text:
Juventus' secret plan to get Man United target Arturo Vidal fit for the season [Tuttosport]
- August 5, 2014
- Comments0
The front page of Tuttosport on Tuesday gives off the impression that Manchester United fans hopes of signing Arturo Vidal this summer are likely to fall by the wayside.
After weeks of chitter-chatter in the British, Italian and Chilean press suggesting that Vidal may be on his way to Old Trafford, the Chilean midfielder travelled with his Juventus teammates to Indonesia this week.
With pre-season now in full swing, Vidal's health is once again a topic of conversation, with the midfielder's knee known to be a problem.
Tuttosport now report that Juventus have a "secret plan" for Vidal. Moreover, the headline on the paper claims that Juventus aim to get their Chilean back in top form, and Massimo "Allegri is ready to hand over the keys to the team" to Vidal.
Taking a step back at the broader picture, the understanding from Tuttosport is that not only is Arturo Vidal not moving to Man United, but the midfielder is now set to be the man whom Juventus build their team around next season.
Latest on Arturo Vidal
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- Arturo Vidal rules out moving to Arsenal because of fans' obsession with Twitter ...
- Arsenal are no longer interested in Arturo Vidal, according to the BBC & Telegraph
- Arturo Vidal to Arsenal rumours refuse to go away: Deal 90% done according to Cope
- Everything you've read about Arturo Vidal to Arsenal over past 24 hours has been ...
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They feel bad, so they found you these other deals to make up for it.
Select Local Merchants
Lincoln Park ZooRead MoreFounded in 1868, Lincoln Park Zoo is a world-class zoological institution nestled in the heart of Chicago's historic Lincoln Park district. The zoo is open every day of the year and offers free admission to provide entertainment and education for visitors of all ages. In addition to architectural treasures and award-winning progressively designed animal habitats, the zoo is also a conservation haven, operating one of the country's largest zoo-based conservation and science programs.200 N Cannon Dr.Chicago, IL US
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ABF U-Pack MovingRead MoreYou Pack. We Drive. You Save. That's U-Pack Moving in a nutshell! U-Pack is the perfect moving solution when you want the convenience of full-service movers combined with the cost savings of a rental truck. You pack and load, and we drive--it's that simple! U-Pack has nearly 300 locations to help you move nationwide.1900 E Lincoln HwyLynwood, IL US
Dahn CorporationRead MoreScattered across nine states, Mini U Storage's secure facilities make frequent cameos on the reality TV show Storage Wars. At each location, rows of storage units in a vast range of sizes house personal goods, business items, and vehicles. Specialty climate-controlled units can help preserve sensitive heirlooms or souvenirs from historic snowball fights. The Mini U Storage staff further assists clients in the moving process by offering complimentary rental trucks, along with boxes, moving supplies, and locks for purchase.4747 W Calumet-Sag Rd.Crestwood, IL US
STE Cleaning ServicesRead MoreHaving a home can be hard work. The masterminds behind STE Cleaning Services recognize this, and so they've created a roster of services that stretch throughout both the life and square-footage of residences, from move-in to move-out and from floor to ceiling. Carpet-cleaning services?which come complete with pre-vacuuming, pre-spot treatments, and furniture moving?ensure that residents have a perfectly clean surface on which to spill their chocolate milk. The company's maintenance and construction teams answer distress calls from homeowners, whether they need to fix a hole in a wall or build a new addition. And STE's staffers can make move-in and move-out as painless as possible, transporting beloved pieces of furniture or incredibly lightweight boxes of books from one home to the next. In-between moves, they can clean bathrooms, bedrooms, and kitchens to keep homes sparkling.2300 West 159th StreetMarkham, IL US
Caravan Beads of ChicagoRead MoreThroughout nearly two decades of collecting and reselling beads, owner Charlene Steele has gathered more than one million types of baubles including Swarovski crystals, semi-precious stones, and Miyuki seed beads and Delica beads. In addition to a variety of beads and jewelry components from Czech glass buttons to crystal pendants, customers can drape themselves in the shop's finished jewelry. To learn techniques for making their own treasures, patrons can attend beginner to advanced classes, in which knowledgeable instructors teach students how to wire wrap a stone or preserve a small photo or dried flower in a resin pendant. The shop hosts private parties for kids and adults alike.3339 N Lincoln Ave.Chicago, IL US

subset: commoncrawl


text:
Find houses for rent, apartments for rent and various leased accommodations in Davanagere .Contact landlord or property manager in Davanagere directly.
RentHome.in is the best way to find your next home rental or small apartment from the convenience of your home computer or office in Davanagere.
Available Rental Accommodations in Davanagere
Relevant Searches
Leased properties in Davanagere, Davanagere rented home, flat for rent Davanagere, residential houses for rent in Davanagere, Davanagere guest house hostels, paying guest accommodation in Davanagere.
If you have any question / requirement related to Davanagere rented homes and houses than feel free to contact us.
Useful Resources
Davanagere, (Kannada:ದಾವಣಗೆರೆ) often spelled Davangere, is a city in the Indian state of Karnataka, lying at the western part of South India.
Davanagere was famous for textiles, with the DCM [Davanagere Cotton Mills] brand being famous in India. Davanagere was a major cotton textile exporter during the
Bapuji Institute of Engineering and Technology (BIET) is one among the top ten self-financing Engineering Institutions in the state of Karnataka, providing high
Introduction Edit. Description of Davanagere. Davanagere City is a district head quarter located on NH4 at a distance of about 260 Km away from Bangalore.
The Shivagangothri Campus located at Tolahunase, 11 Kms. from Davangere houses the post graduate departments of Commerce, Biochemistry, Microbiology
Posted in main |
India apartment rentals, sublets, India temporary housing. Apartments for rent sublets / leasing, roommates India.India home exchange, India apartment rentals, house swap and list your house for rent when on sabbatical, leave or vacation


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Understated and refined track featuring subtle sweeping pads with catchy ,interweaving pulsing melodies. Underpinned with effective stylish contemporary drums, percussion and bass. Great for Film/Tv advertising, corporate branding/presentation, reality tv , new product launch and contemporary drama.
composer: Denis Woods | duration: 114 seconds | size: 19.32 Mb
|Price: $44.95
Big and powerful orchestral hybrid track great for trailer, battle, modern, champions and drama. Sounds like it could be a big and dramatic cue from Inception
view available edits and loops for this track
composer: Vincent Pedulla | duration: 67 seconds | size: 11.70 Mb
|Price: $49.95
Acoustic guitars, bass and harmonica. Light and ironic moods, full relaxing. Ideal for humor video.
composer: Ilya Truhanov | duration: 33 seconds | size: 5.62 Mb
|Price: $24.00
Happy song, with a village/ Wild West atmosphere. Played by - banjo, flute, violin and more ..
composer: Y.Aharon Konsman | duration: 139 seconds | size: 23.56 Mb
|Price: $35.00
Beautiful acoustic guitar track. The piano and percussion comes later as a duet with the guitar. Very soft, very inspirational
composer: Raphael Costa | duration: 136 seconds | size: 37.23 Mb
|Price: $24.95
Bring a happy vibe to your project with this lighthearted piece that embraces a child's ability to be carefree and enjoy the simplicities of youth, enhanced by great vocal riff melody and a great drums beat.
This Track includes 5 versions:
view available edits and loops for this track
composer: Adi Goldstein | duration: 60 seconds | size: 9.75 Mb
|Price: $40.00
This upbeat and driving electro track features rhythmic piano, fun synths and a head bobbing beat. Sets a great catchy, poppy, happy vibe. Also exudes a hint of wonder and awe. Well suited for commercial or corporate projects.
composer: Joseph Rusnak | duration: 171 seconds | size: 28.68 Mb
|Price: $66.00
Energizing instrumental Pop Anthem in the style of Katy Perry, Ke$ha, Pitbull and Nicki Minaj. Very catchy synth riffs and energetic, pumping trap-style beats! Great for corporate presentation/branding, new product launch, infomercial, documentary, website, film and tv advertising, parties, celebration, fashion shows or other cool and trendy events.
view available edits and loops for this track
composer: Tim Besamusca | duration: 137 seconds | size: 23.12 Mb
|Price: $35.00
An easy-going traditional instrumental country shuffle. Features drums, bass, piano, acoustic and electric guitars. This music bed has no melody, so it is idea for voice-over for commercials, and also for source background music for a scene in a country bar or club.
composer: Gary Wolk | duration: 176 seconds | size: 29.49 Mb
|Price: $29.95
Epic and majestic adventure music in the style of 'Lord of the Rings' and 'Game of Thrones'. Featuring a full orchestra with strings, brass, celesta and percussion.
view available edits and loops for this track
composer: Hakan Eriksson | duration: 121 seconds | size: 20.39 Mb
|Price: $20.00
Beautiful and evocative jazz featuring classical (nylon string) guitar with piano, vibes, bass and drums. Mellow and introspective with a light and carefree touch.
view available edits and loops for this track
composer: Abbas Premjee | duration: 114 seconds | size: 19.23 Mb
|Price: $69.99
Atmospheric middle eastern track that builds from a soft and ambient beginning with Cumbus, Saz, The Ney, Duduk. Perfect for visuals that require emotional feeling, passion and warmth.
view available edits and loops for this track
composer: Sungtae Kim | duration: 79 seconds | size: 13.38 Mb
|Price: $34.99


