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Dindayal Femicare Forte Capsule: A herbal, non-hormonal tonic for women that keeps you active and energetic throughout the month besides improving your complexion and general sense of well-being.
Femicare Forte Capsule: Is a proprietary Ayurvedic medicine, manufactured by Dindayal Industries Ltd, Gwalior. It is used in the treatment of menstrual disorders.
Femicare Forte Capsule uses: It is indicated in Ayurvedic treatment for irregular menstrual cycle, excessive vaginal bleeding, painful periods, cervical erosion, habitual abortion, PID, Leucorrhea.
Effect on Tridosha – Balances Vata and Pitta.
Femicare Forte Capsule Dose: 1 – 2 capsules, 2 times a day, or as directed by Ayurvedic doctor.
How long to take? It can be taken for upto 2 -3 months, based on doctor's advice.
Presentation : 20 capsules
Femicare Forte Capsule Ingredients:
Each capsule contains extracts derived from
Ashoka – Bark of Saraca indica– 100 mg
50 mg of each of
Haritaki – Chebulic Myrobalan fruit rind – Terminalia chebula
Daru Haridra – Tree Turmeric (stem) – Berberis aristata
30 mg of
Lodhra – Lodh tree (bark) – Symplocos racemosa
20 mg of
Mocharasa – Extract of Bombax malabaricum
Amra – Mango – Mangifera indica – seed kernel
Musta – Nut grass (root) – Cyperus rotundus
Jiraka – Cumin – Cuminum cyminum
Maricha – Black pepper – Piper nigrum
Sarpagandha – Rauvolfia serpentina
Gokshura – Small caltrops (whole plant) – Tribulus terrestris Linn.
Raktachandana – Pterocarpus santalinus
10 mg each of
Chandana – Sandalwood – Santalum album
Kiratatikta – Swertia chirata
Pradarantaka Lauha – An Ayurvedic medicine used in heavy periods – 60 mg
Madhumalini Vasanta Rasa – 30 mg
Muktashukti Pishti – Powder of Pearl Oyster – 20 mg
Kaheraba Pisti – 10 mg
Rajata Bhasma – Bhasma of Silver – 5 mg
Processed with Vasa – Malabar nut tree (root / whole plant) – Adhatoda vasica,
Doorva – Bermuda grass – Cynodon dactylon
Dhataki – Fire Flame Bush (flower) – Woodfordia fruticosa
Devadaru – Himalayan cedar (bark) – Cedrus deodara
Pattanga – Caesalpinia sappan
Side effect:
This medicine may not help in people with scanty menstruation, since most of the ingredients are styptic in nature. It would help in heavy periods.
This medicine should only be taken under medical supervision.
keep out of reach and sight of children.
Over-dosage or taking medicine for a longer period of time, than prescribed may cause delayed menstrual cycles.
Store in a cool dry place.
Take this medicine only till the pre
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Buy Ayurvedic & herbal medicines online.Shop for ayurvedic product, herbal supplements and healthcare products. Swasthyashopee is an online store for all ayurvedic products, ayurvedic cosmetics. We cater most popular brands like Dabur, Baidyanath, Unjha, Emami, Zandu, Ranbaxy..
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The information presented at this page is not intended to replace the services of a health practitioner licensed in the diagnosis or treatment of illness or disease. Any application of the material in this text is at the readers discretion and sole responsibility. If you have a persistent medical condition or your symptoms are severe please consult a physician. Statements on this web site about health conditions and remedies have not been evaluated by the U.S. Food and Drug Administration.


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Five-year tax holiday for the hotels in NCR
The Centre has decided to give a five-year tax holiday to new hotels coming up before March 31 this year in the NCR region.
Chandigarh: The Centre has decided to give a five-year tax holiday to new hotels coming up before March 31 this year in the NCR region to incentivise completion of projects ahead of the Commonwealth Games, Union Secretary of Tourism Sujit Banerjee said.
"This exemption would be given to 2, 3 and 4 Star hotels and not to the 5 Star hotels," Banerjee said in Gurgaon last evening.
The tax holidays are meant to be an incentive for the hotel industry to complete their projects before the Commonwealth Games when an estimated 1 lakh tourists are expected to arrive in India.
For these tourists about 40,000 hotel rooms would be required while there are only about 10,000 hotel rooms in and around Delhi, he said.
He said to fill in the huge gap of about 30,000 rooms, Haryana is chipping in with a commendable 6,700 rooms, the Delhi Development Authority with 2,300 rooms and Uttar Pradesh would provide 2,000 rooms.
In addition about 11,000 hotel rooms would be available from guest houses at Paharganj and Karol Bagh in Delhi and another 3,000 rooms from the bed and breakfast scheme in Delhi and Haryana, he said.

subset: commoncrawl


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When No. 14 Gonzaga lost four of five games to close 2008, some observers wondered if this much-ballyhooed Zags team was overrated.
Probably not.
The Zags have won 16 of their 17 games in 2009, many by ridiculous margins. They head into this weekend's West Coast Conference tournament as the favorite, and are nearly certain to make their 11th straight NCAA tournament.
While they lack a dominant superstar, the Zags have so many strong players that slowing their offense is difficult. And this team is strong on defense.
The latest example was Tuesday's 90-40 demolition of South Carolina-Upstate. While no one expected the Spartans to win, the domination was total. The Zags held them to 29 percent shooting and forced 26 turnovers, while committing only four themselves. They outscored the Spartans 37-2 off turnovers, 36-18 in the paint and 16-2 in transition.
"They are a team that can make the Final Four," Upstate coach Eddie Payne sighed afterward, echoing many preseason predictions.
With a returning core of all-WCC players like Jeremy Pargo, Josh Heytvelt and Matt Bouldin, plus emerging stars Austin Daye and Steven Gray, and sharpshooter Micah Downs, expectations were high that this might be the first Zags team since the 1999 squad to get past the Round of 16.
Heytvelt is certainly doing his part, averaging 15 points and nearly seven rebounds per game. Bouldin is averaging 13 points, Daye 12, and Pargo and Gray nine each.
The Zags won their first seven games, climbing to No. 4 in the polls. Then in the span of two weeks they lost to Arizona, No. 1 Connecticut in overtime, Portland State and Utah to fall out of the Top 25.
That's not uncommon for the Zags, who play a brutal non-conference schedule to make up for playing in the lightly regarded WCC.
But the Connecticut loss especially seemed to sap some confidence. Pargo, the reigning WCC player of the year who flirted with turning pro, suddenly began struggling with turnovers and shooting. The nadir was a 77-70 home loss to unheralded Portland State, only the third Gonzaga loss at the McCarthey Athletic Center since it opened in 2004.
The turnaround began with an overtime win at Tennessee on Jan. 7. Then Gonzaga waltzed through the WCC, winning 14 games by an average of 20 points. The sole stumble was a blowout loss to No. 5 Memphis in the Spokane Arena on Feb. 7.
The Zags went undefeated in the WCC for the third time in six seasons, and the late run was particularly fueled by Heytvelt and Pargo.
"He was beating himself up a little bit," after Connecticut, Gonzaga coach Mark Few said of Pargo. "He's playing better."
The Zags have a bye into Sunday's semifinals of the WCC tournament in Las Vegas, a break that makes Few nervous. Last year the Zags struggled to beat Santa Clara and San Diego after a similar bye.
"They were a little bit tight," Few said of his team.
He thinks the Zags might face Santa Clara or San Diego in the semis, two teams that like a slowdown game.
The Zags are pleased the tournament is in Las Vegas, a neutral site. For years, the WCC tournament was held at a predetermined home court of a WCC team, meaning the Zags got no home court advantage from winning nine straight WCC regular season titles.
"Finally it's fair to the student athletes," Few said. "It's never been fair."
The tournament is nearly sold out, and a large number of Gonzaga fans are leaving frozen Spokane to enjoy some time in the sun.


