.
About Wyeth Pharmaceuticals
Wyeth Pharmaceuticals, a division of Wyeth , has leading products in the areas of women's health care, cardiovascular disease, central nervous system, inflammation, hemophilia, oncology and vaccines. Wyeth is one of the world's largest research-driven pharmaceutical and health care products companies. It is a leader in the discovery, development, manufacturing, and marketing of pharmaceuticals, vaccines, biotechnology products and nonprescription medicines that improve the quality of life for people worldwide. The Company's major divisions include Wyeth Pharmaceuticals, Wyeth Consumer Healthcare and Fort Dodge Animal Health.
CONTACT: Media, Gerald Burr of Wyeth Pharmaceuticals, +1-484-865-5138;Investor Contact, Justin Victoria of Wyeth, +1-973-660-5340
Web site: http://www.wyeth.com/http://www.prevnar.com/


text:
With the 110th Congress back in Washington for its second session, the general aviation community was girding last month to continue its opposition to user fees to fund the FAA.
The House of Representatives has already passed a four-year FAA reauthorization (funding) bill, but the Senate has not. Through much of the fall, three Senate committees with jurisdiction over the measure–Finance; Appropriations; and Commerce, Science and Transportation–bickered over various provisions.
In December, the Appropriations and Finance committees removed one of the hurdles to passage. According to a December 11 letter from the Finance Committee to the Appropriations Committee, Finance agreed to drop a provision that would create a separate account for new revenue dedicated to modernizing the ATC system.
The Finance measure would have made depositing the money into the account mandatory, sending it directly to the FAA to spend and bypassing the appropriations process.
But the Finance Committee is still fighting with the Commerce Committee about user fees. The Senate FAA reauthorization bill (S.1300)–drafted by the Commerce Committee–would require general aviation, in particular business aircraft, to pay a new $25-per-flight surcharge on all jet and most turboprop aircraft operations.
The Finance Committee's draft (S.2345) would raise the jet fuel tax to 36 cents per gallon, with no additional charges for aviation gasoline or commercial fuel taxes. But it does not contain the $25 charge. Without that fee, the Senate FAA reauthorization bill would more closely mirror that of the House.
In an address to an AOPA Pilot Town Meeting in Fort Lauderdale, Fla., last month, Sen. Bill Nelson (D-Fla.) said that in his opinion, "User fees are dead." He said the Senate is working hard to reach final agreement on the issue of FAA funding.
He reminded pilots that he had coauthored an amendment to strip user fees from S.1300. Although it failed by one vote, Nelson said it "signaled that user fees are dead and it signaled that they are going to be out of the final reauthorization bill. When this bill gets to the floor, I don't expect that I'll have to offer the amendment again."
While the debate over the $25 turbine user fee stalled the Senate measure, on December 20 President Bush signed a $14.6 billion FAA appropriations bill for Fiscal Year 2008 as part of the omnibus funding bill for all federal agencies. In essence, it extends the FAA's existing funding and tax structure through the end of this month.
"While it's encouraging to hear Senator Nelson's assessment, in politics it isn't over until the final vote," said AOPA president Phil Boyer. "The airlines haven't given up on user fees and more taxes on general aviation, and you can bet their lobbyists will be buttonholing senators as soon as they're back in Washington."


text:
About the Book
This book highlights Ali's time at the 2010 pageant, during which time she gave up her title and crowned the new Miss South Carolina Teen.
Features & Details
- Category Arts & Photography
Large Square, 12×12 in, 30×30 cm
40 Pages
- Publish Date Dec 11, 2010
- Tags miss south carolina schloarship program, miss south carolina organization, Miss America Organization, Miss America, pageant, pageantry, crowns
See More
Log in or create an account to post a comment.
0 Comments
Delete this comment?
See More


text:
Kevin S. Scholl
7902 Trumpetvine Lane
Mechanicsville, VA 23111
804.789.0539 (home)
804.229.4331 (cell)
Kevin Scholl is a user experience (UX) professional living and working in central Virginia, USA. With over a decade of experience, he specializes in the architecture, design, and development of small- to medium-sized Web sites and application interfaces. He emphasizes clean, attractive solutions with special attention to human usability.
Kevin is also a big motion picture fan, and continues to grow a large collection of DVDs. Among his favorite genres are science fiction and suspense, though he enjoys movies of all kinds.
This site is a journal of sorts, relating various bits of professional and personal information for your reference and enjoyment.
The realm of visual communications is about conveying a message. The most visually-stunning presentation is useless if the person looking at it cannot determine its purpose or realize its information. My mantra plays to those ideals: Be creative but maintain simplicity.
I strive to design and build clean, aesthetically-pleasing interfaces using accepted industry standards. The results emphasize usability and accessibility, as well as ease of maintenance.
The following is an alphabetical listing of companies and organizations for whom I've completed work across various disciplines in recent years.
Select a Web project below to view further details.
In addition to traditional Web-related services, Kevin develops and maintains customizable modules for the jQuery Javascript library, including:
Kevin has also developed a CSS-based dynamic interface framework utilizing a variables file to dynamically build style properties, allowing for easy layout and color scheme changes.
The brick walls are there to give us a chance to show how badly we want something.
The following titles have been added to the library within the past few weeks.
(No new titles added recently...)
The library contains 1051 titles.
Copyright ©2007-2012, Kevin Scholl. All Rights Reserved. Hosted by ipower.


text:
NetWellness is a global, community service providing quality, unbiased health information from our partner university faculty. NetWellness is commercial-free and does not accept advertising.
Tuesday, August 22, 2017
Breast Cancer
Stereotactic mammotome biopsy
i was called back for this test but because of the fact that i would have to lie on my stomach for 30 to 45 minutes i chose to go to a surgeon.they said i had 3 clusters all attached. i hope its nothing serious but any way i just want them removed they dont belong there.
I generally do recommend that calcifications be biopsied stereotactically. There is much less scarring and often these are benign and do not require surgery. If cancer is diagnosed, then one operation can be performed. If they are excised surgically, then a second operation will be required if cancer is diagnosed.
Doreen M Agnese, MD
Clinical Associate Professor of Surgery and Internal Medicine
College of Medicine
The Ohio State University


text:
Reading Play Worship Guitar Review To Learn Christian Guitar Lessons Fast, Are You Ready?
I would like to introduce the complete review of Play Worship Guitar program to you in a 5-part article as follows:
- What Is Play Worship Guitar?
- What Are The Contents Of The Program?
- What Are The Features Of This Online Guitar Training Course?
- How Much Does It Cost?
- Is It Guaranteed The Program Will Work For You?
What Is Play Worship Guitar?
Play Worship Guitar is an e-guide that teaches people on mastering worship guitar playing techniques and worship songs. As a musician, a 20-year worship song enthusiast, Aaron Anastasi conducted this guidebook to help many people enjoy this wonderful hobby. Actually, this is a customized program which he offers guitar lovers.
To get more information about this program, why don't you keep reading Play Worship Guitar review and discover more features of this system and benefits you are getting from it?
What Are The Contents Of The Program?