text:
Being clean on the inside is just as important as being clean on the outside, which is why doctors recommend that you eat at least one natural sponge a day. De-gunk your trunk with this Groupon.
The Deal
- $59 for one colon-hydrotherapy treatment with a doctor's initial consultation (a $145 value)<p>
Decisions about health care should not be made in haste. Consultation required as all candidates may not be eligible. Eligibility will be determined on an individual basis applying the accepted and prevailing standards of practice. Non-candidates will be refunded.
Organix Med Spa
The versatile staff members at Organix Med Spa perform a comprehensive list of health and wellness services and pride themselves on treating each patient with personalized care. Massage therapy and acupuncture sessions nurture bodies as a whole rather than catering separately to the wishes of disparate arms or heads. Organix Med Spa's nonsurgical chiropractic methods, which are customized to suit each spine's needs, assuage achy backs irritated by the feeble prodding of traditional medicine. In addition, the expansive therapy center boasts an organic med spa with an array of soothing treatments, such as facials and waxing.
The Gist
What some people are buzzing about:
"Nice place, The services were great. The technicans were nice too. The rooms weren't what I'm accustom to as it relates to spa services, like the linen, etc...."
"Nice place, The services were great. The technicans were nice too. The rooms weren't what I'm accustom to as it relates to spa services, like the linen, etc. Overall, it was nice and i would go back. "
"Give it a try, you will love the staff and ambience. Great place to relax and enjoy a nice day at spa."
"the facial with Cindy is awesome. The one that gives the messages needs work."
2001 Midwest Rd.
Ste. 110
Oak Brook, IL 60523
+16306200029Get Directions


text:
You can search for possible plumbing contractor within your location by beginning an informal online search. You will find online directories where you'll find numerous plumbing companies shown with their contact numbers and handle. You can inquire the character of service and charges cited by each one of these companies by setting an immediate contact.
A superb thought during the time of emergency is to employ a company that particularly deals with plumbing emergencies. There are fairly quantity of businesses that supply emergency services at acceptable costs. You do not have to pay an additional for their providers. Also you can contemplate requesting your relatives and pals to urge you few reliable plumbing services in your region. They might have used few solutions when they met up with plumbing emergencies.
Among the most critical characteristics of a home or building is its plumbing system. Without plumbing, a home would be a mere shell. For individuals interested in chasing this deal as a way of work-in Northwest UK, there are certainly a variety of plumbing programs which are easily obtainable.
Indeed the career is not far better if you're planning routine work such as for instance having a boiler served or even a toilet installed. Finding A quote can be described as a problem – getting the task started a whole lot more difficult.
Maybe it's because they're considered "dirty" as their occupation does indicate a specific amount of literal dirt. It may also be because people genuinely believe that being in the plumbing service is actually a horrible task to enter into for your money, which is not the scenario as plumbers are often very well settled. Regardless, the actual fact remains that registered plumbers are extremely beneficial and society, as a whole, wants them.
Draining and insulating all outside sinks and pipes within your home plumbing program ahead of the winter sets in is a must as any water that gets stuck in those pipes may freeze, develop, and fracture, break, or broke the pipes. Getting these outdoor pipes will help you avoid working with property problems and annoying plumbing repair.
Generally, key plumbing materials that are very essential at that time of construction involves sinks, faucets, bath tubs, baths, water heaters and dishwashers. All of these goods can be purchased in industry in different kinds. You can find numerous sizes and shapes to select from. We are providing you solid pieces of advice here, but do be aware that some are more important to understanding celui-ci.
But that can vary slightly, and it really just will depend on how you want to use the information. But we are not done, yet, and there is always much more to be revealed. The last half of the article will offer you a lot more solid info about this. We think you will find them highly relevant to your overall goals, plus there is even more.
A comprehensive plumber training degree is essential before you call yourself a competent plumber. Before you join a formal plumber training program, you've to cross an over-all test which assesses your ability to focus on basic numerical calculations and word problems.
Obtaining good tradespeople can be a headache. Great plumbers are notoriously difficult to discover. Some don't answer their phone if you call, others merely don't turn-up. Some can charge an extortionate amount for a comparatively straightforward job.
This will not only save time at that crucial time but will also help an individual to prevent stress situations and quick conclusions. Before looking out for a plumber a very important factor should be produced certain. People should only decide for licensed plumbers.
There are various courses available for pretty much every theme in plumbing and do it yourself home improvement. Plumbing lessons not just cover repairs but safe handling of components and tools, they also get into depth in regards to the different programs in plumbing. You'll be able to learn to deploy and repair bathrooms, sinks and bathrooms. The water pipework of a home or building will not look arcane for your requirements.
Many jobs do not climb up the building, while an significant actions to improve the construction of each functions in. You must run to fulfill with that valuable. Cancellation of common building construction, electricity provide, oxygen packaging, decoration, color, security devices, many of these jobs can be developed. Such forwards we're concerned with the water pipe as generally understood.
Apart from this, someone can also check with real estate agent who got that house for you to have the useful contact numbers for experts like plumber and electrician. Once the contact number of that localityis plumber is acquired, the next phase is to contact that plumber.


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Citizen prompts arrest for DUIMay 07,2013
Citizen prompts
arrest for DUI
staff report
MIDDLESEX A close call on Route 12 over the weekend prompted what amounted to a citizens arrest that ended when a Burlington woman was taken into police custody and cited for drunken driving.
According to Vermont State Police, a Middlesex man called to report he had just narrowly avoided a head-on collision with another vehicle while driving on Route 12 shortly before 6:30 p.m. Saturday.
Police did not name the man, but said he turned around, caught up to the vehicle and was able to get the driver, Carrie Peck, 39, to pull over.
The man allegedly told police he believed Peck was impaired and when they arrived at the scene she was taken into custody, processed for DUI, and released on a citation to appear at the criminal courthouse in Barre on May 23. Peck is expected to be charged with driving under the influence at that time.MORE IN This Just InMONTPELIER Vermonts Division of Fire Safety says public fireworks displays put on by trained... Full StoryMONTPELIER A Vermont state trooper has been honored for rescuing an 82-year-old woman from her... Full StoryMONTPELIER Vermont specialty food producers took home gold at this weeks Summer Fancy Food... Full Story
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Stock Photo:
Analog Stereo Open Reel Tape Deck Recorder with large reels
- Image ID: 96223397
- Copyright: Viktorus
High quality images curated from over 150 countries.
Over 50 million photos, vectors, illustrations, and more.
Simple and flat pricing by image or by month.
Over 230,000 images downloaded every day.


text:
Howard Johnson Inn Monroe
No prices found through our partners. Please contact the business directly or check some of our recommended alternatives.
- Suites
- Continental Breakfast
- Swimming pool
- Free High-Speed Internet
- Fitness/Health center
- Free parking
Travelers also recommend:
TripAdvisor Reviews Howard Johnson Inn Monroe
Travel Blogs from Monroe
We're in Nashville!
We just got to our room. Had to pick up our tickets to see Little Big Town in the morning by 5 o'clock. Barely made it. Traffic is horrible downtown. Everyone getting into town plus the CMT Awards tonight. Having dinner and relaxing tonight. Fun starts tomorrow! Sent from Outlook on ...
Way to go Ohio
We roll into double-sin city smooth as silk on new pavement welcomed by 100 foot Jesus and escorted on each flank by travelling salesmen, phones lazily dangling from their ears. There must be more of them because every exit offers an assortment of mid-scale inns and motels, sprouting mushroom-like along the edges of the interstate and fed by an even bigger assortment of fast food outlets, some with signs reaching into the sky, taller than ...
Epilogue and Notes on Culture and Misc.
... are fake roadblocks in various portions of the country when the police shut down at night.
You are at the equator and in most church areas at an high altitude (6000 to 8000 feet elevation-except Mombasa on the coast) and the sun is close. You burn quickly and easily so bring sun screen if you will be outside very much.
If you want to buy things, use the local members, as foreigners pay a much higher price.
Game parks charge about ...
Leaving Home and work on the jet...
... Visitor Information System that certifies PDSOs - Principle Designated School Officials, and that would be me for ABC. Kathy DeCampos, ABC assistant will be a DSOs –Designated School Officials.
If certified, we will be able to fill out I-20s for international students giving them a much greater chance of getting a F1 student Visa. The Designated Officials will prescreen applicants, and fill out the I-20 nonimmigrant form ...
Final thoughts
... br>
On this trip while in Australia and New Zealand we didn't stay one single evening in a hotel. All our nights were in people's homes. We appreciated getting to know and spend more time with our ministers and members in this manner.
In Australia we met with all our ministers and their spouses as well as most of the deacons and deaconesses We were at three ...


text: Election 2008: Issues Mormons Divided On Same-Sex Marriage Issue October 31, 2008 California's Proposition 8 would overturn gay couples' recently granted right to marry. The Church of Jesus Christ of Latter-day Saints asked congregations to work to pass the measure. Some Mormons are troubled by the church's prominent role in a political fight. Listen Loading… Playlist Download Embed Embed Close embed modal <iframe src="http://www.npr.org/player/embed/96405866/96510649" width="100%" height="290" frameborder="0" scrolling="no">


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And too few insights gleaned from them
Circular RNA Surprise
February 28, 2013|
Previously enigmatic circular RNAs have been found to influence gene expression by binding to and blocking another class of regulatory RNA, the microRNAs.
February 28, 2013|
Previously enigmatic circular RNAs have been found to influence gene expression by binding to and blocking another class of regulatory RNA, the microRNAs.
December 17, 2012|
The Carribean island, with the help of researchers using creative ways of getting the message out, has rallied behind sequencing the genome of an endemic parrot.
December 10, 2012|
A new initiative lead by the UK's National Health Service aims to sequence the genomes of as many as 100,000 patients, a project that will cost £100 million.
November 30, 2012|
Researchers are getting closer to detecting abnormal tumor DNA circulating in the bloodstream.
November 28, 2012|
In the largest microbial eukaryote genetic sequencing effort ever attempted, researchers are investigating the transcriptomes of 700 marine algae species.
October 22, 2012|
J. Craig Venter plans to develop a machine to find and sequence DNA on Mars, but another genomics mogul, Jonathan Rothberg, may beat him to it.
September 26, 2012|
A large Chinese sequencing center's purchase of Complete Genomics, a California-based DNA services company, ensures the valued technology will remain on the market.
July 25, 2012|
The first company to attempt to cheaply sequence the genomes of 100 people aged over 100 announced its intention to compete for the 2013 X prize.
July 3, 2012|
A new algorithm brings together data from second - and third - generation genome sequencers.
June 28, 2012|
Genome sequence analysis confirms mobile genetic elements are a mutagenic mechanism in a variety of cancers.