text: Rebate! 2 Mags-N-Bag! Sig Sauer M11-A1 Desert Tan 9mm Pistol M11-A1 For Sale from DEGuns_net | Positive feedback: 95% View | Verified Seller | 895 Completed Sales View Sellers Items /UserImages/177672/957411745 11550290.jpg /UserImages/177672/957411745 11550291.jpg Description: Questions Call or email Mon-Sat 8:00am-7:00pm This item is new in the box comes with everything included from the manufacturer If item is not as described we do have a 3 day return period TRUSTED BY ELITE MILITARY PERSONNEL. UPGRADED FOR YOU. PISTOL, COMPACT, 9MM, M11 Issued to US Naval Aviation and key units in every branch of the Armed Services, the M11 is proud to serve. Upgraded with a stainless steel slide, a Short Reset Trigger and three 15 round flush fit magazines, the M11-A1 variant features the same phosphate coated internals, SIGLITE® night sights and MIL-STD UID label of the standard-issue M11. This Desert version features Flat Dark Earth coated alloy frame and stainless steel slide. A 10% Giftcard can be applied to this product! Read More Condition: New In Box Brand: SIG SAUER UPC: M11-A1 Caliber: 9mm Luger Location: NE Trades Accepted: Yes, give us a call! Read More Shipping Notes: 402-875-6500 Read More Share Item Item #: 957411745 SKU: 798681448739 Stock No.: M11-A1-D DEGuns_net Seller Since: Dec. 2014 Seller Level: Trusted Seller This seller is an FFL City: Lincoln State: NE Phone: 402-875-6500 Price: $979.99 Buy It Now Add To Cart Please Login/Register to Order Login or Register to buy This Seller Accepts Online Payment Shipping: See Description (or Arrange local transfer) Accepted Payment Methods: Returns: No Returns Send Message to DEGuns_net You must login or register to send a message to this seller. Contact Seller as Guest Message To Seller Email * That Email Address is already in use. If you typed your email correctly, you are already registered and you should login or click here for the password recovery system. First Name * Last Name * Phone * Address 1 * Address 2 City * State * -State- AK AL AR AZ CA CO CT DC DE FL GA GU HI IA ID IL IN KS KY LA MA MD ME MI MN MO MS MT NC ND NE NH NJ NM NV NY OH OK OR PA PR RI SC SD TN TX UT VA VI VT WA WI WV WY Zip * Already have an account? Login Your Recently Viewed Items


text:
Naspers focuses on fuelling organic growth
by June 23, 2014 7:06 am0
"We report robust consolidated revenue growth of 26 per cent, driven by both the internet and pay-television businesses. This growth was fuelled by development spend of 7.7 billion rand– up 79 per cent on last year – devoted particularly to ecommerce and digital terrestrial television (DTT)," Naspers said in a statment.
Consolidated revenues grew 26 per cent to 62.7 billion rand for the year ended March 31 2014, compared to 49.8 billion rand in the previous comparative period. The growth was largely boosted by growth in the company's internet businesses. The weak rand also contributed to the growth boost.
"Net interest on borrowings increased to 1.2 billion rand from 636 million rand in 2013 due both to the rand depreciation and increased borrowings utilised to fund acquisitions and growth," Naspers added.
[DATA NPN:Naspers Limited] is a mass media company based in South Africa that deals in electronic and print media.
Consolidated operating profit declined to two billion rand from four billion rand, and profit before tax showed a slight increase from 9.2 billion rand in 2013 to 9.4 billion rand for the period under review.
The company's prominent businesses Tencent and Mail.ru nevertheless reported strong growth. Tencent is an instant messaging platform operator in China, while Mail.ru is an operator of a Russian instant messaging service.
Naspers acquired a 46.5 per cent stake in Tencent in 2001, and 30 per cent interest in Mail.ru. It added an additional 2.6 per cent interest in Mail.ru in 2007.
"Tencent consolidated its leading position in communication and games in China, while strengthening its stance in ecommerce. Revenue for the year was 60 billion renminbi, up 38 per cent, while non-GAAP profit attributable to shareholders was 19 per cent higher at 17.1 billion renminbi," Naspers explained.
Revenue for Mail.ru for 2013 was reported at 27 billion roubles, up 30 per cent year on year, while group aggregate net profit rose 36 per cent to 11.4 billion roubles.
Revenue for Naspers's ecommerce segment grew 64 per cent to 20.3 billion rand, and revenue for the company's pay television segment grew by 20 per cent to 36.3 billion rand. Investments in DTT services also resulted in trading profits creeping up at a slower 13 per cent to 8.5 billion rand.
"The print media segment experienced a tough year with flat revenues and declining margins. Media24 managed small revenue growth of 1 per cent, but trading profit declined by 7 per cent. Abril had a poor year, as revenues declined and restructuring lagged," Naspers explained.


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09-18-03, 10:09 #1Registered User
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Unanswered: Oracle DBA with Some Cold Fusion experience
We are looking for a Oracle DBA with some cold fusion experience. Candidate needs to be US Citizen/Green Card Holder. Client is a fortune 500 company located in Chantilly, VA. If you are interested, please email me resume with rates, contact nos
Kiran Gowda
Magnum Software Services, LLC
(248) 895-8049


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ROBERT SIEGEL, host:
Take a second and imagine one of those yellow smiley faces. Now imagine 50 billion smiley faces floating in a drop of water. That is what scientists made using DNA. It turns out that in addition to holding our genetic code and being a basic building block of life, DNA is also great for making incredibly tiny structures.
NPR's Nell Boyce reports.
NELL BOYCE reporting:
For years chemists have been using DNA to build really simple shapes like cubes that are a thousand times thinner than a human hair. They've done it by laboriously designing small snippets of DNA that will hook themselves up into the desired form. But a new method for building things with DNA is much faster and easier, even a high school student could think up a shape, like a star or a snowflake, and then make a DNA version within a week.
Paul Rothemund came up with the idea at the California Institute of Technology in Pasadena.
Mr. PAUL ROTHEMUND (California Institute of Technology, Pasadena): Even by the time I was making smiley faces, I didn't really believe that the method worked as well as it did.
BOYCE: Rothemund's trick is this: instead of custom designing small snippets of DNA so that they sit together in a certain way, he borrows a single long strand of DNA from a harmless virus.
Mr. ROTHEMUND: And we take that very long, single strand of DNA, it's about 7,000 letters long, and then we add to it about 200 short DNA strands that I call staples.
BOYCE: The staples bring two distant parts of the DNA strand together so that it folds.
Mr. ROTHEMUND: We actually fold the DNA into any shape that we want, right? So in the case of the smiley face that I made, I actually fold the DNA into a disc, but then leave three holes for the two eyes and for the mouth.
BOYCE: Rothemund has developed a new computer program that can analyze a shape, figure out the right folding pattern, and then tell you what DNA staples you need to make that shape.
Mr. ROTHEMUND: It's really easy and fun, actually, to make whatever you want at the nano-scale. You design it in the computer, you order the DNA sequences, they come in the mail, you add a little bit of saltwater, you heat it up and cool it down and then, you know, and hour and a half later, it's ready to look at under the microscope.
BOYCE: In this week's issue of the journal Nature, Rothemund shows off some of his DNA art. One nano-creation is a tiny map of the Americas. But the real goal of this work isn't tiny maps. Rothemund says that in the future, tiny DNA shapes could serve as scaffolds for quickly building nano-structures made of metals or other materials. Those could be useful for things like faster computers. Nell Boyce, NPR News.
SIEGEL: Images of DNA art are at our web site, NPR.org.
Copyright © 2006 NPR. All rights reserved. Visit our website terms of use and permissions pages at www.npr.org for further information.
NPR transcripts are created on a rush deadline by Verb8tm, Inc., an NPR contractor, and produced using a proprietary transcription process developed with NPR. This text may not be in its final form and may be updated or revised in the future. Accuracy and availability may vary. The authoritative record of NPR's programming is the audio record.


text: Evaluation of the Completeness of the Nursing Process for Patients Having Gastrectomy Using Electronic Nursing RecordsIhnSook Park, Cheong-suk Yoo, +8 authors Mi-ok ParkNursing Informatics2009The purpose of this study was to evaluate recording completeness of the nursing process. We compared nursing statements documented at the time when the Electronic Nursing Record (ENR) system, based on the ICNP, was implemented in 2004 with those documented in 2007. The ENRs for 35 gastrectomy patients in each year were selected for evaluation. The selected… (More)


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Choose The Best Packers as well as Movers
Feb 10, 2016 • By punepacker • • 54 Views
Are you seeking a very easy relocation? Do you discover it hard to pick from a lengthy list of packers as well as moving companies in? You do not have to fret concerning it. We will aid you strike the very best deal with exceptional packers and moving companies, adapt your every demand. Currently, you can easily simply by maintaining a few simple points in your mind to hire the best packers and also moving companies. This post will certainly take you through the little details, or you may ignore the problem.
There are hundreds of companies on the site discussed in the testimonial. All you should do is figure out the actual review of all individuals. The website, which publishes just by their real customer evaluations, as well as generally allows an individual that published the evaluation of human communication. Try not to be fake evaluations, which could have been posted just to enhance the number of reviews misdirecting.
Interaction with the packers and moving companies Movers in Sherman
Before employing any type of packers and moving companies, make certain they remain in both the resource and also target last destination. This will certainly not only alleviate the moving process for you, but also offer you the guarantee about the company. If possible, try to fulfill the packers and also movers as well as advance word on the whole moving process with them. This will help the top quality of their making you a far better understanding of the solution. It is better to take some time and decide points, as opposed to thrill right into something.
Inspect Items
See to it that the involvement of an array of solutions supplied by them no surprise costs. It makes every little thing clear right from the beginning, so there is no extra expense in between pop-shifting procedure. Also see to it to pack a good thing needs to be looked after. Seeking insurance prices quote to strike the best deal. By doing this, your home or business will certainly be transferred quickly to a new location without any kind of difficulty. If unboxing, you discover any damage to your job, to complete the nine months distribution cases. Preferably as soon as possible record any type of such point, in order to quickly obtain an acknowledgment from the pioneer.
Discover added solutionsSherman Tx Movers
You can additionally extra solutions which might be supplied by them as a couple of first days of cost-free storage area. You can additionally make recommendations on each of the chosen moving knowledge in your use. This will certainly help you to confusion as well as difficulties mixed-up were improved and also smooth relocation, no diving.
Comply with these straightforward actions, Not only will you have the ability to prevent the issues associated with the moving procedure, yet likewise have the ability to pick the very best packers and moving companies can provide you transfer to a new area assisting hand. The writer is widely known company expert and service author. His job has actually been released in leading service publications. With his writings, he commonly aids to locate the very best Packers and also movers ever before person.Movers in Sherman