Play Worship Guitar provides you with everything you need to learn guitar, and play songs of Christian artists. This course works for people of all ages, including guitar learning beginners, and guitar lessons for adults. The following are the main contents of the program that you are going to learn:
- Learn Power Chords, teaching how to open chords and make it easier to play any song
- Finger Picking Patterns, guiding how to make any song sound beautifully
- Discover Octaves, guiding how to play any song
- Warm-Up Exercises that help you loosen up your muscles
- New Chord Voicing, revealing how to cruising up and down your guitar neck
- Dynamic Guitar Techniques, helping you transform any chord progression to impressive
- Different Techniques, including dynamic sliding, pull offs, hammer on in order to make songs come alive
- New Chord Voicing, revealing how to cruise up and down your guitar neck
- Dynamic Guitar Techniques, helping you transform any chord progression to impressive
- Different Techniques, including dynamic sliding, pull offs, hammer on in order to make songs come alive
- Guitar Accuracy Exercises, designed to help you develop your guitar skills
- Learn all the parts of the guitar and the function of these parts
- How to hold the guitar, and posture for developing proper fundamentals to play guitar faster
- Dictate The Mood You Convey By the Chord You Play , guiding you on how to play with your real emotions
- Find Any Note On The Guitar, showing the tips and techniques to memorize every note on the guitar
- How to play longer without fatigue
- How to help your solo hand play fast
- Muscle memory exercises to recall chords easily
- Chord changing exercises to help you stop getting stuck
- Dynamic strumming patterns
What Are The Features Of This Online Guitar Training Course?
The following are typical features of this online training course. Let's have an overview if it!
- Complete A to Z guitar learning system
- Learn this course from a 20-year Christian worship leader
- Get my Songwriting Boot camp
- New worship song lessons each month
- Downloadable chord charts
- 2-camera video lessons
- Unlimited access to the support team
- 24/7 access to Play Worship Guitar
- Works on any computer with internet access
- iPhone and iPad compatible to play on any device
How Much Does It Cost?
The Play Worship Guitar review exposes that this course comes with a very low price of just $17.00, but you can get it today for a 21-day trial with $1. You don't listen wrong. Only $1, it's not a joke!
If you don't cancel the purchase after those 21 days, you will still access to great guitar lessons for just $17/month (cancel anytime).
Besides, the author offers you the following bonus items:
- Bonus #1: Online Chord and Scale Generator
- Bonus #2: Guitar Tuner
- Bonus #3: Online Metronome
- Bonus #4: The Play Worship Guitar Songwriting Boot camp
- Bonus #5: The Play Worship Guitar Guide To Leading Worship
- Bonus #6: The Play Worship Guitar Daily Devotional with 365 Days
Is it attractive to you to get started now?
You can check out some feedbacks that were released by customers of the Play Worship Guitar course.
Is It Guaranteed The Program Will Work For You?
Yes! This program comes with a risk-free 100% money back guarantee. For any reason you are not happy with this system, you can ask for a refund within 60 days of your purchase. No questions asked!
Now, it is the time for you to make a decision on purchasing. Are you going to learn this worship guitar training program? If you get any unclear information about it, notice me by leaving your feedback at the end of this post. I also welcome all your comment. Share it with me!


text:
Genital chlamydia infection is the most commonly diagnosed sexually transmitted infection in the UK. C. trachomatis genital infections are usually caused by strains which fall into two pathovars: lymphogranuloma venereum (LGV) and the genitourinary genotypes D–K. Although these genotypes can be discriminated by outer membrane protein gene (ompA) sequencing or multi-locus sequence typing (MLST), neither protocol affords the high-resolution genotyping required for local epidemiology and accurate contact-tracing.
Principal Findings
We evaluated variable number tandem repeat (VNTR) and ompA sequencing (now called multi-locus VNTR analysis and ompA or "MLVA-ompA") to study local epidemiology in Southampton over a period of six months. One hundred and fifty seven endocervical swabs that tested positive for C. trachomatis from both the Southampton genitourinary medicine (GUM) clinic and local GP surgeries were tested by COBAS Taqman 48 (Roche) PCR for the presence of C. trachomatis. Samples tested as positive by the commercial NAATs test were genotyped, where possible, by a MLVA-ompA sequencing technique. Attempts were made to isolate C. trachomatis from all 157 samples in cell culture, and 68 (43%) were successfully recovered by repeatable passage in culture. Of the 157 samples, 93 (i.e. 59%) were fully genotyped by MLVA-ompA. Only one mixed infection (E & D) in a single sample was confirmed. There were two distinct D genotypes for the ompA gene. Most frequent ompA genotypes were D, E and F, comprising 20%, 41% and 16% of the type-able samples respectively. Within all genotypes we detected numerous MLVA sub-types.
Citation: Wang Y, Skilton RJ, Cutcliffe LT, Andrews E, Clarke IN, Marsh P (2011) Evaluation of a High Resolution Genotyping Method for Chlamydia trachomatis Using Routine Clinical Samples. PLoS ONE 6(2): e16971. https://doi.org/10.1371/journal.pone.0016971
Editor: Stefan Bereswill, Charité-University Medicine Berlin, Germany
Received: October 1, 2010; Accepted: January 11, 2011; Published: February 11, 2011
Copyright: © 2011 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the MRC Sexual Health and HIV Research Strategy Committee G0601640 to INC and PM. All views expressed are those of the authors and not necessarily those of the MRC nor Health Departments. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Genital chlamydia infection is the most frequently diagnosed sexually transmitted infection in the UK. The number of infected individuals with chlamydia identified through testing continues to increase annually (http://www.hpa.org.uk/Topics/InfectiousDiseases/InfectionsAZ/STIs/STIsAnnualData/AnnualSTISlideset/). C. trachomatis is traditionally divided into at least 15 'serovars' which are linked to different disease pathologies. As the majority of women, and a significant proportion of men, diagnosed with this infection are asymptomatic, there is a large pool of prevalent undiagnosed infection which provides a reservoir for further spread (http://www.bashh.org/guidelines). The spread of infection is determined by patterns of sex partner selection, the relative risk of infection is associated with particular social networks and the existence of bridge populations [1]. Genital chlamydial infections are caused by two 'pathovars': lymphogranuloma venereum (LGV: L1,L2 and L3 - invasive and disseminating) and infections associated with serovars D to K, which are non-invasive and can cause epididymitis, cervicitis, urethritis, salpingitis and pelvic inflammatory disease [2]. Sequelae following infection can be severe, such as infertility in women (including pelvic inflammatory disease and ectopic pregnancy) (http://www.bashh.org/guidelines).
Standard multi-locus sequence typing (MLST) approaches which target a limited number of relatively stable and non-selected loci such as house-keeping genes [3], [4] have been developed for global surveillance and show a medium-level resolution across all chlamydia strains at the genotype/serovar level. But MLST as performed by Pannekoek et al [3] is insufficiently discriminating to resolve closely related C. trachomatis isolates. An example of the limited resolution of MLST is the grouping of LGV strains (belonging to serovars L1, L2 and L3) to a single cluster of identical MLST-type using the scheme of Pannekoek et al [3], which was developed using a small number of European isolates and the two complete published genomes available at the time [5], [6]. The more recent MLST scheme developed by Dean et al [4] is only able to discriminate L2b (the proctitis-causing variant of L2) from the rest of the LGV group, and does not discriminate between L1, L2 and L3. However, the purpose of MLST schemes in highly homogeneous bacterial populations is to describe population structure and diversity, and not to operate at the level of an ultimate strain typing scheme [7]. There is growing evidence of a small but meaningful variation at the genome level [8], therefore methods of high resolution genotyping are required to reveal more discriminating data. Such data is important for the monitoring of therapy and contact tracing including route-cause analysis, and study of sexual networks.