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Orebfuscator - Anti X-RAY:
Version: 3.0.5
This plugin is used to counter X-RAY Client mods.
It modifies data that are sent to clients to hide blocks of your choice, such as Ore, chests, dungeons, etc.
It does not manipulate blocks in the world file, thus is safe to use.
ProximityHider is a feature that hides chests that are far from players.
Forum link
Configuration Guide
Dropbox link removed due to Bukkit regulations. Latest version should be on BukkitDev, if not, then the dropbox link is still in the forum thread.
Please also download ProtocolLib and install it in the plugins folder. Download link: http://dev.bukkit.org/server-mods/protocollib/
- Advanced algorithm that hides ore, chest and and anything you specify in the configuration
- Spout compatible, but optional
- No modifications to CraftBukkit.jar is needed
- Customize the blocks you want to hide
- HIDES DUNGEONS and other blocks that are in the dark
- Different hiding mode, or scrambling.
- Extensive configuration. Change updating methods depending on your bandwidth and processing speed.
- Hide hidden chests and furnaces until a player is close to it.
Install: Copy "Orebfuscator.jar" to your plugins folder, and restart/reload your server.
See this: https://github.com/lishd/Orebfuscator/wiki
See this: https://github.com/lishd/Orebfuscator/wiki
See this: https://github.com/lishd/Orebfuscator/wiki
Source Code
Here you go! (Hosted on Github)
- This plugin utilises Hidendra's plugin metrics system, which means that anonymous information of your OS and setup will be collected and sent to mcstats.org
- Opting out of this service can be done by editing plugins/Plugin Metrics/config.yml and changing opt-out to true.
- This plugin uses an Auto-updater to fetch the latest version from BukkitDev automatically. It can be disabled in the configuration file.
- AFFORESS, major help on code optimization and spout support!
- raphfrk, helped with packet processing
- Many donators!
- None ATM
- A command that generates the world cache for the existing chunks.
Version 3.0.5
- Updated to MC1.8.3.
Full history: https:github.com/lishd/Orebfuscator/wiki/Version-History
CPU testing with Initial radius and Caching:
A quick donation is always appreciated :D
Forum link
chris_61 Apr 06, 2015 at 13:15 UTC - 0 likes
Why is this showing putting tnt all over my map?
tremby90 Apr 01, 2015 at 04:52 UTC - 0 likes
can this be used to stop people from downloading your world? using world downloader mod
BootyCrafter Apr 01, 2015 at 01:58 UTC - 0 likes
titanrat Mar 30, 2015 at 13:31 UTC - 0 likes
Doesn't work with spigot 1.8.3 No errors in console.
craigbrian Mar 29, 2015 at 05:27 UTC - 0 likes
@lishid Please make this compatible with Spigot 1.7.10-1.8 specifically spigot1649.jar https://www.dropbox.com/s/9nbgzooxsgccy4p/spigot1649.jar?dl=1
Mirror: https://github.com/Sarabveer/CraftBukkit-Spigot-Binary/blob/master/spigot-1.7.10-R0.1/spigot-1.7.10-R0.1-1649.jar?raw=true I can't get any version to work :( I even tried to take your GitHub code in and do it myself but it's over my head.
Bobcat00 Mar 29, 2015 at 01:11 UTC - 0 likes
You can get a 1.7.9 version here - http://dev.bukkit.org/bukkit-plugins/orebfuscator/files/
Server: lanapug.com - Survival/PVP, 7 Mob Arenas, Creative World
Web site: www.bobcat00.com
NickGamerGR Mar 28, 2015 at 20:50 UTC - 0 likes
Please release an 1.7.9 version!
TheD4n Mar 26, 2015 at 02:54 UTC - 0 likes
Users keep getting blocks not loading and the only fix so far is to clear orb cache and relog. Any idea why this is happening?
advm301 Mar 23, 2015 at 17:01 UTC - 0 likes
HELP! Just a few weeks ago this was working for me until I uninstalled it because of lagg. I have since then reinstalled it and now it does absolutely nothing! I all the commands work such as /ofc clearcache and /ofc engine changes and when i do /plugins i see it in the list and it is green to indicate that it works but whenever i use an xray i can see every ore! I need help why wont this work again!
Bobcat00 Mar 21, 2015 at 01:24 UTC - 0 likes
I'm also seeing fake TNT where there are exposed stone blocks adjacent to torches. I can provide a screenshot if needed.
- Date created
- Sep 24, 2011
- Categories
- Last update
- Mar 18, 2015
- Development stage
- Mature
- Language
- enUS
- License
- GNU General Public License version 3 (GPLv3)
- Curse link
- Orebfuscator
- Downloads
- 1,693,628
- Recent files
- R: Orebfuscator 3.0.5 for 1.8.1 Mar 18, 2015
- R: Orebfuscator 3.0.4 for 1.8.1 Jan 08, 2015
- R: Orebfuscator 3.0.3 for 1.8.1 Jan 08, 2015
- R: Orebfuscator 3.0.2 for 1.8.1 Jan 06, 2015
- R: Orebfuscator 3.0.1 for 1.8.1 Jan 06, 2015


text:
Fire officials issued a reminder today that the period for opening burning of vegetative materials within areas protected by the Reno Fire Department and Sierra Fire Protection District remain closed due to dry conditions.
The Reno Fire Department and Sierra Fire Protection District suggest property owners consider non-burning techniques for vegetation management including composting and chipping vegetation.
Burning is only allowed with a permit and during publicized open burn seasons. The open burn season generally begins March 1, when conditions allow, and continues through the declaration of fire season.
If weather and fuel conditions permit, a short open burning period may be allowed later this spring. Washoe County District Health Department Air Quality Regulations prohibit burning from November 1 through March 1.
For more information about obtaining a burn permit contact the Reno Fire Department's Burn Permit Hotline at 328-3659 or visit www.reno.gov and select "Fire Department" from the Department drop-down menu. Information regarding open burning in the Sierra Fire Protection District can be obtained by calling 849-1108 extension 5 or by emailingSierraFireBurnPermit@
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- Regents plan investigation of Chancellor Klaich - July 3, 2015


text:
- Recent
- Top 25
New! Custom date formats in the API
Format your dates however you like
May Hack Day
Apple Watch app, pricing page designs, modules and more...
April in review
A retrospect of techy trinkets from the month
We'll be at AWS Summit London
Come meet the GoSquared team and see our talks
Building Two-factor Authentication
Behind the scenes: How we built a robust 2FA system
How to save precious bytes on a Node.js server
Micro-optimisations can sometimes make a (relatively) big difference
Running T2 instances on EC2
How to save a boatload of money by running lots of tiny instances
Impact of Facebook downtime on global traffic
How Facebook's outage affected social traffic around the world
Deprecation of insecure HTTP API
Long live HTTPS!
Top 25
Powered by the GoSquared API.
Powered by the GoSquared API