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Know more about this business than we do? Cool! Please submit any corrections or missing details you may have.Help us make it right
Be the first MLive.com user to add photos or videos of Vantimmeren Stables
Vantimmeren Stables was founded in 1994, and is located at 7600 Lincoln St in Allendale.
Vantimmeren Stables can be found at Lincoln St 7729. The following is offered: Farming. The entry is present with us since Sep 9, 2010 and was last updated on Nov 14, 2013. In Allendale there are 4 other Farming. An overview can be found here.


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Hwasangorae Trailer (2014)
23 October 2014 Adventure, Animated, Action 70 mins
It's 2070 and our planet is ravaged by earthquakes and volcanic activity. Nowhere on Earth is hit worse than the Korean peninsula. Ha-jin, a sly, savvy orphan, prowls the rubble of a broken, barely functioning Busan wracked by daily tremors. Her petty crimes land her in jail, and from there the clutches of human traffickers — and finally far out to sea, rescued (or rather, kidnapped?) by a ragtag band of pirates seeking the precious crystals at the heart of a volcanic island. To reach their prize, they'll need to defeat the island's fearsome guardian, the legendary volcano whale. And to do that, they'll need Ha-jin's special talent for summoning whales. But is her rare power a blessing, or a curse?
Watch the official Hwasangorae 2014 trailer in HD below or find more Hwasangorae videos on Vidimovie.


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April 2016expanded
French Innovators: Luncheon discussion with Antoine Bello & Jonathan Benhamou
The French-American Foundation is launching a New York-focused series of discussions with successful French entrepreneurs based in the United States from diverse sectors such as technology, telecommunications, finance, biotechnology, arts and culture, and the culinary world. These entrepreneurs will share their stories and explore the challenges and advantages related to the creation or expansion of new business in the United States compared with France, among them:
- Access to financing and networks
- Differing notions of success and failure
- Managing expectations with respect to achieving profits and growth
- The role of incubators
The first event will feature a conversation between Antoine Bello, Founder of Ubiqus, business angel and writer, and Jonathan Benhamou, CEO and co-Founder of PeopleDoc, moderated by William J. O'Farrell, co-Founder, CEO of Body Labs, Inc. and Adjunct Professor at Columbia Business School.
Antoine Bello
Antoine Bello was born in Boston in 1970. A dual French and American citizen, he lives in Purchase, NY.
While studying at HEC Business School, Bello founded Ubiqus, a company offering reporting and summary services to meeting planners. Ubiqus grew at a brisk pace during the next 15 years, to reach approximately 350 employees and 45 M€ in revenue in 2007, when it was sold to Finama Private Equity.
Bello has been a business angel for almost 20 years, both in France (e.g. Finexkap) and in the United States (e.g. Dollar Shave Club, AdoreMe).
Bello is now a full-time writer, published in France by Gallimard. He is the author of eight novels, including the Falsificateurs trilogy, Eloge de la pièce manquante, and Roman américain. His works have been translated in a dozen languages. They will soon be available in English.
Jonathan Benhamou
Jonathan Benhamou co-founded PeopleDoc, a leading provider of cloud-based HR Service Delivery solutions, in 2007 in France. In 2014, Jonathan moved to New York to oversee the global expansion of PeopleDoc. The same year, Gartner recognized PeopleDoc as a "Cool Vendor" in the Human Capital Management space.
Jonathan helps other young innovative companies and technology start-ups as a member of the delegation of Young Entrepreneurs of the G20. He has been recognized as a Technology Young Leader - an honor awarded by 20 of Europe's top technology leaders, including Apple, Google, Intel, Oracle, and Cisco. Jonathan holds a Master's degree in Business from HEC Paris.
Organized with the generous support of:
Official airline of the French-American Foundation
Baker & McKenzie LLP
452 Fifth Avenue (entrance on 40th Street)
New York City


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While it may not be difficult to find replacement parts for Subaru vehicles, these days the necessity for nothing but the highest quality are required to repair your car or truck. Car aficionados know that a variety of optional equipment is built into each Subaru, and know that only top quality repair SVX parts will suffice when your car or truck needs fixing. Obviously, you didn't buy your SVX unless you wanted to buy dependability and great value both rolled up into one vehicle. You purchased your SVX because its adventure and youthfulness struck a chord inside you; so why would you be okay with anything but the best when it comes to the highest quality auto parts?
Achieving maximum fuel efficiency while reducing emissions on your car or truck depends on the quality and performance of the parts you use. Your Subaru SVX engine, as great as it is, isn't 100% efficient and it uses a PCV valve to control its positive crankcase ventilation. Usually located on your valve cover or cylinder head, your Subaru SVX PCV valve captures and re-routes exhaust blow-by from the combustion process and sends it back through the intake. This is much better than simply venting it into the atmosphere, which is what all engines did before they were equipped with a positive crankcase ventilation valve.
Given all the hassles associated with fixing broken parts on your car or truck, let www.carpartsdiscount.com be the most authoritative website you go to make purchasing it easy. At Car Parts Discount, we have real customer support agents available with enough expertise to help you buy the correct Subaru SVX part for your project or repair. When your Subaru requires a top quality replacement PCV Valve, there's no need to waste time looking anywhere other than the authority, Car Parts Discount. When it comes to Subaru SVX PCV Valve parts, you can find them for 1997, 1996, 1995, 1994, 1993, 1992 models by clicking the appropriate year below.


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Openads.org ranks
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102 Page views for daily.
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$222 USD for it's visitor traffic that has the potential for CPC and Display-based advertising.
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With Openads you're free to choose banners, campaigns and terms that suit you. And our highly sophisticated tracking system means you can gather statistics and optimise campaigns, all from the same simple interface. The result of this flexibility and repo
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The Ultima Dragons 25th Anniversary Bash: Last Day to Pledge on Kickstarter
Okay, technically, there's actually about a day and a half — 35 hours as of this writing — to contribute to the funding of the Ultima Dragons 25th Anniversary Bash on Kickstarter. Still, that crowdfunding campaign ends on October 21st, which is tomorrow, so…close enough.
The campaign is doing quite well; it has exceeded its goal ($14,950 USD) and has raised (again, as of this writing) $21,601 USD. If you're at all curious as to what the money will go toward, Gallara discussed that in an update on Kickstarter, complete with charty goodness:
Extra funds are, of course, being put toward stretch goals. Several of these have already been achieved:
- UNLOCKED 10/17/16! $17,000 – The Avatar himself, JC Shakespeare who played the role of the Avatar in Ultima IX, will be joining us! He will offer free 15-minute "Counseling of the Avatar" sessions as he is a real-life counselor! JC will be at the party!
- UNLOCKED 10/18/16! $18,000 – Free Schlotzsky's/Cinnabon feast on Sunday (served in Disneyland Hotel picnic area) for all ticketholders and their children! If you haven't included an add-on for this already, you don't need to. If you have, thank you! Your add-on helps feed others in attendance.
- UNLOCKED 10/18/16! $18,500 – Pizza picnic on Saturday (served in Disneyland Hotel picnic area) for everyone who attends! All you need is a Fellowship Hall ticket, or be 13 or younger and a child of one who has one, and you will get to partake of pizza.
- UNLOCKED 10/19/16! $20,000 – Goody bags for everyone in attendance, all ticketholders and their children!
Further financial stretch goals are as follows:
- $23,000 – Gourmet buffet dinner (served in Fellowship Hall) for everyone on Saturday night!
- $25,000 – Gourmet breakfast for everyone on Saturday!
- $27,000 – Everyone attending partakes of all fancy meals no matter what level they backed at!
- Anything above that – prices of future tickets go way down & more loot for everyone!
And, of course, there are also some participatory stretch goals as well:
- 100+ Ankhs claimed – The Ankh normally comes on a black leather cord, but once 100 ankhs are claimed a brown leather option opens up!
- 150+ Stickers claimed – If this goal is reached (those pledging Wyrmling and above), everyone receiving a UDIC car sticker will get TWO – one in RUNIC!
- 200+ backers – We'll throw extra UDIC branded items in the shipments of those who are unable to attend and getting mailed their rewards!
- 36+ Kids in attendance – every kid gets a free t-shirt! (Kids must be accompanied by their parent or guardian.)
So, even if you can't make it out to Disneyland in February (I'm not sure I can), I'd encourage you to pledge to this campaign…both because you can still get some great UDIC-branded stuff, and because every little bit helps out pretty much everyone else — both attendees and those that can't make it — in some way.