An "MLST" system has been described [9] that shows some promise for discriminating strains, however this does not follow the rules of MLST (exclusive use of housekeeping genes and small amplicons), therefore this should really be considered as multi-sequence typing (MST). There has been only one attempt at developing rapid, high resolution genotyping [10], which targets short, mutable sequences and thus facilitates detailed resolution of local diversity at high efficiency in short periods of time (the latter incorporating variable number tandem repeat (VNTR) analysis). VNTRs are repetitive sequences of the same nucleotide or motif, and variation in DNA polymerization errors in different strains gives rise to variation in length between strains [10]. Multi-locus VNTR analysis (MLVA) of C. trachomatis VNTR sites and ompA appeared to facilitate a highly discriminatory genotyping survey of C. trachomatis from clinical samples from Aarhus in Denmark [10]. To evaluate this scheme unbiased, the MLVA-ompA approach previously (and incorrectly) called "omp1-VNTR" [10] requires independent analysis on clinical samples in other laboratories. The original Aarhus study [10] which utilizes four PCR targets returned data for all samples collected, whereas most studies are only successful in analyzing a percentage of samples collected, and the scheme has not been assessed independently using routine clinical samples to study epidemiology in a typical host population. This scheme has been compared to the MLST scheme of Pannekoek et al [3] and the MST scheme of Klint et al [9] in a retrospective study of a small number highly selected set of samples which had been cell-culture adapted in Moscow collected in 2005 [11]. Amplification conditions were highly optimized as the chlamydial DNA was prepared from semi-purified elementary bodies: the approach in the Moscow study is therefore not applicable to routine clinical samples.
Our aim was to evaluate independently the MLVA-ompA approach. To facilitate this we used routine clinical swab samples from women in the local C. trachomatis diagnostic laboratory to test the success rate of MLVA-ompA analysis for all four markers. Our selection of samples positive for C. trachomatis was a commercial nucleic acids amplification technology (NAAT) test (COBAS Taqman 48, Roche) which uses highly conserved DNA targets. The fact that only swabs were collected resulted in men being excluded from this survey, because in the case of the COBAS Taqman 48 (Roche) system urine samples can also be processed and this is the preferred sample chosen by men. Therefore the study in Southampton was limited to women. Our approach resulted in a six month survey in which primary swab samples from women paired with DNA extracts with commercial NAATs C. trachomatis positive results were collected, cultured and genotyped. A far greater diversity in MLVA-ompA genotypes in comparison to using ompA alone was found in our sample set including a new genotype (which was a J variant) that could not be isolated in cell culture.
Materials and Methods
Clinical samples
Endocervical swabs collected between February and July 2009 from patients presenting at the GUM clinic in Southampton, and also samples referred from GP surgeries were analyzed for the presence of C. trachomatis using the commercial NAAT incorporating the Tecan DNA extraction system and COBAS Taqman 48 (Roche) real-time PCR protocol in the Health Protection Agency (HPA) Molecular Diagnostics Laboratory, Southampton. Crude DNA extraction, real-time PCR and analysis were performed according to the manufacturer's instructions. DNA extracts, which gave "strong" positive results according the cycle threshold (CT< or = 30), were processed for high resolution genotyping as described below. The primary swab sample from which the DNA extract was made was retained, so that the transport medium (M4RT) could be used to inoculate tissue culture for isolation of live C. trachomatis. Although this real-time PCR protocol is not a validated quantitative assay, the CT values of positive extracts were taken to indicate a reasonable level of C. trachomatis carriage in that sample for use in further PCR and sequence analysis as well as having a reasonable likelihood of providing a culturable inoculum. As an additional criterion to qualify for the latter process, we excluded swab samples and their paired DNA extracts that were >3 days old from time of sampling, as samples older than this did not usually contain culturable inocula. DNA extracts were stored at −80°C prior to MLVA-ompA analysis. The paired transport medium samples were stored at 4°C for <4 days following collection prior to isolation in cell-culture. Approval by ethical committee and patient consent were not obtained as this was considered a standard evaluation of an existing method. Furthermore the samples that we used had been discarded following routine diagnostic analysis, and they were unlinked and anonymised so as to permanently protect patient confidentiality.
Culture of isolates
The transport medium (M4RT) contained gelatin, gentamicin, and amphotericin B, and although specifically designed for transport and long term storage of viral specimens, we investigated the potential to culture C. trachomatis isolates directly from the clinical swabs rather than collect separate swabs for the specific purpose of this study, so as to maintain a collection of local isolates and facilitate further in-depth whole-genome and biological studies. This was possible because M4RT is designed to preserve organism integrity rather than act as a precursor to lysis, which occurs with some other commercial systems. It should be clarified that we defined the ability of an isolate from a sample to be cultured as being capable of at least two growth-passages through tissue culture, visible at each stage as inclusions by phase contrast microscopy, thus enabling a perpetuated collection of local isolates which may be used in future studies.
A 24–well tray of McCoy cells was prepared the day before infection. For infection, the cell culture medium was removed from the cells and 1 ml of the transport medium containing potential C. trachomatis isolates was added to a well. The 24–well tray was then centrifuged at 754×g for 30 min to facilitate infection. The transport medium was then replaced with 1 mL fresh Dulbecco's Modified Eagle Medium containing cycloheximide (1 µg/mL), gentamicin (20 µg/mL), and vancomycin (10 µg/mL), and the cells incubated at 37°C for 72 h. After incubation, if any viable C. trachomatis isolates were present they could be viewed by light microscopy as inclusion bodies. Positive samples were then scraped up using a sterile pipette tip, mixed with glass beads for 1 min to break open the host cells, centrifuged at 110×g for 5 min to remove any cell debris, and then the supernatant was added to an equal volume of storage buffer (4SP) and stored at −80°C.
Amplification of VNTR and ompA sequences
VNTR loci and ompA sequences were amplified by PCR from the DNA extracts from positive swab samples (as determined by the COBAS Taqman 48 method) as follows: The ompA gene was amplified using primers PCTM3 and NRI according to Lan et al [12] (Table 1). As a contingency, alternative primers OMPSeqF and OMPSeqR were used which amplified a 718 bp fragment of ompA in cases where PCR of the 1,019 bp amplicon proved not possible (Table 1). Sequences encompassing the three VNTR loci were amplified using primers CT1291F and R, CT1299F and R and CT1335F and R according to Pedersen et al [10] (Table 1). The PCR reaction components were: 1×HF buffer, 200 µM each of dioxynucleotriphosphates G, A, T and C, 250 nM each of forward and reverse primers (Table 1), 2–4 µl of DNA sample, 0.5 µl of Phusion High-Fidelity DNA polymerase (New England Biolabs UK, Hitchin, UK), and H2O to final volume of 50 µl.
The PCR was performed using the Veriti 96 well Thermal Cycler (Applied Biosystems International, Warrington, UK). The general PCR program was: 2 min at 98°C, followed by 35–40 cycles of 20s at 98°C, 30 s at 56–64°C and 45 s at 72°C, with a final extension step of 7 min at 72°C, and then held at 16°C. For ompA PCR, 35 cycles and 56–58°C annealing temperature were used. The PCR completed forty cycles for the VNTRs; the annealing temperatures were 56–59°C for CT1291 and CT1335, and 62–64°C for CT1299. The PCR products were purified using Wizard SV Gel and PCR Clean-up System (Promega, Southampton, UK).
Sequencing reactions on purified PCR products were carried out by a commercial company: Gene Service (http://www.geneservice.co.uk). The sequencing primers for CT1291, CT1299 and CT1335 are internal primers CT1291inF, CT1299inR and CT1335inR, respectively. The sequencing primers for ompA are NR1 (longer PCR) or OMPSeq2R (shorter PCR) (Table 1).
MLVA-ompA sequence analysis
OmpA sequences were genotyped via comparison to the NCBI database via BLAST, and sequence types were assigned using reference strains according to the following strain names and accession numbers: D, D/UW-3/CX NC 000117; E, E/BOUR DQ06428; F, F/IC-CAL3 DQ064287; G, G/392 DQ064288; I, Ia/870 DQ064291; J, J/UW-36 DQ064292; K, K/UW-31 DQ064293.