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Wonderland Trail
Wonderland Trail is located near the Bass Harbor Lighthouse on the western side of Mount Desert Island. Not a terribly long or hard trail, you walk throught the woods through pine trees and rosebushes and end up on a gorgeous craggy beach. I say it's off the beaten path because there were a lot less people here when we visited which was right in the middle of the day. You wont see nearly as many people over on the western side of the Island as you will over on the Loop road.
Along Route 102A, you will see signs and a little pulloff parking "lot" . The trail is next to it
- National/State Park
- Adventure Travel
- Hiking and Walking
Mt. Desert St. Cemetery
Bar Harbor can get a wee bit crowded so best to ditch your car as soon as you get into town and stroll some of the backstreets. One nice quiet place is the cemetery on Mt. Desert Street. Though right in the middle of town, it seems not a soul bothers to check it out. All the more reason you should. ;)
- Historical Travel
- Road Trip
- Architecture
A nice end point on our climb
The climb up Precipice was challenging and created a little bit more excitement for us. It was really worth the extra effort to go up the way we did. The views from the top were fantastic. We could see Bar Harbor and some nearby islands below with a cruise ship just off shore. The mainland of Maine and the open ocean were also on the horizon.
- Adventure Travel
Get adventurous and go up the challenging way
It was kind of my decision to take this way up but my brother and Diana from Hungary were troopers. They had never done this type of climb before. They were a bit unsure at times because there were some steep dropoffs and a slip could mean a nasty fall, but they perservered. A good way to go is to take it slow and really pay attention to where you're putting your hands and feet as you climb. This is not a route you want to take if it is raining or there is an electrical storm. The ledge surfaces get very slippery and you could slide right off the edge. Being exposed on a ledge could also attract lightening to you. Check out weather conditions before you go.
- Adventure Travel
Climb the Precipice Trail
This may not be off the beaten path for some, but it is certainly not the way most people would choose to get up Mt Champlain. When starting out at the trailhead, there's a warning that this is a non-technical climb. You won't be getting up this way without using your hands. The ascent is very steep and involves rungs and ladders. There are exposed cliffs and ledges and you'll also need to crawl a bit through some boulders. If you have a fear of heights don't attempt this.
- Adventure Travel
Have a look at the trees
Yes, I know, some may think this is boring
then they should be there in autumn.
Every tree his own firework!
- Adventure Travel
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Nature's Alchemy 100% Pure Essential Oil Lemon -- 2 fl oz
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Lady Hi-Liner basketball has made the State Tournament for the first time since 2005.
East Region Tournament No. 4 Valley City took top seed West Fargo to the wire in Friday night's 59-47 semifinal, then edged No. 7 Wahpeton 57-50 in Saturday's State qualifier for a date with West Region winner Bismarck in Thursday's 1 p.m. State quarterfinal in Minot.
vs. West Fargo https://www.facebook.com/media/set/?set=a.499283313515429.1073741940.116...
vs. Wahpeton https://www.facebook.com/media/set/?set=a.499286650181762.1073741941.116...
March 7 - 9 Weekend Sports Action
Eastern Dakota Conference Girls Basketball Tournament Semifinals
(4) Valley City vs. (1) West Fargo, 5:30 p.m. Fargo Civic Center [Wear blue]
Eastern Dakota Conference Girls Basketball Tournament
* (4) Valley City vs. [(2) Shanley - (3) Fargo Davies winner if Hi-Liners win Friday] in EDC Tournament Championship, 6 p.m. Bison Sports Arena
* (4) Valley City vs. [(6) Fargo South - (7) Wahpeton winner if Hi-Liners lose Friday] in a loser-out State Qualifier, 11 a.m. Bison Sports Arena
Fairhill Properties won all four games against Dirty Johnny's
Dacotah Bank won 3 of 4 from Wesley Acres
Bettin's Greenhouse and Farmers Union Insurance split 2-2
High Team Game and Series
Dacotah Bank 741 and 2,143
High Individual Game
Rhonda Nelson
High Individual Series
Betty Marler 478
High Averages
Cheryl Olson 162, Doris Weller 155, Mary Tangen 154
Farmers Union 24-16
Dirty Johnny's 22-18
Bettin's 22-18
Dacotah Bank 21-19
Fairhill Properties 16-24
Wesley Acres 15-25
Below are the All Maple River Conference Boys and Girls Basketball selections, as voted on by conference coaches.
Both the Maple Valley boys and girls were this year's conference champions.
Boys Basketball All Maple River Conference
Barnes County North
David Undem
TJ Steckler
Daniel Lindgren
Graham Hurlburt
Jared Kittelson
Finley-Sharon/Hope Page
Ethan Jacobson
Lucas Montag
Maple Valley
Brady Behm
James Richman
Riggs Nudell
Northern Cass
Cody Springer
Hunter Ward
Taylor Kyllo
Valley City State head football coach Dennis McCulloch announced on Thursday the addition of three more high school freshmen who have signed letters of intent to attend VCSU and play football for the Vikings this fall: wide receiver/defensive back Tyler Ukestad of Jamestown, wide receiver Curtis Marden of Powers Lake, N.D., and linebacker Riley Fredrickson of Wasilla, Alaska.
Read this story in Friday's Times-Record.
Valley City State baseball opens the 2014 spring season this weekend with Saturday and Sunday doubleheaders at Midland University in Fremont, Neb.
Games begin at 1 p.m. and 3 p.m. both days.
The Saturday forecast is iffy, calling for temperatures near 35 degrees and mostly sunny skies. The forecast for Sunday is sunny with a high near 58 degrees.
Fans can watch this weekend's games at http://www.livestream.com/midlandathletics.
Valley City State senior forward Brittany Lehner was named First Team All Conference Tuesday night by the North Star Athletic Association league office.
A 5-foot-10-inch forward from Watertown, S.D., Lehner is one of five players to receive First Team honors in women's basketball after averaging 18 points and 7.2 rebounds per game this season and shooting 59.1 percent from the field, the fourth-best shooting percentage in NAIA Division II basketball.
Class B Girls Regional Tournament Schedule
Region 1 Tournament
Maple Valley (2nd place District 2) vs. North Sargent (3rd place District 1), 4:30 p.m. March 10 at North Dakota State College of Science in Wahpeton, N.D.
Region 3 Tournament
Barnes County North (4th place District 5) vs. South Border (District 6 winner), 6 p.m. March 10 at Hazelton-Moffit-Braddock High School
LaMoure/Litchville-Marion (District 5 winner) vs. Linton/HMB (4th place District 6), 3 p.m. March 10 at Hazelton-Moffit-Braddock High School
Valley City State senior guard Aaron Duske and junior guard AJ McDonald were named to the North Star Athletic Association Men's Basketball All-Conference Team, the NSAA announced Tuesday.
Duske, of Buffalo, Minn., one of five First Team All-Conference selections, averaged 14.9 points and seven rebounds this season and reached 1,000 career points earlier in the year. He graduates with 1,375 points, which ranks him seventh in school history, scored 20 or more points in five games, and 19 times he reached double figures. Duske was the only senior on the team this year.
Eastern Dakota Conference Girls Tournament No. 4 seed Valley City figured out No. 5 seed Fargo North's 2-3 zone early in Tuesday's semifinal and rode junior guard Claire Olson's double-double to a 65-50 win as three Hi-Liners in all scored double-digit points.
Valley City faces No. 1, Class A second-ranked West Fargo in the semifinals at 5:30 p.m. Friday at the Fargo Civic Center. With a win, the Hi-Liners enter the EDC Championship at 6 p.m. Saturday at North Dakota State University. With a loss, Valley City plays a State qualifier Saturday at 11 a.m. at NDSU.


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Originally written on Fox Sports Arizona | Last updated 12/29/11
Two winding paths of misfortune have led to the intersection of need for the Phoenix Suns and opportunity for Michael Redd. The Suns are limping toward the close of their opening week at 0-2. Redd, whose impressive career has been sidetracked by knee injury, may -- if he's not eventually reduced to limping through the compacted schedule -- be able to help. His signing was announced Thursday. This means that in their quest to identify a temporary go-to scorer, the once-mighty Phoenix offensive machine is required to take the "where do we go now?" approach. For positive spin, we give you a partial resume for Redd, a 32-year-old, 6-foot-6 wing who has spent each of his 11 NBA seasons with the Milwaukee Bucks: The 43rd selection in the 2000 NBA Draft, Redd pumped in more than 20 points per game six times in Milwaukee and has a career average of exactly 20 points per game. He was chosen over then-rookie LeBron James for the Eastern Conference All-Star team in 2004 and earned an Olymp...
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The Society for Biomaterials will hold its 2015 Annual Meeting (SFB 2015) in Charlotte, NC, USA on 15th-18th April 2015 at the Charlotte Convention Centre.
Dates and deadlines:
Registration is open, sign up now to avoid disappointment.
The abstract submission deadline for poster presentations has passed and abstracts are no longer being accepted.
Browse the programme:
The programme for SFB 2015 is live, so why not take a look. Key speakers include: Ashutosh Chilkoti, Duke University and Stuart B. Goodman, Stanford University. For a full list of speakers visit the website.
The theme of this year's meeting is Driving Innovation and the Race to Translation; a tribute to the city's rich motor racing history. In keeping with that theme, the meeting will be organised into five thematic areas:
1. Translation: Focused on translating research from the lab to the clinic, advancing biomaterials in their product development life cycle
2. Biofabrication/Biomanufacturing: Manufacturing techniques e.g. nanoprinting, rapid prototyping, microfluidic based printing, etc.
3. Multi-Functional Biomaterial Design: Drug/gene delivery, biosensing, and complex tissue regeneration, etc.
4. Biocompatibility and Immune Engineering: How to harness/modulate the immune response (innate and adaptive) for biomaterial design or technology translation
5. Other topics of interest to the biomaterials community
For full details visit the SFB 2015 website.


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Rated 5 out of 5 stars
Please please make this work with Firefox Nightly 8.0a1...please..This review is for a previous version of the add-on (1.0.4).
Did you changed the maxVersion in install.rdf? I only test betas, sorry. But if Mozilla continues with this release cycle you'll soon see a proper version ;)


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It has been a month since Jay Leno returned to his seat behind the desk of "The Tonight Show," following a prolonged three-way battle between himself, former "Tonight Show" host Conan O'Brien and NBC. Since O'Brien closed up shop, he has started one of the best Twitter feeds on the Internet and is about to embark on a national tour (cleverly dubbed the Legally Prohibited From Being Funny on Television Tour ). Meanwhile, Leno surged ahead of longtime rival David Letterman in the ratings, and he remains willing to speak out about the fiasco that unfolded at the beginning of the year.
"The View" host Joy Behar was a guest on "The Tonight Show" on Tuesday, and in a behind-the-scenes video blog she filed for CNN, Leno spoke frankly about what happened at NBC.
"Conan got screwed," he said. "I got screwed. I mean, this is TV. The reason show business pays a lot of money is so when you get screwed, you've got something left over. If you're a nurse or a cop and you get screwed out of your pension, you're screwed out of your pension. On TV, at least you walk away with some money."
Leno told Behar that he feels bad about how O'Brien was treated and hopes that the spirit of competition between them will continue. "Conan was treated terribly, and I was treated terribly. I think Conan will come back and he'll be strong, and we'll all compete against one another. It should be me against Letterman against Conan against [Jimmy] Kimmel, and then we'll see who wins."
Behar then called Kimmel "a bit of a turncoat," referring to the incident in January when he went on "The Jay Leno Show" and slammed the host . But Leno is letting that water flow under the bridge. "What are you gonna go? Welcome to showbiz," he said. "All comics are friends. When you go on stage, your job is to blow the other guy off the stage. I get it! That's the way it works. You don't impede someone else's opportunity, but when you go out there, that's what you do."
Are you watching "The Tonight Show" with Jay Leno as host? Let us know in the comments!