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London, ONT.: September 14, 2010 -- Leaf's rookie Andrew Crescenzi tries to put the puck past Ottawa Senators goalie Robin Lehner during the teams final game of the 2010 NHL Rookie Tournament at the John Labatt Centre in London, Ontario, September 14, 2010.
(GEOFF ROBINS for National Post)
(GEOFF ROBINS for National Post)
- Copyright
- Copyright: Geoff Robins
- Image Size
- 3565x2543 / 1.7MB
- Contained in galleries


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I love the Steelers but my brother hates them because they are from Pittsburgh and so are the penguins from hockey and we like the capitals sooo yeah
winners of the superbowl
Well whether they win or not, I think a person should only like a team depending on where they came from, what they think about the team, what they think about the teams cooperation, and the behavior. Just my opinions
Well I have never been to Pittsburgh and missed the chance to see them play a few years ago when they came to Seattle but nonetheless I am still a Steelers fan. Why? I became a fan back in the days of Bradshaw et al and the Steel Curtain and have been so since.
I hate the steelers... they only won the superbowl against the seahawks, because the refs made poor calls throughout the entire game... the hawks should have won 24-14 instead of losing 21-10... those were perhaps the most blind refs I have ever seen


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Happy New Year 2012
December 28, 2011 by staff
Happy New Year 2012, We are all looking forward to a new year but many are fearful of the coming of 2012. Some believe that the upcoming year is marking the end of an historic period. Many think that this year will be a spiritually changing one and far too many believe it will mark the end of the world. This idea has been boosted in popular culture through movies, books and websites detailing how and why this year will mark the end.
The apocalypse was a recurring topic of excitement when I was a growing up in a remote Native community on the James Bay coast. It seemed like whenever a significant date or the announcements of a mystic or religious leader was heard, everyone talked about an end date. When news spread of the end, everyone lived at a more frenzied pace and more people attended the local church. Those end time believer's plans were tossed to the wind. What was the use of thinking about the future if the end was coming? There was a general sense of fear and apprehension for the future. I have lived through countless apocalypses at this point. I grew so tired of them by the time I was a teenager that I started wishing for an apocalypse for the apocalypse.
When it comes to the upcoming year 2012, most people cite the Mayan long count calendar, a number of years calculated to be a 5,125 year period, according to the Mayan system. This time period actually ends on December 21, 2012 in our modern calendar and it is this date that is falsely explained as the moment when our world will end.
When Europeans first read about Mayan culture and writing, they did so with a Euro perspective. One of the strongest of European ideas were based on Christianity and the image of an end of the world and a catastrophic apocalypse. It was with this frame of mind that a German scholar named Ernst Forstemann, in the early 1900s interpreted some Mayan writing to describe a catastrophic flood and a 'destruction of the world'. This scholar made no reference to the Mayan long count calendar, December 21, 2012 or even if the events he described took place in the past or would happen in the future. Succeeding researchers in Mayan culture made reference to his ideas and later attributed the events to the end of the Mayan long count calendar. This was an untruth.
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Some people just need a high-five, in the face, with a chair. Or just be extinct. As simple as that, you know.
|All you need to know is that you will be doing a big favour to this universe if you SHUT your big big mouth
Source: http://www.someecards.com/usercards/viewcard/MjAxMi0yNmRlMjliNTExNjdlNjMy
Remember: do not believe in everything people tell you so easily before hearing or knowing the full story. Some people can be blinded by their jealousy and ego that they turned into mean, deceiving, and wicked people. It's a small world after all--even a smaller world from where we came - a place filled with profoundly malicious and malevolent souls. They who see things not as what they are but as what they perceive.
Oh, and please, put install some filters inside your brain, will you? Don't just naively believe in practically everything you heard.
Anyway, I used to care about what you think of me (I swear I did care), but for now (and hopefully forever), I just don't because: please refer to this post (link).
Right, here you go. Till next time XOXO


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Deion Jones was born and raised in Louisiana and played college football at LSU. At the age of 10, he and his family had to relocate to Houston due to Hurricane Katrina.
On Monday night, the second round pick would step inside the Mercedes Dome for the first time with "NOLA" written on his cleats. Playing in front of friends and family in his home state, Jones would deliver a huge play while the Saints were in the redzone.
There was no better time or place for Jones to record his first pick-six going, 90-yards. He also would snap Drew Brees' passing streak of 305 passes without an interception.