VNTR sequences were analyzed manually using the original fluorescent traces and by comparison of the alphabetical sequences to those of D/UW-3/CX, accession number NC 000117. The assignment of MLVA-type was carried out according to the rules described in Pedersen et al [10], namely, a number was assigned to each different VNTR locus based on the differing number of mononucleotides. Therefore the three sequenced VNTR loci gave rise to a corresponding three-digit code according to the order: CT1335; CT1299 and CT1291. Where new MLVA types were discovered that were not defined in Pedersen et al [10], we assigned new numbers which were added to the C. trachomatis MLVA database (http://www.klinmik.dk/genotyping/MLVA_types.asp).
Preliminary Pilot study
In December 2008 we began our preliminary primary study, collecting DNA samples. In the first instance we collected nine DNA samples during that month. Of these, only seven gave a full set of amplicons compatible with the VNTR and ompA PCR products capable of yielding sequence data. Therefore we concluded that a proportion of our samples in the main study may not yield full genotyping data. To address this possible loss of data, we designed a study in which we collected crude DNA samples paired with the primary swab used to collect the sample, this swab was preserved in virus transport medium (Micro Test M4RT Transport medium, Remel, Lenexa, Kansas, USA). From the latter, we attempted to culture live isolates of the C. trachomatis present in the positive swab samples. We found that swab samples lost viability if more than three days (at 4°C) had elapsed between time of sample taking and receipt following confirmation of positivity. This contingency would allow us to extract DNA from cultured isolates and enable genotyping analysis data to be added to the data collected from the crude DNA isolates.
Clinical samples
In the six month period between February and July 2009 a total of 5339 female swab specimens were submitted to the HPA laboratory and from these 268 (4.9%) provided a positive result. From these 268 there were 111 positive samples that did not meet our inclusion criteria leaving 157 paired positive DNA extracts with swab samples which did comply with our sampling criteria. From these 157, 46 samples originated from GP surgeries and the other (111) samples originated from the GUM clinic. The age distribution of the samples demonstrated most specimens occurred in patients who were 16–24 years of age (117/157, with a median of 20 years of age in the 16–24 range; Table 2).
MLVA-ompA sequence analysis
Prior to this study we tested a collection of Taq DNA polymerase types, and due to its high fidelity concluded that we should use Phusion Taq DNA polymerase (New England Biolabs UK, Hitchin, UK) for all PCR reactions. This is a high fidelity enzyme which we employed for several reasons, including to eliminate the possibility of PCR error in amplification of tandem repeats of monothymidines of greater than eleven base-pairs [13]. Direct genotyping from the DNA extracts used for the NAAT test that deployed the three VNTR loci and ompA was possible for 85 of the samples. The ompA sequences were aligned with 18 known reference ompA sequences from the GenBank sequence database [14]. The genotypes which most regularly produced a positive genotype were; D, E and F, comprising 20.4%, 40.9% and 16.1% of the genotyped positives respectively (Figure 1). Genotype D could be sub-divided into two divergent types by comparing the differing sequences available on the NCBI database. Therefore the proportion that were genotype D (20.4%) comprised 11.8% of the total which aligned with D/UW-3/CX, and 8.6% of the total which aligned with D/IC-CAL3. No genotype H was found.
Isolates were cultured (i.e. were capable of at least two passages through tissue culture) from 68 of the paired M4RT samples that originated as primary swab samples; 49 of these were amongst the 85 that were directly genotyped (see Table S1 for full results). All of the 89 samples that were classed as non-culturable did not show any growth in tissue on the primary passage. Of the 68 samples which were subsequently found to be culturable, use of the MLVA-ompA typing scheme on the primary DNA samples allowed us to identify 26 distinct types including six of genotype D; nine of genotype E; four of genotype F; four of genotype G; three of genotype Ia; and one each of J and K. Of the 19 samples that were not directly genotyped but were cultured, it was possible to extract DNA and fully genotype 8 samples (the incomplete data for all 19 isolates is shown in Table S2). To achieve this, we had to use the "short fragment" PCR and sequencing option because no result was obtainable using the long fragment option. Only where we could attain good quality sequence data for variable domains II and IV of ompA did we accept the identification data as accurate. Therefore we gained a further eight of nineteen cultured samples to add to our total of MLVA-ompA genotyped collection. Consequently we returned a total of 93 fully MLVA-ompA genotyped samples. The age-distribution of the 93 specimens analyzed by MLVA-ompA was similar to that of the 157 total specimens received, in that most specimens occurred in the patients 16–24 years of age (Table 2).
Of the 93 fully genotyped specimens, six profiles appeared to have a multiple (mostly two) of VNTR sequences in one sample (Table S3), i.e. sequence data suggested the presence of more than one VNTR type at the same locus in these samples (evidenced by a succession of two nucleotides detected at the same point on the sequencing chromatogram). In one specimen, a mixed infection according to the ompA sequence was suspected. In this case, the ompA gene was cloned and sequenced, revealing that this sample contained genotypes E and D/UW-3/CX. Isolates that were cultured then genotyped belonged to genotypes D, F, G, J and K (Figure 1).
Throughout the analysis of ompA, various nucleotide polymorphisms in comparison to database/known sequences were noted (see Tables S4 and S5). Of the four genotype J samples, two had twelve identical nucleotide changes in the ompA sequence compared with J/UW-36 . Ten mismatches distinguished D/IC-CAL8 from D/UW-3 (data not shown).
There were different MLVA types within each genotype, including the two genotype D variants (Figure 2). Overall, the combination of the ompA and MLVA as a genotyping panel gave, for this set of results, a Simspon's discriminatory index [15] of 0.96. Of the eight samples which could only be genotyped following culture (Table S3), five (MLVA-ompA types: 3.2.4-K; 3.6a.3-D/UW-3; 12.5a.2-G; 3.4a.2-J; and 8.8.2-F) were unique genotypes which had no replicates that were also detectable directly from the original DNA samples. Some MLVA signatures were common to different ompA types, for example 8.5.2 was found in ompA types D (both sub-types), E, F and J; whilst 8.6.2 was found in D/IC-CAL8, E, and F. In most cases however, MLVA signatures were unique to ompA types. As well as defining some new VNTR sequences for the three loci (Table 3), we further noted that in some cases there were differences in the flanking regions between two samples where the VNTR locus sequence itself was identical. For example, VNTR CT1299 variant 4 (Table 3) has a tandem repeat of ten cytosine residues (10C). In our survey, we found three versions of CT1299 variant 4 with 10C, with three different sequences between the conserved flanking regions, namely 10C (variant 4), 10C-T3C (variant 4a) and CT-10C-T3C (variant 4b). We therefore extended the signature numbering system of Pedersen et al [10] to enable differentiation by inclusion of the eight-base flanking regions. Two samples which were genotypes E and J, had a new CT1299 variant (which we have designated as variant 9; Table 3). This variant is common in LGV strains, which may indicate a closer linkage of particular genital tract strains to LGV than others.
For each sample, locus CT1291 PCR mostly produced either of two different amplicon sizes (∼225bp or ∼510bp), the former amplicon being common to genotype E samples. CT1291 variant 1 (Table 3) was exclusively found in the ∼510bp amplicons. CT1291 is found within a gene coding sequence and this variation may represent a significant difference in lineages associated with variant 1 which has a large indel site between the primer-annealing sites in comparison to the variants found in the ∼225bp amplicons. Furthermore, one sample displayed evidence of deletion of locus CT1291.
Of the two different sizes of CT1291 PCR products (∼225bp or ∼510bp), the 225bp fragment aligned with the downstream half of the ∼510bp fragment. All ∼510bp PCR products had the same variant (variant 1; Table 3) - most found in genotype E (32 out of 34 CT1291 variant 1 samples). One sample failed to generate a VNTR sequence despite amplification of a ∼280 bp amplicon. Sequence analysis revealed this amplicon to have a sequence homologous to the upstream region only of the ∼510bp version of CT1291 PCR products.