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Men's / Boy's Tennis
Get the latest Louisville Catholic Sports tennis news, scores, stats, standings, and other high school tennis announcements on LouisvilleCatholicSports.com. Catch up with news about DeSales, Holy Cross, St. Xavier, and Trinity tennis along with more coverage of the local Catholic high schools on LouisvilleCatholicSports.com.
LEXINGTON, Ky. — Brandon Lancaster put his name in the KHSAA tennis record books Saturday when he became the 19th player in state history to win a second straight singles championship. Lancaster was aware of the company he was joining and was appreciative to be a member of such a... +Read More


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Obtaining an accurate sequence alignment is fundamental for consistently analyzing biological data. Although this problem may be efficiently solved when only two sequences are considered, the exact inference of the optimal alignment easily gets computationally intractable for the multiple sequence alignment case. To cope with the high computational expenses, approximate heuristic methods have been proposed that address the problem indirectly by progressively aligning the sequences in pairs according to their relatedness. These methods however are not flexible to change the alignment of an already aligned group of sequences in the view of new data, resulting thus in compromises on the quality of the deriving alignment. In this paper we present ReformAlign, a novel meta-alignment approach that may significantly improve on the quality of the deriving alignments from popular aligners. We call ReformAlign a meta-aligner as it requires an initial alignment, for which a variety of alignment programs can be used. The main idea behind ReformAlign is quite straightforward: at first, an existing alignment is used to construct a standard profile which summarizes the initial alignment and then all sequences are individually re-aligned against the formed profile. From each sequence-profile comparison, the alignment of each sequence against the profile is recorded and the final alignment is indirectly inferred by merging all the individual sub-alignments into a unified set. The employment of ReformAlign may often result in alignments which are significantly more accurate than the starting alignments.
We evaluated the effect of ReformAlign on the generated alignments from ten leading alignment methods using real data of variable size and sequence identity. The experimental results suggest that the proposed meta-aligner approach may often lead to statistically significant more accurate alignments. Furthermore, we show that ReformAlign results in more substantial improvement in cases where the starting alignment is of relatively inferior quality or when the input sequences are harder to align.
The proposed profile-based meta-alignment approach seems to be a promising and computationally efficient method that can be combined with practically all popular alignment methods and may lead to significant improvements in the generated alignments.
Keywords:Multiple sequence alignment; Iterative refinement; Dynamic programming
The alignment of multiple DNA, RNA or Protein sequences is of major importance for a variety of biological modelling methods, including the estimation of the phylogenetic tree of the sequences and the prediction of their structural, functional and/or evolutionary relationships [1]. In addition, the recent advances in rapid, low-cost sequencing methods, have resulted in the accumulation of large amounts of molecular data to be processed, making thus the need for fast and accurate multiple sequence aligners even more imperative [2].
A widely used approach to cope with the Multiple Sequence Alignment (MSA) problem, is the employment of a computational formulation comprised of two major components, namely an objective function [3] able to quantify the degree of similarity of a given alignment and an optimization procedure that targets at identifying the optimal alignment based on the underlying objective function [4]. Concerning the former component, the Sum-of-Pairs scoring model (SP) [4,5] remains amongst the most popular choices [6-8].
The maximization of the SP score is usually performed using dynamic programming. For the pairwise alignment case, an optimal (numerically but not necessarily biologically) solution can be found within reasonable time. However this does not hold for the multiple sequence alignment case, where it has already been shown [9-12] that obtaining the optimal alignment using the SP score is NP-hard. To overcome the computational intractability of the MSA problem, a large number of efficient heuristic algorithms have been proposed with the most popular being the progressive alignment approach [13-16].
In progressive alignment the sequences are initially placed on a bifurcating tree according to their degree of similarity. Then, they are progressively aligned in pairs following the formed guide tree in a bottom-up order until its root is reached. At each step, two nodes of the tree (i.e. two sequences, a sequence and an alignment or two alignments) are aligned by a standard pairwise alignment algorithm, and the deriving subalignment is retained to be used at a subsequent step. One important aspect of the progressive alignment strategy is the "once a gap, always a gap" rule, first introduced in [13]. Based on this policy, once a group of sequences is aligned, all gaps in the alignment are replaced by a neutral 'X' symbol ensuring that all subsequent pairwise alignments will be consistent with the pre-existing alignment of the group [17]. This rule by definition implies that once a group of alignments has been built up, they will remain fixed even in the view of new sequences that could potentially improve the overall alignment. Consequently, early errors in the progressive alignment steps are accumulated and propagated to later alignment stages compromising thus the alignment quality.
This problem is often tackled by using iterative refinement techniques [18,19]. In iterative refinement, one sequence (or a group of sequences) is iteratively subtracted and realigned against the alignment of the remaining sequences. Via this sequence-profile or profile-profile realignment, a new alignment is obtained which is then used for the next iteration of the algorithm. The refinement terminates when a fixed number of iterations is reached or when the alignment remains unchanged between consecutive iterations [17]. Although these methods are very efficacious at curating early alignment errors, they only partially address the "frozen subalignments" issue, since at each iteration of the algorithm only one sequence (or a group of sequences) is realigned whereas the alignment of the remaining sequences is kept fixed.
In this article we present a variation of the aforementioned iterative refinement strategy where all sequences may be simultaneously and independently realigned against a summarization profile that encapsulates all the starting alignment information. The process begins by constructing a standard profile that summarizes all the initial alignment information. Then, a series of individual (and possibly concurrent) sequence-profile pairwise comparisons takes place, recording the way that each sequence is aligned against the profile. The new alignment is then indirectly inferred by merging all the individual subalignments into a unified group. The proposed approach is implemented as part of a newly introduced meta-aligner under the name ReformAlign (Reformed Alignments) and is freely available to the public from http://evol.bio.lmu.de/_statgen/software/reformalign/ webcite under the GNU General Public License (version 3 or later). We call ReformAlign a meta-aligner, in the sense described in [20], meaning that an initial alignment is required and it can be used with a variety of alignment programs.
As currently ReformAlign can only align DNA/RNA sequences, for the needs of our performance evaluations real nucleic acid datasets of variable length and average sequence identity rates were used. Our experimental results demonstrate that the suggested profile-based modification of the classic progressive alignment and iterative refinement strategies is able to overcome the challenges posed by the propagation of early pairwise alignment errors and that ReformAlign is an efficient, well suited approach that may improve on the performance of a vast variety of existing alignment software.
Alignment strategy
ReformAlign aims at improving on the quality of an existing alignment by providing the sequences with an additional opportunity to be individually re-aligned against a standard profile that efficiently summarizes the starting alignment information. The idea is that via this re-alignment step, early alignment errors caused by "frozen" subalignments may be fixed, delivering thus better results in terms of alignment accuracy.
Towards this end, in ReformAlign the alignment of the sequences is performed in two steps. The first step involves the construction of a non-probabilistic profile from an existing alignment, whereas during the second step all the sequences are individually realigned against the profile that derived from the first step. The new alignment is finally indirectly reconstructed by merging all the individual sequence-profile subalignments into a unified group. Due to the nature of the latter step, all pairwise sequence-profile comparison can be performed in parallel, improving thus the aligner performance in terms of execution time.Upon completion of the latter step, a new (reformed) alignment is derived which is very often different to the starting one. Since this alignment may be also susceptible to further improvement, the whole process can be re-initiated using the reformed alignment as starting alignment. The algorithm terminates when either the alignment between two successive runs remains unchanged or a pre-defined maximum number of iterations is reached. A diagrammatic overview of the ReformAlign alignment logic flow is provided at Figure 1.
Figure 1. Logic Flow diagram of the ReformAlign alignment strategy. The algorithm creates an initial profile based on a starting alignment deriving from an established aligner. Then, all the sequences are aligned against the formed profile to obtain the reformed alignment. Notice that in this step, in case a new insertion is detected, the algorithm automatically switches to a profile fine-tuning mode in order to modify the profile to also account for the new insertion(s). After all sequences have been successfully aligned against the profile, the deriving alignment is indirectly inferred by merging all the estimated subalignments into a unified set and it is then freed from uninformative void columns (i.e. columns consisting exclusively of gaps). The process is repeated using the reformed alignment as starting alignment until the alignment between two successive runs remains unchanged or a predefined maximum number of iterations is reached.
A. Profile construction step
In the profile construction step, a popular aligner is employed to infer a starting alignment for the examined group of sequences. Then, a summarization profile is constructed upon the starting alignment, having as many position as the columns in the alignment. Each position in the profile is characterized by a series of < Residue, Weight > pairs sized to fit the number of distinct residues that appear in the corresponding column of the alignment. The "Residue" part in each pair corresponds to the label of a particular residue in the alignment column whereas its "Weight" represents the number of times that this particular residue appears in that column.