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Friday, May 29, 2009
Rhymes and Reasons
So you speak to me of sadness
And the coming of the winter
Fear that is within you now
It seems to never end
And the dreams that have escaped you
And the hope that you've forgotten
You tell me that you need me now
You want to be my friend
And you wonder where were going
Where's the rhyme and where's the reason
And its you cannot accept
It is here we must begin
To seek the wisdom of the children
And the graceful way of flowers in the wind
For the children and the flowers
Are my sisters and my brothers
Their laughter and their loveliness
Could clear a cloudy day
Like the music of the mountains
And the colors of the rainbow
They're a promise of the future
And a blessing for today
Though the cities start to crumble
And the towers fall around us
The sun is slowly fading
And it's colder than the sea
It is written from the desert
To the mountains they shall lead us
By the hand and by the heart
They will comfort you and meIn their innocence and trusting
They will teach us to be free
For the children and the flowers
Are my sisters and my brothers
Their laughter and their loveliness
Could clear a cloudy day
And the song that I am singing
Is a prayer to non-believers
Come and stand beside us
We can find a better way
~Words and music by John Denver
In these tough times where so few tower over the many who are fearful for the fate of their livelihood. In these times where we look at the real question: How did we allow this to happen? At this time where so many are at the brink of hopelessness, having had to surrender a house, and face gouging credit card debt; at a time where medical attention is getting increasingly scare for the majority, and the ability to provide food for a family is an increasing concern; at a time where political figures make doubtful promises.
Now, at this time listen carefully to the words of this song (Rhymes and Reasons). It will take you to a place of inner simplicity. It will return you to a place that is not of this world. It will return you to a Self that can readily utter what really matters in our lives. It will draw you to place within where your heart and mind are filled with the presence of God. When you arrive at that place listen to what God has to say to you. DON ¤¤
Friday, May 22, 2009
Faithful Attentiveness
Native to Asia is the Chinese bamboo tree. Compared to most other trees the Chinese bamboo grows in a particularly unusual fashion. It does not emerge from the ground until four years after the seed is planted. Then in the fifth year, at a truly amazing rate of growth, the little shoot pierces the soil and rockets to an amazing height. In a matter of five or six weeks it can grow to a height of 90 or more feet. Some Asians say that an observer can literally watch the Chinese bamboo tree grow right before their eyes.
Being faithful to our True Nature is not unlike the development and growth of the Chinese bamboo tree. You may wait for what is seemingly an indefinite time period to realize the truth about who you are. You may be waiting for the light to draw you to the point of realization, where you can fill your awareness with real love and happiness.
Faith is key, and faith invariably requires waiting with both assurance and patience.
The Chinese bamboo tree does not remain dormant for four years. Rather the reason that growth from the ground up is not observed is because the tree uses four years to develop a vast supportive root system below the ground. Tree growers are however endlessly faithful to what will one day occur. They realize that the tree, in order to grow tall and fast, must first spread its roots deep and wide. For if the tree was to first grow above the ground without developing a hardy root system, little more than a slight wind would bring it down.
If you want to experience a state of consciousness that provides abundant love and happiness, then like the Chinese bamboo you must remain faithful to what you know will one day transpire. You must remain attentive to your intention, and be faithful to the outcome. Whether it is a matter of uncovering the God within, or discovering the meaning of your life, the universe once hearing your request, will begin to produce the required root system.
The bamboo growers are very attentive to the development of the tree. Without expecting to see any visible signs of growth they continue to nurture the fledgling trees with water and fertilizer. And although there are no apparent signs of life, growers realize that the tree is doing what it needs to do below the soil so the above ground plant will
one day grow and flourish. And when the tree finally burst through the soil it does so at an accelerated rate.
The universe intervenes with our loving intentions in much the same way. If we keep our minds focused on the outcome and remain faithful to God's powers, then at one point when all necessary elements are in perfect balance and harmony, the way to our intentions will open up at an accelerated rate. The appropriate friends, acquaintances and teachers will appear. Also the right situations will develop. These may seem as if they are chance occurrences or coincidences, but they are not. The guiding wisdom of universal love is doing what it knows how to do. It is drawing us to what we are intended to receive.
Be thoughtful about what you request. The energy created by Divine Love is of a higher frequency than conventional ego transmitted energy. Ego energy tries to attract to itself that which it believes that it is lacking. In the realm of Divine Consciousness we are one with the mind of God. We are unified with the Mind that lacks nothing. So when making a request of that mind you are asking for your intention to be fulfilled as it relates to a divine orientation. I am not speaking of request that are specifically centered around ideas like earning a larger paycheck of getting a good deal on a car that you want. I'm speaking of requests that are beyond the dimension of getting things and people to match up to our desires. If you are experiencing financial or transportation issues then ask God to guide you through love to bring you the solution.
Ask that you experience love in a specific facet of your life. For example if you are experiencing job related concerns and would like for the wisdom of God to prevail. Then make that specific request. Keep it in the forefront of your mind. But, don't meddle. Allow God to do His work. He will send you messages and messengers. Have faith. Be attentive to your intention by meditating. Quiet the concerns and problems that arise in your mind. Let go of all ego-based remedies for solving your problems. Be patient. The tree will sprout. You will be given a direction and your concerns will be handled.
If you are looking for the meaning of your life then ask the question: What is the meaning of my life? After, ask God to allow you to see the answer through love. Once you have asked, be assured that a messenger will deliver the response. You will hear the answer. But before that answer arrives make sure you tend to the tree. Hold loving thoughts in your mind. Avoid doubtfulness or making judgmental perceptions of yourself and others. Nurture your question with love, kindness, peacefulness and compassion. One day a sprout of knowledge will appear and from that sprout you will see new growth. Your True Nature will be revealed and you will be on your way to discovering the meaning of your life. And in that knowledge the
understanding of your Authentic Self will be revealed to you. Don¤¤
Friday, May 15, 2009
The bridge of Meditation
When I begin meditation, I frequently place an image in my mind that creates a sense of peacefulness. The brilliant pink lotus flower is an excellent representation of peace. To me it is a symbol of calmness and helps create a sense of inner-stillness. The effortlessly floating lotus is a metaphor for the practice of meditation. It rests quietly, full of its own beauty, wanting nothing, thinking of nothing, judging nothing.
A bridge allows easier accessibility to two landings that are otherwise separated by a chasm. In my life I am often aware of the consciousness that is represented by love, and compassion. Often I am reminded by the Holy Voice to remain non-judgmental and see only Love. The frequent reminders makes it none-the-less difficult to, at times, cross over, and just relinquish one form of consciousness for the other. The cross over can be resisted by old thought patterns. It seems like when I'm in a certain frame of cognition I just cannot bring myself to a place in the mind where I want to make the effort to be peaceful, calm and accepting. Rather I want to clutch the anger and judgment. In my mind, I see the other side. I realize the benefits that I will enjoy by crossing over from one reality to the other. But! But I hesitate. And then I ask myself: Why am I reluctant?
The answer I believe is centered in the concept of control. To walk the bridge from one state of reality to the other would mean relinquishing control. On one side of the bridge is the domain that I have controlled for many years. When I set foot to the bridge I realize that I am moving toward another domain, one in which I must surrender both my offensive and defensive weapons. This is a place in which weapons of the psyche have no use. This is Divine turf, a place where I am asked to surrender my ego-based thoughts and instead extend love and offer forgiveness.
Maybe at some point you also experience difficulty in bringing yourself to inner peace and joy. Maybe you, like me, realize that there is a peaceful place that waits, but you are resisting the walk over the bridge. Maybe you want to hold onto those toxic thoughts for a while.
Here are some things I do to help myself. I first place in the center of my mind an image that brings me to a happy state of mind. Then I move my mind to an image that represents calmness. As these images are interrupted by thoughts that encourage me to turn back, I visualize letting those taunting thoughts go.
Like a balloon filled with helium drifts high into the sky, I too encourage these hindering thoughts to drift off. I see myself taking one step at a time, crossing the bridge ever so slowly. As my mind begins to settle down, I focus on my breath, taking notice as each breath arises and falls.
Again ego-based thoughts will try to creep in and disturb this meditative practice. When they do, I mentally perceive myself letting the thought go. Over time, the anxiousness subsides and divine consciousness prevails. Again I become calm, relaxed and one with love. God fills my heart.
Friday, May 8, 2009
Receiving the Light & Treasures of the Light
At some point in our early lives many of us loose our memory of the Light. Rather our awareness is diverted and we are restricted by a reality that wants us to respond to its ever-persistent demands and expectations. In simple terms, we forego the wisdom of God, the Light, for satisfaction of self-serving desires.
Wondering in the dark causes loneliness and a sense that something is missing. And so, many come to a point in their lives where they eventually search for truth and in doing so acknowledge the Light, and recognize its divine power. When initially entering this new awareness, folks begin to see into the flawed conventional reality of self. They begin to view this reality from an objective point of view, much as if who they are is playing on a theatre screen, and they are the detached viewer.
In entering this transitional point many folks claim to see the self from a distant and removed vantage point. They realize that a powerful attraction, something beyond conventional reality is drawing them toward it. Something of a divine transpersonal nature is pulling them away from their pursuits of self-indulging wants and cravings.
Worldly desires become more and more meaningless during this transitional period.
Followers of the Light realize that the magnetic force of divine wisdom is redirecting their awareness and that they are part of Something that is Ultimate and Wise.
Once this recognition is acknowledged then what is to be done next? How does one immerse one's self in this new reality? How do those who have entered this brink, evolve into the Divine Self and share in Divine Consciousness. The answer is: we must first be clear about our intention, then remain faithful and attentive to that intention.
I'm not speaking of intentions that are based on firstly satisfying self-gratifying expectations, wants and desire. I'm not referring to intentions that are pinpointed to getting a new Bentley or affording a wardrobe overhaul.
Intentions that emanate directly from self-serving worldly cravings are rooted in the idea that we lack something and will thusly be happy once we get whatever we believe is missing.
Those desires are seeded and propagated in a reality that restricts both self-awareness and our spiritual transformation. I am speaking of intentions that call forth love, happiness, and the fullness of life. These intentions will bring forth into our lives everything that is required to nurture, develop and support their wellbeing.
In asking that God guide you and ingratiate you with fullness, willingly release your worldly appetites. Ask that God take control of your daily comings and goings. Ask that you experience Love, and it is love that you shall receive. Ask that you be happy and you will be given the where-with-all and guidance to be a happy being. Ask that you experience a life of fullness and you will enjoy the abundant flow of love and happiness that comes your way. "Ask and you shall receive." Ask, then allow God to do His thing. Get out of the way. Ask to receive the Light and you shall be the Light of the world. Don¤¤
Treasures of the Light
Last Sunday at The Center for Happy Beings, the theme of our talk was fear. We spoke of how we involve fear in our everyday lives, and also how fear can take control our behavior and the direction of our lives. We looked at how to transcend our fears, and get beyond the hurt that fear has caused us. Then we participated in a guided meditation where we silently and individually returned to a critically fearful place in our past, and allowed the fearful emotions to dissipate and then diminish. It was a great day for the Light. Each Sunday morning is an adventure in finding new treasures of the Light. db¤
Any transition serious enough to alter your definition of self will require not just small adjustments in your way of living and thinking but a full-on metamorphosis - Martha Beck
From within or from behind, a light shines through us upon things, and makes us aware that we are nothing, but the light is all. – Ralph Waldo Emerson
An age is called "dark," not because the light fails to shine but because people refuse to see it. ~James Michener
Even a thought, even a possibility, can shatter and transform us.- Nietzsche
Enjoy a wonderful and happy month of May – remember you are the
Light. db¤
Discover the Love Within.
Take the Pathway to Self-Transformation. Your butterfly is awaiting!
What is necessary to change a person is to change his awareness of himself. -Abraham Maslow
It is difficult to see the picture when you are inside of the frame. -Author Unknown
It is a paradox of life that some of the best people we ever meet are those who have suffered a great deal. Their lives are like still waters that run very deep. There's a quality of inner goodness that has been wrought through all the pain.- James Jones