This study showed that in an urban UK population, there is a greater genetic diversity among circulating C. trachomatis strains than can be measured by ompA-based genotyping alone, the (previous) gold standard for C. trachomatis genotyping. This revealed a diversity measured by differences in MLVA, but also showed that there is a diversity of MLVA types unique to specific ompA genotypes. In addition we noted that there was a diversity of ompA types found within certain common MLVA types, for example MLVA type 8.5.2 was distributed between ompA types D (both sub-types), E, F and J. This strongly suggests evidence of recombination, i.e. mobility of an element containing ompA supporting recent observations of Dean et al [4].
This report describes the first independent study of the MLVA-ompA genotyping scheme applied directly to routine clinical samples and used as a tool for genotyping in a survey of C. trachomatis-positive specimen-swabs from women. It also demonstrates that it was possible to preserve isolates (capable of undergoing more than two passages in tissue culture) based on using the swab supernatant, giving an isolation rate of 43%. An estimation of diversity according to assignment of different genotype by the MLVA-ompA definition indicates we have at least 26 different live variants (out of 68 culturable isolates in total) as an ongoing collection across the genotypes D, E, F, G, Ia, J and K. Further genomic analysis of these and others of the total of 68 culturable isolates may reveal a greater diversity in this collection, for example it is possible that two isolates with a MLVA-ompA identity of 8.7.2-F may differ significantly at other loci. Furthermore, the stability of VNTR loci should be taken into account when assigning identities. VNTR sequence may vary over only a few generations, therefore such loci may only be useful for once-only assessments of local epidemiology and not suitable for repeated sampling of a population. There is some evidence that these loci may be stable [10], but this is based upon recurrent or persistent infections in patients and not using tissue culture to grow defined generations of a single strain against which to measure stability. The incidence of genotypes that could only be cultured in this evaluation (Table S3) raises the possibility that culturing may be selective, and that certain genotypes are more suited to culture–recovery than others. The use of swab samples which employ M4RT as the transport agent for the COBAS Taqman 48 (Roche) diagnostics assay is fortuitous in that the presence of gentamicin is of benefit to chlamydial tissue culture as it helps control contamination and at the correct concentrations does not inhibit chlamydial growth [16]. It is recommended that tissue culture of isolates should be commenced within 24 hours of collection of a swab sample [17]. In our case, the time taken to allow delivery of the swabs from the GUM clinic and GP practices to the HPA Molecular Diagnostics Unit was without a reliable cold chain which adversely affected our isolation rate. Furthermore we set high criteria for isolation hence our isolate rate of 43% reflects a realistic figure with routine samples. It may be possible in future studies to obtain a greater number of isolates by freezing samples immediately after processing at −80°C so as to increase the probability of preserving viable elementary bodies.
The high numbers of samples from the 16–24 age-group (79%; Table 2) reflects the published figures for the UK South Central Strategic Health Authority (SCSHA) highlighting this to be the most at-risk age group in Southampton, which has one of the highest rates of sexually transmitted infections in the country (hpa.org.uk). In 2009 there was a 38% increase in total C. trachomatis cases in the SCSHA since 2000, and of the 3028 female cases in this region, 2286 fell within the 16–24 age-group.
The sampling algorithm meant that it was likely that swabs with a large load of target were mainly taken. This may have had the affect that samples arose from a proportion of the infected population which sheds large numbers of EBs, possibly symptomatic. Although the algorithm in this study was designed to ensure culturable samples, future high resolution genotyping studies should focus on DNA alone and thus bacterial load will be irrelevant, allowing more samples from asymptomatic patients to be collected.
The distribution of ompA genotypes was similar to that found in a survey of this marker in the UK (Cambridge) in that E was the most common genotype detected, and D and F were the next most common genotypes [18]. Interestingly, the Cambridge survey also reported no genotype H detectable from females. The abundance of genotypes D, E and F reflects many studies [19]–[21]. By contrast, Ikryannikova et al [11] showed the most abundant ompA genotypes to be E, G and K. This most likely reflects the very small sample size and the pre-selection of only culture-adapted strains. Culture-bias is known to underestimate certain strains in epidemiological studies, especially in difficult-to-culture pathogens [22].
Only one sample in our study was found to represent a mixed infection (i.e. more than one different genotype in the same sample), confirmed by cloning the amplicons into vectors and analyzing the sequences of the two separate clones obtained. The mixed infection proved to consist of genotypes equivalent to laboratory C. trachomatis strains D/UW-3 and E/Bour.
Comparison of C. trachomatis genomes has demonstrated that different chlamydia strains share >99.5% sequence identity [5], [23], therefore sequence differences are often apparent between different isolates only at the single nucleotide level. MLVA has many advantages for epidemiological studies of bacterial pathogens with highly conserved genomes [24]. The strength of MLVA is that it targets single, or small-number, nucleotide deletions or insertions in the tandem repeat locus. This allows for high definition differentiation between isolates in monomorphic bacterial populations but it does not indicate evolutionary relationships [25]. However, in the case of C. trachomatis epidemiology, MLVA helped ascertain that whilst infected partners may appear to have the same strain owing to mutual transmission according to ompA analysis, MLVA data showed that one partner could have the same ompA type but a different MLVA type, indicating links to infection outside the partnership post-mutual transmission [10]. A weakness of MLVA is that it effectively relies on "mistakes" made by the host DNA polymerase during replication, therefore although useful in a localized survey covering a selected time-period, drift in tandem repeat insertions or deletions (indels) in the same C. trachomatis strain may reveal disparities when comparing surveys taken at different times from the same population. A key factor in our experimental design was the choice of a high quality, high fidelity thermostable polymerase therefore we have very high confidence in the sequencing results. Our diversity index value for the MLVA-ompA scheme (0.96) reflects that measured by Ikryannikova et al [11] when combining the three VNTR markers and ompA. In our survey, although we observed a wide diversity of MLVA types particularly in genotype E, there may be drift (e.g. plus or minus one nucleotide of the VNTR) in the locus represented by the middle digit of the MLVA code. For example there were a number of results for VNTR locus CT1299 suggesting this might be a site which undergoes variation with replication of a single clone. This was manifested in samples which appeared to be single infections according to pure sequence data for three of the four loci, but with one locus showing evidence of several sequence variants within the tandem repeat. This was confirmed by cloning and sequencing VNTR amplicons where this phenomenon was apparent. An example of the results showed the presence of both 9C and 10C loci occurring in a sample with otherwise identical remaining loci. Examples of such single-locus mixed variants were found in all three VNTR loci. This could either represent genuinely different VNTR sequences, or the same VNTR sequence in the Southampton population experiencing drift of the middle locus (i.e. it is possible that the VNTR sequence in the same strain may vary by minus or plus one nucleotide with different generations). Several MLVA-ompA genotypes were present in large clusters indicating specific sexual transmission networks, for example there were twelve samples that were 8.5.1-E, seven 8.6.2-F, and seven 8.5.2-D/IC-CAL8 (Figure 2 and Table S3). These may represent interesting clusters of cases, that might indicate specific sexual networks in terms of lines of transmission. Future studies that would allow demographic data to be collected along with the samples may be able to answer questions regarding contact tracing (which is acknowledged as only allowing an incomplete picture of a network due to its reliance on verbal cooperation of patients [26]) using analysis of such clusters.
There are several important uses for being able to type C. trachomatis to a high definition once an acceptably sensitive and robust genotyping panel is available: Partner notification in tracing transmission patterns in sexual networks; connection of clinical presentation and pathogenicity to genotypes; organ and tissue tropism association with genotypes; aiding forensic investigations; discrimination of persistent infections [10] epidemiology and detection of emerging strains. To establish a reliable high resolution genotyping system, the first step is to test the system against a local population to review the fidelity of resolution [26].