B. Sequence alignment step
In ReformAlign, the formation of the final alignment is performed indirectly by aligning all the sequences against the profile that derived from the first step. All sequence-profile alignments are performed using a standard pairwise alignment algorithm and for each sequence its alignment against the profile is recorded. By joining all deriving pairwise alignments into a unified group, it is possible to infer the final alignment of the examined sequences. In contrast to traditional iterative refinement approaches where at each refinement only one sequence (or a group of sequences) is realigned, in the proposed approach all the sequences have a chance to align differently against the formed summarization profile.
Intuitively, the majority of the pairwise alignments that will come up from this step will have at most as many columns as the number of positions of the formed profile. Nevertheless this is not always the case. For example, it might happen that a particular residue is aligned as a mismatch in one of the columns of the starting alignment, but during the sequence alignment step the score obtained by misaligning this residue against the corresponding profile position might be smaller than the penalty for opening a new gap in the alignment. In cases where the profile has to be updated in order to accommodate new insertion(s), ReformAlign automatically switches to a profile fine-tuning mode. As soon as the profile has been successfully updated, ReformAlign restarts the sequence alignment step using the new profile.
Another important aspect of the suggested meta-alignment technique is that since the alignment of the sequences may alter during the sequence alignment step, it might happen that none of the sequences aligns against one or more of the positions of the final profile, leading thus to the derivation of void (all gapped) columns. To fix for this issue, upon completion of the sequence alignment step, the program checks for uninformative void columns and completely removes them from the reported alignment.
C. Iterative refinement
After the sequence alignment step has been completed, a new alignment is produced. In case the reformed alignment is different to the starting one, ReformAlign may be iteratively applied to further fine-tune the produced alignment(s). The iterative refinement step terminates when the pre-defined limit of iterations is reached or when the deriving alignments between two successive runs of the algorithm are identical.
Dynamic programming and scoring system
The dynamic programming scheme employed by ReformAlign for the sequence-profile alignment task is the Gotoh's [21] affine-gap penalties variation of the Needleman-Wunsch global alignment algorithm [22], as described in [17].
Regarding the scoring scheme for assessing the similarity between a position in a sequence and one from the profile, in contrast to the average function encountered in various alignment models such as ClustalW [6], we employed an additive variant where the observed counts of the residues are used instead of their averaged frequencies, as follows:
where A is the current alphabet (e.g. Α = {A, C, G, T} for DNA alignments), Ca and Cb represent the observed counts (tallies) of residues a and b respectively and Suba → b corresponds to the score matrix value for aligning residue a to b.
The main reason for selecting aforementioned scoring scheme is that the use of the observed counts instead of the relative frequencies allows for a better discrimination between well-conserved columns. For example, a profile position exclusively comprised of a several Cytosine residues will have exactly the same score as another profile position formed by a single Cytosine, if the averaged sum of pairs scoring scheme was used, but the score for the former case would be significantly higher if the additive scoring model was employed instead.
Alignment parameters
The choice of a proper values for the alignment parameters is essential for the multiple sequence alignment problem, since it has been proven [23,24] that it may significantly affect the decision of the "optimal" alignment among the numerous alternatives. These parameters may significantly vary among different alignment approaches and they are usually decided by cross validation testing.
In ReformAlign, the alignment parameters that have to be defined are the substitution scores and the gap opening and extension penalties. However, it would be desirable in our strategy that each residue of the sequences could inexpensively skip any low-scoring profile positions in order to align itself against another higher scoring position. Furthermore, based on the fact that by definition the profile already incorporates enough positions where every residue of the sequences could possibly be aligned, opening a new gap in the profile should be more costly than opening a gap to the sequence. Consequently, in ReformAlign four distinct types of gap penalties are considered: a penalty for opening a gap in the profile (HGOP), a penalty for extending a gap in the profile (HGEP), a penalty for opening a gap in the sequence (VGOP) and a penalty for extending a gap in the sequence (VGEP). As in ClustalW, terminal gaps (TGOP and TGEP) are not penalized in our approach either.
An additional parameter constraint deriving from the inherent properties of our model is that the substitution scores should be substantially higher than the gap penalties. In case this restriction is not met, it might happen that many sequences require a profile update during the sequence alignment phase, resulting thus in very large profiles containing a lot of poorly supported positions. To avoid such cases it is recommended that substitution scores are always set to (relatively high) positive values whereas gap opening and extension penalties are set to negative values.
Regarding the substitution weights, ReformAlign currently uses a modified version of the HOXD substitution matrix [25] where all scores are increased by a positive "bonus" value, so that the aforementioned condition is met and to further reward (mis)matches compared to gaps. Nevertheless, increasing all substitution scores by the same "bonus" value results in understating the differences between matches and mismatches. For this reason, each score value is then increased by a re-adjustment coefficient (Coeff).
The default bonus value and re-adjustment coefficient applied to the score matrix as well as the default gap opening and extension penalties were decided based on a greedy parameters search that was performed using MUSCLE [8,26] as the starting aligner and a separate training set with alignments from the data-set 1 of a previous version of BRAliBase (i.e. BRAliBase II) [27]. For each test in the training set, the percent APSI value of the initial alignment was calculated and then more than 300,000 parameter combinations were greedily assessed by comparing each time the deriving (reformed) alignment to the expected one (reference alignment) in terms of alignment accuracy. The results of this cross-validation analysis suggested that the score matrix bonus value and the gap penalties should be re-adjusted according to the APSI value of the initial alignment based on the subsequent formulas:
where APSIp is the percent Average Pairwise Sequence Identity calculated over the initial alignment.
The aforementioned scheme for defining the starting parameter values was used for all the experiments outlined in the present study and is automatically employed by default in ReformAlign, unless more appropriate starting values are defined to the program by the user.
Finally, ReformAlign follows ClustalW's paradigm [6] by heuristically updating the gap opening and extension penalties during runtime, in an attempt to deliver superior alignment results. In particular, the penalties are modified based on the following criteria:
Dependency on the lengths of the sequences
Both the gap opening and gap extension penalties are modified depending on the ratio between the length (number of residues) of the sequence to be aligned and the largest sequence in the set. Intuitively, gaps should be penalized harder for shorter sequences in order to avoid having alignments with too many and/or sparse gaps. The calculation of the starting gap penalties is performed according to the following formula:
where GP represents all types of gap penalties (HGOP, HGEP, VGOP and VGEP), M is the length of the largest sequence in the set and N is the length of the sequence to be aligned.
Position-specific penalties based on the profile conservation rate
During the computation of the dynamic programming matrix, the gap opening penalties (HGOP and VGOP) are modified in a position-specific way depending on the conservation rate of the profile. If a position in the profile is well conserved (the weights for one or more of its pairs are set to relatively high values), then this implies that the specific position is strongly supported by many sequences (probably even by the examined sequence itself – based on the starting alignment) and thus the penalty for opening a new gap against this profile position should be higher compared to weakly supported positions. Consequently, the gap opening penalties per position are calculated as follows:
where GOP represents the gap opening penalties (HGOP, and VGOP), PCRi is the Profile Conservation Rate for the ith position of the profile calculated as a fraction of the maximum weight value among all < Residue, Weight > pairs contained in the ith position of the profile divided by the sum of all weights of the pairs in the ith position and SSij is the substitution score for aligning the ith position of the profile against the jth position of the sequence.
The idea behind the multiplication of the profile conservation rate by the substitution score is to linearly increase the gap opening penalties for conserved columns, so that gaps would be favored only if subsequent matches of the sequence against ensuing positions of the profile would score high enough to sufficiently compensate for the very costly gap that was opened.
In order to assess the efficiency of ReformAlign in improving the quality of existing alignments, testsets from the BRAliBase 2.1 RNA alignment database [27,28] and the DNA SMART database [29] were used.
BRAliBase 2.1
BRAliBase (Benchmark RNA Alignment database) is a collection of RNA alignments taken from the Rfam database. It was initially introduced in [30], but since then it has been further enriched with additional alignments [27,28], leading to its current version v2.1 which contains in total 18,990 aligned sets of sequences (packed in sets of 2, 3, 5, 7, 10 and 15 sequences) with an Average Pairwise Sequence Identity (APSI) rate ranging between 20% and 95% [28]. For the needs of the present study we limited our analysis to the BRAliBase alignments composed of 7, 10 and 15 sequences per alignment.
The DNA Reference Alignment Benchmarks database [29] was proposed in 2007 as a collection of DNA reference alignments for the assessment of MSA applications. It is comprised of multiple DNA sequence alignments (MDSAs) corresponding to protein alignments of the BAliBASe [31], OXBench [32], PREFAB [8] and SMART [33] benchmarking datasets. For our experiments, we considered all alignments corresponding to the SMART database containing between 20 and 300 sequences per alignment. Since however ReformAlign does not currently support ambiguous characters, we limited our analysis to a total of 264 alignments containing exclusively unambiguous DNA letters.