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WORDS: Drew Petersen
It wasn't his first, and won't be his last, but at the moment, Sam Smoothy's recent Freeride World Tour win may be his most impressive performance yet. The New Zealander was the clear winner at the second event of the year located in the small country of Andorra, a first time host on the FWT. Questionable snow conditions, consequential exposure, and technical airs highlighted the run of the day, and planted Smoothy rightfully atop the podium. Since then, the video of his run has all but broken the Internet. If you haven't seen the video yet, do yourself a favor and click play. Sam joined me on Skype over the weekend to discuss the run, ensuing media attention, and his goals moving forward to the last two FWT events of the season.
POWDER: First off, congrats on the win in Andorra, as well as the attention your run has garnered since then. It's been pretty cool to see your video get so many views.
Thanks, mate. It's kind of crazy; you always work hard to try to create things that people will watch. And then that thing just took off like a rocket. Any win on the FWT is big, but this one—just with the media storm around it—is for sure one of the highlights of my career so far.
The line was absolutely mental. What were you thinking before you dropped in, and then during the run? Walk me through it.
I was just a bit nervous that the snow hadn't softened up enough. I knew that if I aired into that zone and it wasn't soft, I'd be in all manner of trouble. But once I committed to it, it all went by so fast. I was just so far gone and in that moment the thought processes weren't really there. I was just on autopilot. When I came out of that top zone, I just exploded yelling.
What would you say were the cruxes of the line?
I think that left hand turn after the first air was super critical. I just stood on that outside ski and drove it home. Also, when I came into that next section and I had to get onto this little ski width platform to go back left, that was probably the other really tricky bit.
I was coming into it blind, and I knew if I couldn't get enough skier's left off it, I'd just air straight fall line into that wall.
I've heard you say that this line reminded you of tussock skiing in New Zealand. Can you describe what you mean by that?
Tussock is a large, native grass that grows in the alpine. At home we don't get a lot of snow, so the grass often pokes through in late season. We have a lot of fun skiing really skinny lines that involve straight lining tussock sections and bouncing around on the grass. That all kind of culminates in the closing day antics, which is who can ski the gnarliest thing without really any snow in it. That's kind of where I drew my inspiration from for this run.
Some of the headlines on the Internet sharing your video include this question in some form: "Is this the greatest comp run ever?" What are your thoughts on that claim?
I think that's fairly ridiculous really. It was the best run on the day, it's one of my best runs ever, but it's still a small face—it's still just a bit of a laugh. I don't think it's even close to some of the best runs the world has seen, so it's flattering, but it's fairly wide of the mark I think.
In your opinion, what are some of the best comp runs of all time?
Some of the runs that Reine Barkered has done on the Bec de Rosses, for sure. Ditto with Aurélien Ducroz. Just full throttle, full line skiing, but super precise and clean. That's definitely some of the riding I've looked up to over the years. It's just those guys on big faces making big moves in critical places and making it look easy.
What are some of your best comp runs of all time? Where do you rank this Andorra run?
This one would be right up there. I think it was a completely different style, but my win in Chamonix was a pretty good one in 2012. And then I did a pretty rowdy one in New Zealand at Mount Ruapehu once in a qualifier. That one was kind of similar to the run in Andorra. It had this blind air into an exposed zone—you just couldn't fall in—and a couple more airs out. That was in even worse snow conditions (laughs).
Are you pretty excited to head up to Haines for the first ever FWT event in Alaska?
Massively excited, I think everyone is. It's what everyone's been talking about all season on tour. I'm going to Haines for the stop and then coming home to Verbier for the [Swatch] Xtreme and then going straight back to Haines to film with Legs of Steel. I might even leave my toothbrush there or something.
You know Verbier and you know the Bec de Rosses. Do you have anything in mind for a line on the Bec this year?
Yeah, it's kind of hard to tell this far out. I've had this line in Dogleg [Couloir] that I've been looking at. I wanted to do it last year, but I had a crash and I wasn't healthy enough at all. It's kind of a classic, but with a twist. I hope it will get people excited. It's either going to go really well or I'm going to explode.
Now that you've survived the first part of the year, and qualified for Alaska and Verbier, what is your focus moving forward? What are your goals for the rest of the year?
My goals the rest of the year would be pretty basic, non-ambitious goals: winning the next two events and the title. Just simple stuff (chuckles). I'm not sitting too good now, I've had one good run and two bad ones. I'm in tenth, but it's really tight at the top. I'm just going to not think too much about the rankings to be honest. I'm just going to find the best thing I can ski on each day and send it as hard as I can. I'm not much of a tactician. I'm just going to ski hard and go all in.


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Memorial Brick Program
The Memorial Brick Program is a unique opportunity for friends of The National Civil War Museum to honor or memorialize a special individual or moment in time.
Located to the left of the Museum's main entry, the Walk of Valor allows surviving descendants of Civil War veterans a thoughtfully designed place to memorialize their loved ones.
In addition, the Friends Walkway, adjacent to the patio and parade grounds, provides an opportunity to make an expression of support to the Museum by honoring a special person or event.
Bricks may be purchased for as low as $75 each. Each 4"x8" brick includes 3 lines of inscription with 15 characters per line including spaces and punctuations. The 8"x8" brick allows for a NCWM logo or an extra line of inscription or up to 5 lines of inscription.
Bricks are generally installed twice a year. Those purchased by March 1 will be installed by May 31; those purchased by August 1 will be placed by October 31. Notifications will be sent via email once bricks are placed.
Click to download the Walk of Valor order form in PDF (file size is 60k). Friends Walkway order form in PDF (file size is 56k).
Click here to order online


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As the Hudson Valley braces for another heat wave, the Con Edison lockout has reached the two week mark without an end in sight.
Talks broke off a few minutes after 6 p.m. on Friday night and are scheduled to resume at 3 p.m. Saturday, according to spokesmen for Con Edison and Utility Workers Union of America Local 1-2.
On Friday, union leaders acknowledged Con Edison provided long-sought documents detailing company proposals on compensation and health care benefits, but stopped short of saying they believe there's any real progress in negotiations.
Con Edison provides electricity to 3.2 million, including some 350,000 customers in Westchester County. Despite several heat waves, and accompanying strain on the grid, the company has avoided major outages over the past two weeks without the more than 8,000 unionized workers who were locked out without a contract on June 30.
About 5,000 Con Edison managers have stepped in to handle day-to-day operations and repairs, supplemented by workers nonunion contractors from other areas.
The lockout does not impact Con Edison subsidiary Orange & Rockland Utilities. NYSEG and Central Hudson also serve customers in Westchester County.