Although not specified by our experimental design, all the samples were from women, most probably attributable to swabs being unpopular amongst men attending UK GUM clinics. The genotyping system deployed sequence analysis of the ompA gene and three VNTR loci. Although recent evidence reveals that ompA may be more mobile than previously thought [4], it remains a useful target as part of a panel of markers as in the present study because there is a limited resource of database information encompassing all possible genome-based markers for genotyping and ompA genes have been the most studied in terms of C. trachomatis epidemiology. The use of a small number of markers (e.g. three for VNTR) is limiting for investigating phylogenetic relationships between strains, and whilst programmes such as eBurst (http://eburst.mlst.net/) have been used the analyses appear to be of limited use [11]. However, the value of the current MLVA-ompA scheme seems significant based on this and other studies [10], [11] which have shown its value in determining a greater diversity in C. trachomatis populations than measured by ompA alone. Therefore further investigation and development work is required to identify more VNTR markers. High throughput analysis of chlamydia genome sequences may be the ultimate way of generating data for in-depth studies but there remain considerable challenges with this technology and sample quality [27]. A better intermediate typing scheme with greater resolution and strain definition may be possible without the need for complete genome sequencing if the target panel is increased and/or altered to include new loci including meaningful SNPs and markers from other schemes additional to VNTRs. Based on similar criteria the new J samples should be considered a new ompA genotype J subtype.
Supporting Information
Table S1.
Data for directly sequenced samples (n = 85). The samples from which C. trachomatis was isolated are highlight in green (column one).
Table S2.
A: Sequencing results from 19 culturable C. trachomatis samples that did not return a direct MLVA- ompA genotype from the original clinical sample. (This data shows the eight samples that were fully genotyped after isolation in cell culture [green font – column one], and those that were rejected because they did not have good quality variable domain I and II sequence data [black font - column one].) B: The four ompA sequence types (column one) showing the areas of ompA (variable domains) where typing data was obtained. C: ompA types of the samples in S2A– the ompA sequence for sample 32 was not readable.
Table S3.
Distribution of different VNTR types according to ompA genotype (total of each type).
Table S4.
Overall distribution of ompA and mutations therein plus variable number tandem repeat.
Table S5.
Nucleotide changes in ompA sequences.
We are grateful for the support of David Browning, Andrew Tuck & his team of HPA Regional Microbiology Network Laboratory Southampton, and to Brian Spratt and Cynthia Bishop of Imperial College London for useful discussions with us on molecular epidemiology.
Author Contributions
Conceived and designed the experiments: INC PM. Performed the experiments: YW RJS. Analyzed the data: YW INC PM. Contributed reagents/materials/analysis tools: YW EA INC. Wrote the paper: YW RS INC PM. Tissue culture and archiving samples used in this study: LC.
- 1. Aral SO, Hughes JP, Stoner B, Whittington W, Handsfield HH, et al. (1999) Sexual mixing patterns in the spread of gonococcal and chlamydial infections. Am J Public Health 89: 825–833.SO AralJP HughesB. StonerW. WhittingtonHH Handsfield1999Sexual mixing patterns in the spread of gonococcal and chlamydial infections.Am J Public Health89825833
- 2. Cates W, Wasserheit JN (1991) Genital Chlamydial Infections - Epidemiology and Reproductive Sequelae. Am J Obstet Gynecol 164: 1771–1781.W. CatesJN Wasserheit1991Genital Chlamydial Infections - Epidemiology and Reproductive Sequelae.Am J Obstet Gynecol16417711781
- 3. Pannekoek Y, Morelli G, Kusecek B, Morre SA, Ossewaarde JM, et al. (2008) Multi locus sequence typing of Chlamydiales: clonal groupings within the obligate intracellular bacteria Chlamydia trachomatis. BMC Microbiology 8: 42.Y. PannekoekG. MorelliB. KusecekSA MorreJM Ossewaarde2008Multi locus sequence typing of Chlamydiales: clonal groupings within the obligate intracellular bacteria Chlamydia trachomatis.BMC Microbiology842
- 4. Dean D, Bruno WJ, Wan R, Gomes JP, Devignot S, et al. (2009) Predicting Phenotype and Emerging Strains among Chlamydia trachomatis Infections. Emerg Infect Dis 15: 1385–1394.D. DeanWJ BrunoR. WanJP GomesS. Devignot2009Predicting Phenotype and Emerging Strains among Chlamydia trachomatis Infections.Emerg Infect Dis1513851394
- 5. Carlson JH, Porcella SF, McClarty G, Caldwell HD (2005) Comparative genomic analysis of Chlamydia trachomatis oculotropic and genitotropic strains. Infect Immun 73: 6407–6418.JH CarlsonSF PorcellaG. McClartyHD Caldwell2005Comparative genomic analysis of Chlamydia trachomatis oculotropic and genitotropic strains.Infect Immun7364076418
- 6. Stephens RS, Kalman S, Lammel C, Fan J, Marathe R, et al. (1998) Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282: 754–759.RS StephensS. KalmanC. LammelJ. FanR. Marathe1998Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis.Science282754759
- 7. Urwin R, Maiden MC (2003) Multi-locus sequence typing: a tool for global epidemiology. Trends Microbiol 11: 479–487.R. UrwinMC Maiden2003Multi-locus sequence typing: a tool for global epidemiology.Trends Microbiol11479487
- 8. Nunes A, Nogueira PJ, Borrego MJ, Gomes JP (2008) Chlamydia trachomatis diversity viewed as a tissue-specific coevolutionary arms race. Genome Biology 9: R153.A. NunesPJ NogueiraMJ BorregoJP Gomes2008Chlamydia trachomatis diversity viewed as a tissue-specific coevolutionary arms race.Genome Biology9R153
- 9. Klint M, Fuxelius HH, Goldkuhl RR, Skarin H, Rutemark C, et al. (2007) High-resolution genotyping of Chlamydia trachomatis strains by multilocus sequence analysis. J Clin Microbiol 45: 1410–1414.M. KlintHH FuxeliusRR GoldkuhlH. SkarinC. Rutemark2007High-resolution genotyping of Chlamydia trachomatis strains by multilocus sequence analysis.J Clin Microbiol4514101414
- 10. Pedersen LN, Podenphant L, Moller JK (2008) Highly discriminative genotyping of Chlamydia trachomatis using omp1 and a set of variable number tandem repeats. Clinical Microbiology and Infection 14: 644–652.LN PedersenL. PodenphantJK Moller2008Highly discriminative genotyping of Chlamydia trachomatis using omp1 and a set of variable number tandem repeats.Clinical Microbiology and Infection14644652
- 11. Ikryannikova LN, Shkarupeta MM, Shitikov EA, Il'ina EN, Govorun VM (2010) Comparative evaluation of new typing schemes for urogenital Chlamydia trachomatis isolates. FEMS Immunol Med Microbiol 59: 188–196.LN IkryannikovaMM ShkarupetaEA ShitikovEN Il'inaVM Govorun2010Comparative evaluation of new typing schemes for urogenital Chlamydia trachomatis isolates.FEMS Immunol Med Microbiol59188196
- 12. Lan J, Ossewaarde JM, Walboomers JMM, Meijer CJLM, Vandenbrule AJC (1994) Improved Pcr Sensitivity for Direct Genotyping of Chlamydia trachomatis Serovars by Using A Nested Pcr. J Clin Microbiol 32: 528–530.J. LanJM OssewaardeJMM WalboomersCJLM MeijerAJC Vandenbrule1994Improved Pcr Sensitivity for Direct Genotyping of Chlamydia trachomatis Serovars by Using A Nested Pcr.J Clin Microbiol32528530
- 13. Clarke LA, Rebolo CS, Goncalves J, Boavida MG, Jordan P (2001) PCR amplification introduces errors into mononucleotide and dinucleotide repeat sequences. Mol Pathol 54: 351–353.LA ClarkeCS ReboloJ. GoncalvesMG BoavidaP. Jordan2001PCR amplification introduces errors into mononucleotide and dinucleotide repeat sequences.Mol Pathol54351353