Accuracy measures
To assess the agreement between the reconstructed and the reference alignments, the following measures of accuracy were considered.
The sum of pairs (SP) score (aka developer's score - fD[34]) was initially proposed by Thompson et al.[35] and is defined as a fraction of the number of the correctly aligned residue pairs in the reconstructed alignment over the number of aligned residue-pairs of the reference alignment. If the denominator of the fraction is replaced by the number of residue-pairs in the test alignment, then the reverse sum of pairs score (aka modeler's score - fM) is obtained. Finally, the total columns (TC) score is computed by dividing the number of correctly identified columns in the reconstructed alignment over the total number of columns in the reference alignment.
Although these metrics can provide an overview of the quality of the deriving alignments, each one has its own drawbacks. The developer's score fails to penalize over-alignments and could give a great score to an alignment that erroneously aligns non-homologous regions, whereas the modeler's score is not sensitive to detecting under-alignments and thus could possibly give high scores to alignments that systematically fail to align homologous regions (refer to [36] for further details regarding these types of alignment errors). Finally, the TC score is very sensitive to the misalignment of even a single sequence yielding a zero-valued TC Score even if all the remaining sequences in the examined group are properly aligned.
For this reason, two additional metrics were considered: a) the Cline's score (CS) [36], which efficiently addresses the issues of the developer's and modeler's scores by penalizing over- and under- alignments while taking into account minor shifts in the reconstructed alignment compared to the reference and b) the D-POS metric proposed by Blackburne and Whelan [37] which satisfies the conditions of symmetry and triangular inequality that are not met by the SP and TC scores, where at the same time it incorporates information from indels by recording the position where gaps occur. All accuracy measures take their values in the [0,1] range, with the exception of Cline's Score which may also take negative values in case there exist many large shifts. Moreover, in contrast to the remaining metrics, D-POS reports the distance between the generated and the reference alignment and thus lower D-POS values correspond to better alignments.
Since D-POS and Cline's Score can efficiently summarize the quality of the deriving alignments while at the same time being the most immune to deficiencies, the discussion will be mostly based on the experimental results of these two metrics.
Alignment programs
We assessed the effect ReformAlign on the deriving alignments of ten leading methods: ClustalW [38], ClustalO [39], MUSCLE [8,26], MAFFT [40], Kalign [7], GramAlign [41], ProbConsRNA [42], R-Coffee [43,44], PicXAA [45] and Dialign-TX [46]. These aligners implement a variety of alignment techniques, such as progressive alignment [7,38,41], iterative refinement [26,39,40], segment-based alignment [46], probabilistic/consistency-based alignment [42,44] and maximum expected accuracy alignment [45]. Moreover, R-Coffee specializes in the alignment of RNA sequences and could thus provide us with an overview of the way that the proposed sequence-based method may affect initial alignments that use secondary structure information.
Since our main intention was to examine the effect of ReformAlign on existing alignments rather than to analytically assess the efficiency of each aligner on the benchmark databases, we used the default settings for all aligners except for MAFFT which was executed using both the –FFT-NS-i and the –L-INS-i options. Finally, the default value for the maximum number of refinement iterations in ReformAlign was set to 5 for all experiments (an analytical listing of the command-line options used for each aligner is provided at Additional file 1: Table S1).
Additional file 1: Table S1. Command-line options per alignment method.
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Results and discussion
Each benchmark test was given as input to all the considered aligners in order to obtain an initial alignment which was compared against the reference. Then, this alignment was provided as input to ReformAlign and the newly generated alignment was also compared against the reference using all considered accuracy metrics. Via this procedure, we came up with eleven distinct alignment pairs for each benchmark test (Mafft was separately assessed for the FFT-NS-i and L-INS-i options). Furthermore, in order to determine the statistical significance of the differences between the initial and the reformed alignments, Wilcoxon signed rank tests were also performed for each alignment-pair. However, since multiple test cases in the benchmark data may contain sequences of the same families, the samples independency assumption of the statistical test might not be fully met and for this reason the reported results should be interpreted with caution.
BRAliBase 2.1
The results for the 2,218 BRAliBase benchmark tests are visualized in Figures 2 and 3. Moreover, in order to explore the effect of ReformAlign to testsets of increasing APSI, we grouped the benchmarks in four clusters according to the percent identity of the sequences. The average scores for each group are shown in Table 1 and Additional file 2: Table S2 and are graphically depicted at Figure 4.
Figure 2. Scatterplots of the Cline scores per alignment pair for the 2,218 benchmark tests of the BRAliBase dataset. A scatterplot is constructed per alignment pair for each one of the 2,218 benchmark tests. The diagonal line represents the expected plot if the initial and the reformed alignments were identical. Consequently, an accumulation of points above the diagonal line represents improved reformed alignments whereas points below the diagonal correspond to cases where ReformAlign worsened the initial alignment.
Figure 3. Boxplots of the averaged Cline scores of the considered alignment pairs for the 2,218 alignments of the BRAliBase dataset. The boxplots of the Cline scores averaged over all 2,218 test cases of the BRAliBase dataset are given at this figure. The figures are organized in pairs corresponding, from left to right, to the averaged performance of [ClustalW – Ref. ClustalW], [ClustalO – Ref. ClustalO], [Kalign – Ref. Kalign], [Mafft (FFTnsi) – Ref. Mafft (FFTnsi)], [Mafft (Linsi) – Ref.Mafft (Linsi)], [Muscle – Ref. Muscle], [DialignTX- Ref. DialignTX], [GramAlign – Ref. GramAlign], [ProbConsRNA – Ref. ProbConsRNA], [PicXAA – Ref. PicXAA], and [RCoffee – Ref. RCoffee]. The bars at the bottom of each pair represent the percentage of times where the reformed alignments were superior (blue bars), equal (green bars) or inferior (red bars) in terms of Cline score compared to the initial alignments.
Table 1. BRAliBase Cline scores
Additional file 2: Table S2. BRAliBase Results. Averaged D-POS, SP and TC scores for the 2,218 benchmark tests of the BRAliBase dataset. The results are presented averaged over all benchmark tests as well as organized in four groups according to the average pairwise sequence identity of the sequences. For each accuracy metric three figures are provided corresponding to the average scores of the initial alignments (I), the reformed alignments (R) and their respective differences (R-I). Differences that are found to be statistically significant at the .05 significance level are highlighted in bold. For the SP and TC scores, positive average differences indicate that the reformed alignments were superior to the initial ones whereas negative values indicate that ReformAlign (on average) worsened the starting alignments. For the D-POS scores the situation is reversed: negative values indicate an improvement due to ReformAlign, whereas positive values represent a degradation of the starting alignments.
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Figure 4. Average Cline score differences for the benchmark tests of the BRAliBase dataset. Effect of ReformAlign to testsets of increasing APSI (A) and increasing number of sequences per alignment (B). The alignment pairs (from top to bottom, as appearing in the legends) correspond to [Ref. ClustalO – ClustalO], [Ref. ClustalW – ClustalW], [Ref. DialignTX – DialignTX], [Ref. GramAlign – GramAlign], [Ref. Kalign – Kalign], [Ref. Mafft (FFTnsi) – Mafft (FFTnsi)], [Ref. Mafft (Linsi) – Mafft (Linsi)], [Ref. Muscle – Muscle], [Ref. ProbConsRNA – ProbConsRNA], [Ref. PicXAA – PicXAA], and [Ref. RCoffee – RCoffee]. For each value of the horizontal axis, the difference is calculated by subtracting the average Cline score of the reformed alignments to the respective score of the initial alignments that belong to each group. Statistically significant differences at the .05 significance level are denoted by an asterisk (*) at the top-left corner of the respective points. The number of test cases (N) per group is given at the bottom of each value in the x-axis.
Despite the simplicity of the ReformAlign logic, the experimental results suggest that the proposed approach may significantly improve on the performance of almost all examined aligners. In general, the improvement due to ReformAlign seems to be substantially higher for difficult examples (i.e. alignments with relatively low APSI) or for lower-scoring aligners (e.g. DialignTX or GramAlign) and becomes less noticeable when the starting alignments are already nearly optimal leaving thus less room for improvement (Table 1, Additional file 2: Table S2 and Figure 4). The only cases where ReformAlign seems to degrade on average the initial alignments are for PicXAA and ProbConsRNA. Nonetheless, even for these aligners it was found that in a considerable amount of cases the reformed alignments were superior to the initial ones (Table 1 and Figure 3) whereas it may also be observed that the average decrease for both aligners is relatively small and not always statistically significant (Table 1). In addition, although the Cline (Table 1), SP and TC scores (Additional file 2: Table S2) seem to indicate a marginal degradation of the initial alignments due to ReformAlign, according to the D-POS similarity metric the reformed alignments were superior to the starting ones for all aligners participating in our experiments (Additional file 2: Table S2).
Another important observation is that ReformAlign very often delivers alignments which are different to the starting ones (Figure 3 and Table 1), and in the majority of these cases the reformed alignments are superior to the corresponding starting ones. This trend is much more apparent for testsets with relatively low APSI values and becomes less evident for highly similar alignment cases (Table 1 and Additional file 2: Table S2).
The experimental results also demonstrate that the application of ReformAlign to existing alignments does not affect all aligners in the same way and may result in changes in their overall ranking. For example, as it may be seen at Table 1, although initially GramAlign and R-Coffee score on average lower than DialignTX and ProbConsRNA respectively, the situation is reversed after ReformAlign has been applied to fine-tune the respective alignments. The same holds true for the MUSCLE, Kalign and Mafft (with the FFT-NS-i setting) aligners with the latter scoring higher than the other two after the respective alignments have been reformed.