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Accurate determination of orthology is central to comparative genomics. For vertebrates in particular, very large gene families, high rates of gene duplication and loss, multiple mechanisms of gene duplication, and high rates of retrotransposition all combine to make inference of orthology between genes difficult. Many methods have been developed to identify orthologous genes, mostly based upon analysis of the inferred protein sequence of the genes. More recently, methods have been proposed that use genomic context in addition to protein sequence to improve orthology assignment in vertebrates. Such methods have been most successfully implemented in fungal genomes and have long been used in prokaryotic genomes, where gene order is far less variable than in vertebrates. However, to our knowledge, no explicit comparison of synteny and sequence based definitions of orthology has been reported in vertebrates, or, more specifically, in mammals.
We test a simple method for the measurement and utilization of gene order (local synteny) in the identification of mammalian orthologs by investigating the agreement between coding sequence based orthology (Inparanoid) and local synteny based orthology. In the 5 mammalian genomes studied, 93% of the sampled inter-species pairs were found to be concordant between the two orthology methods, illustrating that local synteny is a robust substitute to coding sequence for identifying orthologs. However, 7% of pairs were found to be discordant between local synteny and Inparanoid. These cases of discordance result from evolutionary events including retrotransposition and genome rearrangements.
By analyzing cases of discordance between local synteny and Inparanoid we show that local synteny can distinguish between true orthologs and recent retrogenes, can resolve ambiguous many-to-many orthology relationships into one-to-one ortholog pairs, and might be used to identify cases of non-orthologous gene displacement by retroduplicated paralogs.
The accurate determination of orthology is central to comparative genomics. Pinpointing the origin of new genes, understanding the evolution of new gene families, and assessing the impact of gene and genome duplication events all require the accurate assignment of orthology between genes in distinct genomes. In complex genomes with large gene families this task requires differentiating between genes that have diverged through a speciation event (orthologs) and those derived through duplication events within a species (paralogs). Determination of orthology and paralogy is especially challenging in vertebrates species such as the mammals. Very large gene families, high rates of gene duplication and loss, multiple mechanisms of gene duplication, and high rates of retrotransposition all combine to make the determination of orthology between vertebrate genes difficult.
Given the importance of accurate orthology assignment, many methods have been developed to identify orthologous genes. Most of these methods rely upon analysis of the inferred protein sequence of the genes in question by clustering the results of protein sequence comparisons results in the classification of putative orthologs. Examples of this approach include reciprocal best BLAST hits and more inclusive BLAST based clustering methods. Splitting of these clusters based on relative similarity can distinguish between older and newer duplication events and is implemented in the widely used Inparanoid algorithm [1] and related approaches [2]. While these methods are robust and easily implemented, they rely upon a single character, the protein sequence, for classifying genes into orthologous groups.
More recently, methods have been proposed that use genomic context in addition to protein sequence to improve orthology assignment. These methods have been most successfully implemented in fungal genomes [3,4], and have long been used in prokaryotic genomes [5,6], where gene order is far less variable than in eukaryotes. An interesting implementation of this approach is found in the SOAR and MSOAR algorithms [7,8], which seek to assign orthology by minimizing the recombination distance between two genomes. In most of these approaches, synteny blocks covering some percentage of the genome are used hierarchically with protein coding information to assign orthology between similar genes. Approaches that exploit synteny information can be particularly useful in resolving ambiguous sequence based matches between putative orthologs. Recently, Han and Hahn [9] used local synteny information to identify parent-daughter relationships among duplicated genes. However, it is worth noting that "phylogenetic shadowing" [10] approaches used in genome assembly might lead to a lack of independence between sequence and synteny information.
In this study, we evaluate a simple method for using gene order (local synteny) in the identification of mammalian orthologs. We explicitly compare the relative performance of local synteny and Inparanoid for inferring orthology within this clade and show that local synteny alone is sufficient to identify orthologs with an accuracy comparable to that of Inparanoid. By analyzing cases of discordance between local synteny and Inparanoid we show that local synteny can distinguish between true orthologs and recent retrogenes, can resolve ambiguous many-to-many orthology relationships into one-to-one ortholog pairs, and can highlight possible cases of parental gene displacement by retrogenes.
Several orthology inference methods, such as Inparanoid [1] and OrthoMCL [2], use coding sequence similarity (for example Blastp score [11] or Protdist [12]) as primary orthology signal. In this paper, we use local synteny information to determine orthology. We define the local synteny of two genes as the maximum number of unique homologous matches between their six neighboring genes (three upstream and three downstream immediate neighbors for each gene, see Figure Figure1).1). Homology between two neighboring genes is defined as Blastp E-value < 1e-5.
To validate the use of local synteny for inferring orthology, we evaluated the correlation between Protdist and local synteny using a dataset derived from the Pfam protein family database. Pfam families are highly accurate protein families based on protein domains [13]. We randomly selected 1,000 cross-species homologous protein pairs (homologs belonging to a given Pfam family) from five mammalian genomes: Homo sapiens (human), Pan troglodytes (chimp), Mus musculus (mouse), Rattus norvegicus (rat), and Canis familiaris (dog). To avoid protein family-specific bias in this analysis, we chose one homologous pair from each Pfam family. For each pair we computed Protdist and the degree of local synteny between the two genes. Figure Figure2a2a shows that there is negative correlation between Protdist and the local synteny of these samples (r = -0.67 with p-value < 0.0001). This is not surprising as gene order is conserved between DNA segments resulting from speciation or large-scale segmental duplication events. However, because local synteny is not directly computed based on the coding sequence, we can use local synteny to test hypotheses of orthology between two genes independent of their coding sequence.
Theoretically two non-orthologous genes should not share homologous neighboring genes. However, there is a small probability of homology matches occurring by chance. Moreover rearrangements, insertions, and deletions will lead to loss of local synteny between orthologous genes. In order to account for these events, we wanted to determine an optimal window size and match percentage that could reliably identify orthologs based on local synteny. In Figure Figure2b,2b, we test the impact of the number of matches between six neighboring genes to determine local synteny. Student's t tests comparing Protdist means illustrate that 0, 1, and >1 matches have significantly different Protdist means (p-value ≤ 0.05). In order to choose the threshold of homologous matches, we calculate the false positive rate (FP) and false negative rate (FN) to Inparanoid orthologs and Ensembl orthologs then choose the threshold that minimizes the sum of FP and FN events. For six neighbors, the pairs with more than one homologous match minimizes FP and FN rates (0.152 to Inparanoid orthologs, and 0.151 to Ensembl orthologs (see Additional file 1)). Increasing the window size to 10 or 20 flanking genes does not show a significant difference in detecting orthology (see Additional file 1). Based on these results we define orthology by local synteny when the number of maximum unique homologous matches between the six neighboring genes is greater than one. We will refer to those pairs as syntenic from now on.
Since many orthology detection methods use more complicated algorithms than just coding sequence similarity, a high correlation between Protdist and local synteny (Figure (Figure2)2) is not sufficient evidence that local synteny captures true orthology. For a more rigorous analysis we compared local synteny based orthology to the orthology relationships inferred by six well-known orthology detection methods: Inparanoid [1], OrthoMCL [2], RBH (Reciprocal Best Hit), SBH (Single-way or One-way Best Hit), BLASTP, and orthology data from Ensembl [14]. Since there is no gold standard of orthology, we performed Latent Class Analysis (LCA) [15,16] to estimate the accuracy (sensitivity and specificity) in the absence of a reliable standard. LCA estimates false positive (FP) and false negative rates (FN) based on agreement and disagreement between various ortholog definitions. To minimize sampling bias 10 LCA's were performed on random samples with a size of 1,000 orthologous genes from five mammalian genomes using the same sampling method described in the previous section. For one sample 1,000 Pfam families were randomly selected, then one cross-species protein pair was selected from each Pfam family for analysis by the seven compared orthology inference methods. For all methods based on coding sequence similarity, the longest proteins were used as the representative proteins of the genes. A more detailed description can be found in the methods section.
Figure Figure33 shows that orthology inference based on local synteny yields a lower FP rate than SBH/BLASTP and a lower FN rate than OrthoMCL, reinforcing the interpretation that orthology can be accurately inferred without coding sequence information. However, local synteny has a slightly higher FN rate than the four orthology methods based on coding sequence similarity (BLASTP, SBH, RBH and Inparanoid). This is partially due to the fact that these coding sequence based methods cannot distinguish retrotransposed genes from the original copies unless retrotransposed genes are sufficiently diverse. This might lead to incorrect orthology assignments (retrotransposed copies replace the original ortholog genes) or ambiguous orthology assignments (one-to-many or many-to-many ortholog groups including retrotransposed copies as their members) by these methods. Local synteny also has a slightly higher FP rate than Inparanoid and RBH. This is likely due to the fact that local synteny cannot distinguish DNA-mediated duplicates from the original copies. We analyze these discordances in more detail in the following section.
Because Inparanoid is one of the most widely used ortholog definition methods [17-19] and is purely based on the coding sequence information, we decided to do a more thorough comparison of Inparanoid and local synteny based orthology. Figure Figure44 shows the agreement and disagreement between these two orthology prediction methods. The majority of these samples are concordant between two ortholog predictions (syntenic/Inparanoid (55.1%) and non-syntenic/non-Inparanoid (37.9%)), which agree with the LCA results (Figure (Figure3).3). However, 2.5% of Inparanoid orthologs are non-syntenic, and 4.5% of gene pairs are syntenic but not Inparanoid orthologs.
In order to identify the source of this discordance we employed intron-based evidence. Specifically, for each gene pair used in the test we computed the intron conservation ratio (ICR) between the two genes defined as follows (for more details see Methods):