- 14. Brunelle BW, Sensabaugh GF (2006) The ompA gene in Chlamydia trachomatis differs in phylogeny and rate of evolution from other regions of the genome. Infect Immun 74: 578–585.BW BrunelleGF Sensabaugh2006The ompA gene in Chlamydia trachomatis differs in phylogeny and rate of evolution from other regions of the genome.Infect Immun74578585
- 15. Hunter PR, Gaston MA (1988) Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity. J Clin Microbiol 26: 2465–2466.PR HunterMA Gaston1988Numerical index of the discriminatory ability of typing systems: an application of Simpson's index of diversity.J Clin Microbiol2624652466
- 16. Wentworth BB (1973) Use of Gentamicin in the Isolation of Subgroup A Chlamydia. American Society for Microbiology 3: 698–702.BB Wentworth1973Use of Gentamicin in the Isolation of Subgroup A Chlamydia.American Society for Microbiology3698702
- 17. Chernesky M (2005) The laboratory diagnosis of Chlamydia trachomatis infections. Canadian STI best practice laboratory guidelines 16: 39–44.M. Chernesky2005The laboratory diagnosis of Chlamydia trachomatis infections.Canadian STI best practice laboratory guidelines163944
- 18. Jalal H, Stephen H, Alexander S, Carne C, Sonnex C (2007) Development of real-time PCR assays for genotyping of Chlamydia trachomatis. J Clin Microbiol 45: 2649–2653.H. JalalH. StephenS. AlexanderC. CarneC. Sonnex2007Development of real-time PCR assays for genotyping of Chlamydia trachomatis.J Clin Microbiol4526492653
- 19. Beni BT, Motamedi H, Ardakani MR (2010) Genotyping of the prevalent Chlamydia trachomatis strains involved in cervical infections in women in Ahvaz, Iran. J Med Microbiol 59: 1023–1028.BT BeniH. MotamediMR Ardakani2010Genotyping of the prevalent Chlamydia trachomatis strains involved in cervical infections in women in Ahvaz, Iran.J Med Microbiol5910231028
- 20. Jalal H, Stephen H, Bibby DF, Sonnex C, Carne CA (2007) Molecular epidemiology of genital human papillomavirus and Chlamydia trachomatis among patients attending a genitourinary medicine clinic - will vaccines protect? Int J STD AIDS 18: 617–621.H. JalalH. StephenDF BibbyC. SonnexCA Carne2007Molecular epidemiology of genital human papillomavirus and Chlamydia trachomatis among patients attending a genitourinary medicine clinic - will vaccines protect?Int J STD AIDS18617621
- 21. Petrovay F, Balla E, Nemeth I, Gonczol E (2009) Genotyping of Chlamydia trachomatis from the endocervical specimens of high-risk women in Hungary. J Med Microbiol 58: 760–764.F. PetrovayE. BallaI. NemethE. Gonczol2009Genotyping of Chlamydia trachomatis from the endocervical specimens of high-risk women in Hungary.J Med Microbiol58760764
- 22. Benschop K, Thomas X, Serpenti C, Molenkamp R, Wolthers K (2008) High prevalence of human Parechovirus (HPeV) genotypes in the Amsterdam region and identification of specific HPeV variants by direct genotyping of stool samples. J Clin Microbiol 46: 3965–3970.K. BenschopX. ThomasC. SerpentiR. MolenkampK. Wolthers2008High prevalence of human Parechovirus (HPeV) genotypes in the Amsterdam region and identification of specific HPeV variants by direct genotyping of stool samples.J Clin Microbiol4639653970
- 23. Thomson NR, Holden MTG, Carder C, Lennard N, Lockey SJ, et al. (2008) Chlamydia trachomatis: Genome sequence analysis of lymphogranuloma venereum isolates. Genome Res 18: 161–171.NR ThomsonMTG HoldenC. CarderN. LennardSJ Lockey2008Chlamydia trachomatis: Genome sequence analysis of lymphogranuloma venereum isolates.Genome Res18161171
- 24. Lindstedt BA (2005) Multiple-locus variable number tandem repeats analysis for genetic fingerprinting of pathogenic bacteria. Electrophoresis 26: 2567–2582.BA Lindstedt2005Multiple-locus variable number tandem repeats analysis for genetic fingerprinting of pathogenic bacteria.Electrophoresis2625672582
- 25. Comas I, Homolka S, Niemann S, Gagneux S (2009) Genotyping of Genetically Monomorphic Bacteria: DNA Sequencing in Mycobacterium tuberculosis Highlights the Limitations of Current Methodologies. Plos One 4: I. ComasS. HomolkaS. NiemannS. Gagneux2009Genotyping of Genetically Monomorphic Bacteria: DNA Sequencing in Mycobacterium tuberculosis Highlights the Limitations of Current Methodologies.Plos One4
- 26. Choudhury B, Risley CL, Ghani AC, Bishop CJ, Ward H, et al. (2006) Identification of individuals with gonorrhoea within sexual networks: a population-based study. Lancet 368: 139–146.B. ChoudhuryCL RisleyAC GhaniCJ BishopH. Ward2006Identification of individuals with gonorrhoea within sexual networks: a population-based study.Lancet368139146
- 27. Thomson NR, Clarke IN (2010) Chlamydia trachomatis: small genome, big challenges. Future Microbiology 5: 555–561.NR ThomsonIN Clarke2010Chlamydia trachomatis: small genome, big challenges.Future Microbiology5555561


text:
Goodbye Ratmaze 1.0(original publish date: February 14th, 2006)
Hi! Welcome back to the games.pixeljam.com blog. This week I would like to briefly touch on what's new with Gamma Bros, and then I think I'd like to take some time to reflect on a game Miles and I were developing last year that will probably never be finished that we call "Ratmaze".
So, first things first, a Gamma Bros update. Who are they again, you ask? Well, their names are Buzz and Zap, and they are brothers (by blood, although it's unclear exactly how), and they work together on a space station, doing some very important work (we think)..
They seem to be great with computers and are a bit on the strange side. I'd say Buzz (redhead) is a little more high-strung than Zap, who is more evenly balanced. they've both got girlfriends back on Earth, and the part of their lives that you, the player, will be involved with is getting them home from work safely. I'll get further into the history of these guys, where they made their first appearance, etc. another time.
As for what's new, Miles and I have both fortunately gotten a bunch of client design work recently, and since making a living is of primary importance, and cash reserves are becoming frighteningly low, that's where our time must be spent. We took many months off so that we could produce Gamma Bros and lived off savings & credit. Sadly,we haven't really worked on the game in over a week & I've also been having a rough time with my broken arm, but that's another story. We're hoping of course to find a way to make our living off of creative projects, retro games primarily. We truly want to make many great, fun games, we are just bursting & exploding with ideas and have about 6 or 7 games slated for development after Gamma Bros, including a sequel to Gamma Bros that will be a combo of spaceshooter, platform action game and adventure story. think Galaxian meets Robotron meets Master Blaster. maybe add Bionic commando in there..
Soo, speaking of games on hiatus, let me tell you a little about a little game that we were working on after we put our first large adventure game on indefinite hold. It was called "Ratmaze" and we took it as far as completing the game map, adding some cheese to the rooms, and making the rat controllable to run around (u,l,d,r) the maze. the idea was to make a simple game where you had to do certain tasks to collect all the varieties of cheese in the maze within a specified time limit. The game would have been relatively non-violent, no killing, just lots of dodging, running around and knocking over "enemies" (snakes, bugs, bats, worms, spiders, scorpions, mousetraps, other rats & mice maybe). There would also have been a large main cat that would stick its paws and face into the maze occasionally to thwart the rat's mission, but the cat wouldn't kill the rat, just stun him for a moment, where he would lose precious time.