Finally, as it may be seen at Figure 4A, the effect of ReformAlign for the majority of aligners seems to be more substantial for harder test cases (APSI ≤ 75%) and becomes less noticeable as the average pairwise sequence identity increases. This does not seem to be the case for the highest scoring aligners (ProbConsRNA, PicXAA and R-Coffee) which deliver quite accurate alignments (Table 1 and Additional file 2: Table S2) even for relatively hard test cases leaving thus less room for improvement, and for GramAlign where several test cases from the higher APSI groups seem to violate its grammar-based assumptions resulting thus in lower-scoring initial alignments that are then refined by ReformAlign (Figures 3 and 4, Table 1 and Additional file 2: Table S2).
The analysis of the Cline score differences versus the number of sequences per alignment (Figure 4B) revealed similar results. Probabilistic and consistency-based aligners, which appear to deliver more accurate starting alignments, seem to benefit less (or are even marginally worsened) from the application of ReformAlign compared to optimization-driven or iterative refinement based approaches. The main reason for this might be in the underlying assumptions of these models. In particular, these high-scoring aligners employ sophisticated albeit computationally expensive probabilistic assumptions resulting thus quite often in very accurate alignments. ReformAlign however is based on the ad hoc SP scoring scheme and it may thus happen that accurate starting alignments that meet the probabilistic/consistency based assumptions are slightly degraded by the more arbitrary optimization scheme of ReformAlign, especially in alignments composed of multiple sequences. However, as the experimental results demonstrate, there is a considerable amount of cases where the initial alignments of the Probabilistic Consistency Transformation (PCT) approaches are improved by the proposed post-processing step (Table 1, Figures 2 and 3), suggesting thus that ReformAlign could appear to be useful even for fine-tuning the starting alignments of such sophisticated alignment methods.
A similar analysis was carried out for the benchmark tests of the DNA SMART database. For these experiments R-Coffee (which is specialized for RNA alignments) was removed from the analysis. Since the majority of the test cases (257 out of 264 alignments) were sharing an average sequence identity equal or lower to 55%, the differences between the reformed and the initial alignments for the higher APSI groups were not found to be statistically significant at the .05 level (data not shown). The performance averaged over all 264 test cases per alignment pair is graphically depicted at Figures 5 and 6 whereas the analytic results for the considered accuracy metrics are reported at Table 2 and Additional file 3: Table S3.
Figure 5. Scatterplots of the Cline scores per alignment pair for the 264 benchmark tests of the DNA SMART dataset. For each alignment pair a scatterplot is constructed. Each point corresponds to an alignment test case of the DNA SMART dataset and depicts the Cline score of the initial alignment versus the respective score of the reformed alignment. The diagonal line represents the expected plot if the initial and the reformed alignments were identical.
Figure 6. Boxplots of the averaged Cline scores for the 264 alignments of the DNA SMART dataset. The boxplots corresponding to the Cline scores for the DNA SMART dataset are given in this figure. The figures, correspond, from left to right, to the averaged performance of [ClustalW – Ref. ClustalW], [ClustalO – Ref. ClustalO], [Kalign – Ref. Kalign], [Mafft (FFTnsi) – Ref. Mafft (FFTnsi)], [Mafft (Linsi) – Ref.Mafft (Linsi)], [Muscle – Ref. Muscle], [DialignTX- Ref. DialignTX], [GramAlign – Ref. GramAlign], [ProbConsRNA – Ref. ProbConsRNA] and [PicXAA – Ref. PicXAA]. The bars at the bottom of each pair represent the percentage of times where the reformed alignments were superior (blue bars), equal (green bars) or inferior (red bars) in terms of Cline score compared to the initial alignments.
Table 2. DNA SMART Cline scores
Additional file 3: Table S3. DNA SMART Results. Averaged D-POS, SP and TC scores for the 264 benchmark tests of the DNA SMART dataset. For each accuracy metric three figures are provided corresponding to the average scores of the initial alignments (I), the reformed alignments (R) and their respective differences (R-I). Statistically significant differences at the .05 significance level are highlighted in bold.
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The experimental results suggest that ReformAlign was able to improve on the starting alignments for the majority of the considered aligners. For ProbConsRNA the effect of ReformAlign is dubious: although on average the reformed alignments appear to be marginally superior to the initial ones (Table 2 and Additional file 3: Table S3), they demonstrate a greater variability compared to the starting alignments (Figure 6) and the reported differences are not always found to be statistically significant. Regarding the remaining aligners of our benchmark study, only Mafft (with the L-NS-i setting) and PicXAA are shown to be negatively affected by the application of ReformAlign whereas a noticeable improvement is observed for the rest. The reported degradation of the PCT-based aligners could be due to the sophisticated assumptions of these high performance methods compared to the ad hoc optimization scheme of ReformAlign. Specifically, since the DNA SMART database is composed of alignments which are based on biological features such as the tertiary structure of encoded proteins, the simplicity of the ReformAlign scheme could result more often in degradation of alignments deriving from the more accurate PCT-based methods, compared to alignments generated from progressive and iterative refinement aligners.
In agreement to the results of the BRAliBase experiments, the DNA SMART analysis supports the conclusions that ReformAlign generates alignments that are frequently different (and often superior) to the starting ones (Figure 6 and Table 2) and that the suggested approach does not affect all aligners the same way, resulting thus in changes in their overall ranking (Table 2 and Additional file 3: Table S3).Finally, the assessment of the effect of ReformAlign to testsets of increasing APSI (Figure 7A) and increasing number of sequences per alignment (Figure 7B) did not provide us with conclusive results, mainly due to the fact that there were only a limited number of test cases belonging to the higher order groups, often resulting in statistically insignificant differences for the considered alignment pairs. Nevertheless, the results of Figure 7A indicate a trend that further supports the idea that the effect of ReformAlign is weaker for closely related sequences compared to alignments with lower APSI values, whereas there does not seem to be a clear pattern describing the way ReformAlign affects the examined aligners with an increasing number of sequences per alignment (Figure 7B).
Figure 7. Average Cline score differences for the benchmark tests of the DNA SMART dataset. Effect of ReformAlign to testsets of increasing APSI (A) and increasing number of sequences per alignment (B). The alignment pairs (from top to bottom, as appearing in the legends) correspond to [Ref. ClustalO – ClustalO], [Ref. ClustalW – ClustalW], [Ref. DialignTX – DialignTX], [Ref. GramAlign – GramAlign], [Ref. Kalign – Kalign], [Ref. Mafft (FFTnsi) – Mafft (FFTnsi)], [Ref. Mafft (Linsi) – Mafft (Linsi)], [Ref. Muscle – Muscle], [Ref. ProbConsRNA – ProbConsRNA] and [Ref. PicXAA – PicXAA]. For each value of the horizontal axis, the difference is calculated by subtracting the average Cline score of the reformed alignments to the respective score of the initial alignments that belong to each group. Statistically significant differences at the .05 significance level are denoted by an asterisk (*) at the top-left corner of the respective points.
In this paper we presented ReformAlign, a novel profile-based meta-alignment approach that aims at correcting early alignment errors by giving the sequences a second opportunity to re-align themselves against a standard profile that efficiently summarizes the initial alignment information. ReformAlign is based on the Gotoh's affine-gap penalties variation of the classic Needleman-Wunsch algorithm and uses a refinement scheme according to which the reformed alignment is indirectly inferred by a series of individual and independent sequence-profile pairwise comparisons.
ReformAlign was extensively assessed on the way it affects the alignments of ten leading aligners using benchmark testsets from the BRAliBase and DNA SMART datasets. The results suggest that the majority of aligners showed a notable improvement on the accuracy of the delivered alignments when ReformAlign was employed as a post-processing step. This improvement was more substantial for harder alignment cases with low APSI or when there was still adequate room for improvement on the starting alignments, and became less evident when the initial alignment was already quite accurate or for easier alignment cases with high identity rates.
Finally, it is important to mention that the proposed method does not come to replace other popular alignment techniques. Instead, users may continue to use their aligner(s) of preference (even programs that were not examined in the present study) and then complementarily employ ReformAlign as a post-processing step to examine if the delivered alignment is more appropriate for their analyses.
Availability and requirements
ReformAlign is freely available to the public under the GNU General Public License (version 3 or later). Both the source code and precompiled binaries for Linux and Windows may be downloaded from http://evol.bio.lmu.de/_statgen/software/reformalign/ webcite. Currently ReformAlign can only align DNA and RNA sequences and it is provided as a command line program. Its source code is in C++ and it makes use of the Open Multi-Processing (OpenMP) parallelization API.
For the benchmark tests, the BRAliBase 2.1 RNA alignment database [28] and the alignments from the DNA Reference Alignment Benchmarks database [29] were employed. The evaluation of the alignments accuracy was performed using Robert Edgar's QScore, (v.2.1) multiple alignment scoring software and Blackburne and Whelan's MetAl (v.1.1) [37] command-line utility for calculating metric distances between alternative alignments.
Finally, the alignment programs that were used in our benchmark analysis are as follows: ClustalW2 [38] (v.2.1), ClustalO [39] (v.1.2), MUSCLE [8,26] (v.3.8.31), MAFFT [40] (v.7.149), Kalign [7] (v.2.04), GramAlign [41] (v.3), ProbConsRNA [42] (v.1.1), R-Coffee [43,44] (T-Coffee v.10.00.r1613), PicXAA [45] (v.1.03) and Dialign-TX [46] (v.1.0.2).
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
D.P.L. developed the idea, implemented the software and drafted the manuscript. D.M. planned and supervised the work and contributed to the manuscript. Both authors read and approved the final manuscript.
The authors would like to thank the anonymous reviewers for their insightful comments and Prof. Dr. Nick Goldman and Dr. Ari Löytynoja for their advice. This work was supported by the German Science Foundation DFG through grant number ME 3134/4-1.
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