There would have been secrets too, of course. There's got to be secrets in video games. it's like rule #3 of video game making! Well, that's about all I can tell you about Ratmaze. we've moved on to our Gamma Bros game and will probably leave Ratmaze behind. I loved the little Rat, so he'll likely be seen in some of our games to come, and maybe there's a possibility that we'll try a Ratmaze 2.0 in the future, many games later, but for now we've just got to consider it a learning experience & move on. Perhaps we'll let people run around the relatively empty and unfinished halls of Ratmaze sometime, but it's not that entertaining beyond a minute or two & once you've gotten all the cheese. it's totally unfinished & somewhat lifeless in its current state. check out some more of the screens: (click for larger views, actual game size)
Well, there you have it. Ratmaze. Sorry little Rat buddy. Maybe we'll see you again in the future.
That's all for today. check back in a week or two for more of.. something. oh yeah, please send us email to firstname.lastname@example.com if you'd like to be on our mailing list. We'll let you know when Gamma Bros is ready, and future games as well.
by Rich Grillotti Tags: Gamma Bros


text:
|Anderson, Richard
|Polansky, Marilyn
|Mirsky, Nitsa
|Roussel, Anne Marie
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/26/2003
Publication Date: 11/18/2003
Citation: Anderson, R.A., Polansky, M.M., Mirsky, N., Roussel, A. 2003. Chromium, cinnamon & tea as insulin potentiators & antioxidants [abstract]. Paper No. 026. Interpretive Summary:
Technical Abstract: Dietary factors that may be the most likely to elicit the largest effects on controlling glucose intolerance and diabetes are factors that function not only as potentiators of insulin action but also antioxidants. We have demonstrated that three dietary components that are both insulin potentiators and antioxidants are chromium, cinnamon and tea. Insulin potentiating forms of chromium are well documented to serve as potentiators of insulin and recent studies have also demonstrated that chromium functions as an antioxidant in animal studies and in human subjects with type 2 DM. Water soluble polyphenol components from cinnamon have also been shown recently to not only potentiate insulin activity several-fold but we now have evidence that these compounds also function as antioxidants. The antioxidant activity of the cinnamon compounds was as good or better than any of the antioxidant compounds tested including vitamin C, L-cysteine, glutathione, N-acetylcysteine and -carotene using the ferric-reducing antioxidant power (FRAP) assay. The antioxidant properties of tea catechins are well established with the largest effects of epigallocatechin gallate. We recently reported that tea catechins also enhance insulin function with the largest effects of epigallocatechin gallate. In summary, Cr, water-soluble cinnamon polyphenols and tea catechins all function to not only improve insulin sensitivity but also alleviate oxidative stress and therefore may all be important in the prevention and alleviation of glucose intolerance and diabetes.


text:
Points only keeper League, 24 teams, weekly head to head, 2RW, 2LW, 2C, 4D, 1 G , (goalies 2pts for W +3 for SO) [/I][/B]
Keep 11
LW: Gaudreau, Marchand, Forsberg
C: Seguin, Pavalski, Kuznetsov, Little
RW: Ryan, Eberle, Atkinson, Vrbata
D: Klingberg, Ekman-Larsson, Ristolainen, Gostisbehere, Zaitsev,
G: Allen, Fleury, Murray, Hutton
It Ain't How Hard You Hit. It's How Hard You Can Get Hit and Keep Moving Forward. It's About How Much You Can Take And Keep Moving Forward!
Blades of Steel
10 Team Full Keeper - Pts Only (G,A,W,SO) - Champions (2011, 2013, 2014)
Crosby, Backstrom, Wheeler, Neal, Pavelski, Zuccarello, Fabbri, Oshie, Hoffman, Pastrnak
Chara, Karlsson, Giordano, Vatanen, Streit
Rask, Rinne
Nyqvist, S. Bennett, Burakovsky, Toffoli, Connor, Pulock, D.Pouliot, S.Theodore, Hellebuyck
Not sure what you're expecting, you're talking about a guy with one year left on his deal, injury prone, and past his peak. A guy who is not on the same page as the head coach, a guy who took the captaincy but has hurt feeling when he feels the heat. You're also not losing a ppg player as it's doubtful he ever does that again with his injury history. So that argument really needs to stop being made.
They have to move him. Just take the best offer. As I've said I think they improve by getting rid of him immediately regardless of the return. They won't be set back as he would've left next year anyway and they've done well when he isn't playing. Look at what Boston I got for a Joe Thornton. Didn't matter in the long run.
There is little chance you get a very good, impact player in return for spezza alone. I can see them adding Methot or something in a package though.
A 50 point c like Berglund who plays better defensively is exactly what should be expected. Look what bobby ryan got, a fringe nhl player, prospect, and first. Berglund, Rattie, 1st is similar and that's with a guy with one year left on his deal.
imo they make the playoffs if spezza was injured the whole year this year.
Last five cup winners have not had a ppg centre
If they get Berglund, a 1st, and a prospect I would call that par for the course.
The player has openly stated he wants out. (Or at least the gm has stated this).
And from Ottawa's side they don't just get this. They gain 3.75 million in cap space next season. That's a lot for a budget team like Ottawa. Plus they gain a player that is still an RFA after seasons end.
Ottawa has their number 1 centre in Turris. They need Berglund to be their number 2. Which he is more than capable IMO.
This could be a great move for Berglund too. He showed some pretty good offensive skills at the start of his pro career (Allsvenskan included) so playing in a more open system could help him break out. That and getting solid 2nd line minutes + good PP time.
I wonder if Ottawa could pry Jordan Schmatlz away as the prospect. IMO Berglund, Schmaltz + 1st would be way better than Berglund, Rattie + 1st.


text:
HOME of BEST IN SHOW CHINESE CRESTED DOGS
and PREUVIAN HAIRLESS (large)
Jeanette Bryce & Bernard Cherry
Koreke Kennels breeding physically sound, happy Crested's using the best of New Zealand and International blood lines.
Amun - Chinese Crested Dog Monju - Peruvian Hairless (large)
Binny and Aoede
About our breeding practice: We believe that every puppy born in our kennels deserves the very best start in life that we can provide. Because the dogs we breed are first and foremost companion dogs, We use the best New Zealand and International lines that are available to us in order to produce sound, happy Chinese Crested's. We believe temperament and health are very important considerations when we plan a litter - therefore we health test.
None of our success or breeding would have been possible without the pratical help and mentoring provided by Sally Johnson from Xioma Kennels in Australia; in 2004 we campainged Aust. Grand Ch Xioma Sagebrush. In 2007 it was the turn of Aust Grand Ch Xioma Fire In The Sky aka Billy. He delighted the breed in NZ by becoming the first Chinese Crested to win a BIS at a All Breeds Show. Other imports from the Xioma Kennels to have made their mark at Koreke are: Ch Xioma Prince Of Darkness, Ch Xioma Witches Brew, and Ch Xioma Dragon Of The Smoke. Sally also enabled the NZ visit of Ch Sun-Hee's Live and Love bred by Swedens Sun-Hee's Kennels.
Recent imports from Australia have been with the support of Linda Millot of Saxor Kennels, and Hannele Ståhl of Finlands Zhannels Kennels. Ch Saxor Honney Boo Boo Child and Zhannels Simpatica have settled in. We are looking forward with excitement to see what their addition to our lines will produce.
We are always happy to discuss your questions or requirements for a Pet or Show Crested and will export to the 'right' home. Bernard and I hope you enjoy your visit to this website and would love your feedback.
Thank you
Jeanette Bryce & Bernard Cherry
Christchurch, NZ
Phone : 03 312 9410
Email : [email protected]
70846 Visitors