Figure Figure44 shows ICR histograms for pairs of genes falling in each of the four classes of agreement/disagreement between Inparanoid and local synteny. In both concordance cases, namely for syntenic Inparanoid orthologs (Figure (Figure4a)4a) and non-syntenic non-Inparanoid orthologs (Figure (Figure4d),4d), ICR is in strong agreement with the orthology assignments made by the two methods. Indeed, most of the syntenic Inparanoid orthologs have ICR of 1 (Figure (Figure4a),4a), and the majority of non-syntenic non-Inparanoid orthologs have ICR < 0.5 (Figure (Figure4d).4d). In the two discordant cases (7% of the evaluated gene pairs) intron evidence can be used to resolve the conflicting assignments made by Inparanoid and local synteny. About 3/4 of syntenic non-Inparanoid orthologs (Figure (Figure4b)4b) have ICR > 0.5 and half of non-syntenic Inparanoid orthologs (Figure (Figure4c)4c) have ICR = 0. This suggests that in these cases local synteny based orthology assignments are more often concordant with gene structure evidence (ICR) than those based on coding sequence similarity. However, the ICR histogram of non-syntenic Inparanoid orthologs (Figure (Figure4c)4c) has a bimodal distribution, which might arise from a mixture of FNs from local synteny (pairs with high ICR) and FPs from Inparanoid (pairs with low ICR). We further investigate these cases in the following subsections.
All the non-syntenic Inparanoid ortholog pairs with zero ICR contain one intronless copy and one intron-bearing member. Based on local synteny information and intron conservation ratio, these intronless copies are probably retrotransposed (RT) copies of the original orthologs. Of all the samples, 1.2% are non-syntenic 0-ICR Inparanoid orthologs. Inparanoid likely included RT copies in these orthologous groups because the RT copies have not diverged sufficiently to be distinguished from their parent gene. In 0.2% of samples Inparanoid chose RT copies even though there were other syntenic high-ICR copies. Figure Figure55 shows one of these pairs. In this case, the Blastp score of the dog gene to the RT rat gene (shown as Rat A) is smaller than to the syntenic high-ICR rat gene (Rat B), which caused an Inparanoid miscall, assigning the RT paralog as the ortholog. For the detailed information including Blastp scores and Protdist in tabular format, see Additional file 2.
The rest of non-syntenic 0-ICR Inparanoid orthologs (1.0%) of all samples are classified as one-to-many orthologs by Inparanoid. In many of these one-to-many orthologs, syntenic high-ICR pairs are chosen as "core orthologs" (based on the Inparanoid scores) with additional RT copies added to the orthology group because their protein sequences are still close to those of the original copies. By using local synteny, the RT copies in one-to-many orthologs would be distinguished from the other members. Thus, local synteny based orthology separates RT copies from those generated by speciation or other duplication mechanisms and can be more informative in recovering the evolutionary history of a gene family.
All non-syntenic Inparanoid orthologs with an ICR > 0 (1.3%) are likely to result from the loss of local synteny. They each have one homologous match between neighboring genes (lower than the threshold of being syntenic) and are selected from distant species pairs (i.e. not human-chimp or mouse-rat). Each of these distant pairs is part of larger orthology groups (5-species), in which the counterparts in closer species pairs have higher local synteny (more than 1 match). This loss of local synteny likely results from rearrangements and gene insertions or gene losses in more distant species. See Additional file 3 for an example.
There are 2.3% syntenic non-Inparanoid orthologs with an ICR ≥ 0.5 (Figure (Figure4b).4b). The majority (2.1/2.3%) of syntenic non-Inparanoid orthologs with high ICR are likely distant paralogs: both genes are in different syntenic Inparanoid orthology groups with high ICR. This may result from old DNA-mediated duplication events followed by speciation events without significant local genome rearrangements. Local synteny cannot distinguish between orthologs created by large-scale segmental duplications and by polyploidy events. Moreover, the syntenic non-Inparanoid pairs with an ICR < 0.5 (1.1% of all tested pairs) are also likely distant paralogs, by old tandem duplications with different gene structures resulting in lower ICR.
In the non-syntenic non-Inparanoid case (Figure (Figure4d),4d), where the most of these pairs have low ICR, we still find 2.2% of pairs with ICR equal to 1. All these pairs are likely distant paralogs. Table Table11 contains the summary of all of these cases.
Since local synteny is able to differentiate some speciation and duplication events, a phylogenetic tree (or duplication-speciation history) of ambiguous many-to-many orthologs may be determined using local synteny information. In mouse-to-rat ortholog definitions from Inparanoid, there are 131 many-to-many ortholog groups. The majority (~75%) of many-to-many groups are comprised of DNA-mediated duplicated copies (usually from tandem duplications combined with one or two distant segmental duplication events) while ~20% have true orthologs (confirmed by local synteny and ICR) as well as non-syntenic intronless copies (probably from RT events).
We present two many-to-many Inparanoid ortholog groups where the local synteny determines the order of evolutionary events in the gene family. One of them is an example of orthologs from distant DNA-mediated duplication event(s) followed by possible rearrangements or gene gains/losses, and then speciation event (Figure (Figure6).6). In this ortholog group, there are three mouse gene members, ENSMUSG00000001175 (MGI symbol: Calm1), ENSMUSG00000019370 (Calm3), ENSMUSG00000036438 (Calm3, which referred to Calm3x in the figure to avoid confusion) and two rat gene members, ENSRNOG00000004060 (Calm1), ENSRNOG00000016770 (Calm3). All the genes are on different chromosomes and the transcripts of these (protein coding) genes are known. Since all the cross-species pairwise sequence similarity measures are equal, Inparanoid could not pick distinct orthologs nor could phylogenetic tree-building programs determine the tree (for Blastp results see Additional file 4). Neither could ICR break the tie due to a high conservation of gene structure. Finally, no Ensembl ortholog prediction is made between these genes. However, in the figure, two Calm1 genes (ENSMUSG00000001175 and ENSRNOG00000004060) and two Calm3 genes (ENSMUSG00000019370 and ENSRNOG00000016770) have high local synteny (4 and 5 matches, respectively) and any other local synteny is either 0 or 1. In this specific case, local synteny helps break the tie in sequence based similarity. Using this information we can infer an old segmental duplication (SD) event before mouse-rat speciation giving rise to the Calm1 and Calm3 ancestors followed by rearrangements reducing the local synteny between these two mouse and rat ortholog pairs (Figure (Figure6).6). The third mouse gene (Calm3x) has just one match with mouse Calm1 gene and rat Calm1 gene, and high levels of intron conservation, indicating a DNA duplication event, but we do not have enough local synteny information to tell precisely when the duplication occurred. Also since the mouse Calm3x gene has only one match with the rat Calm1 gene, local synteny does not find this apparent ortholog. For the detailed information in tabular format, see Additional file 4.
Another case where local synteny clarifies the history of closely related groups of duplicates can be seen in a mouse-rat many-to-many Inparanoid ortholog group including RT copies (Figure (Figure7).7). This example contains two members from each species, ENSMUSG00000013701 (MGI symbol: Timm23 referred as Mm1 in the figure), ENSMUSG00000069622 (Timm23 as Mm2) and ENSRNOG00000019811 (Timm23 as Rn1), ENSRNOG00000032900 (TIM23_RAT as Rn2), where each has one known transcript on different chromosomes. Again, neither Inparanoid nor pairwise Protdist analysis could discriminate orthologs due to identical cross-species Blastp measures (for Blastp results see Additional file 5). Ensembl has a bigger ortholog group including these four genes, but no better information about which are orthologs or RT copies. However two intron bearing genes have perfect local synteny (= 6) and an ICR = 1, and two intronless copies do not have any local syntenic match to the two intron bearing genes. Therefore, we can infer that two intron bearing genes are the main orthologs and two intronless genes are RT copies of these orthologs. Furthermore, since intronless genes are not syntenic to each other, we infer that the two intronless genes are the result of two separate RT events on each species lineage. For detailed information in tabular format, see Additional file 5.
The accurate determination of orthology is critically important to comparative genomics. However it has been a challenge to compare the various orthology determination methods without a reliable gold-standard orthology dataset [15]. The statistical technique of Latent Class Analysis allows estimates of false positive and false negative rates from data based on agreement and disagreement between various ortholog definitions. Because error rates estimated in this way may be affected by which methods are included in the analysis, we must consider the FP and FN rates estimated here as relative error rates. Furthermore, these error rates might not reflect rates obtained from a genome-wide implementation of these methods. There is an ongoing effort on standardizing protein datasets for benchmarking orthology determination methods [20] which would help resolve this issue.
In Figure Figure3,3, Inparanoid and RBH agree more closely than any other pair of orthology definitions. This is in accord with the fact that Inparanoid uses the reciprocal best hit to start core ortholog identification. SBH and BLASTP have zero FN rates and higher FP rates than Inparanoid and RBH due to less stringent conditions used for ortholog detection in these methods. The order of FP rates (BLASTP > SBH > RBH) is concordant with the stringency of each method. The FP rate of local synteny based orthology falls between SBH and RBH. This is reasonable considering the fact that SBH and local synteny based orthology cannot distinguish close paralogs from ortholog pairs, but local synteny can separate RT close paralog copies from orthologs.
The FN rate of local synteny based orthology is higher than those of Blastp based orthologies (Inparanoid, RBH, SBH, and BLASTP). This may be due to distant paralogs retaining flanking genes, but diverging in their coding sequences enough to be distinguishable by Blastp. RT miscalls by Inparanoid (e.g. Figure Figure5)5) are also likely to have contributed to the relative FN rate of local synteny based orthology.
Due to the fact that OrthoMCL detected the smallest number of orthologs in any sample (data not shown) the estimated FN rates from OrthoMCL are the highest in this experiment (approximately 0.3). This is opposite from the result of Chen et al. [15] where OrthoMCL and Inparanoid were shown to have lowest estimated FP and FN rates and OrthoMCL has lower FN rates than Inparanoid. The apparent disparity between these results could be explained by the fact that LCA is designed to estimate consensus FP and FN rates without any guarantee that the estimated rates are close to absolute values, and by the difference in the species sets used in the two experiments: our dataset of five mammalian genomes and the comparatively distant seven eukaryotic genomes used in Chen et al.
Synteny information has been used to cluster synteny blocks between two related genomes in order to detect orthologous gene pairs [8,21] and to reconstruct phylogenetic trees [22]. These synteny blocks are generally used as "genomic anchors" [11] or to place gene loss/deletion events on phylogenetic trees [23,24], not as a definitive measure to distinguish close paralogs from distant paralogs. In Zheng et al. [21], one of three methods to define orthologs between human and mouse used a genomic anchor approach. They identified synteny anchors and synteny blocks [25,26] then introduced a local synteny approach for the anchor-poor regions by accepting the pairs of genes flanked by previously identified ortholog pairs. With this approach they found 11% more orthologs than by RBH alone.
Another similar approach to local synteny was used to reconstruct phylogenetic gene trees from coding sequence similarity and local gene order [22]. In the algorithm SYNERGY, Wapinski et al. measure a synteny similarity score for a pair of genes by counting the neighboring genes in syntenic blocks. However, SYNERGY has not been tested in mammalian genomes.
Finally, MSOAR [8] uses combinatorial optimization on global gene order to identify orthologs based on minimal rearrangement scenarios. However, Fu et al. point out that the global optimization might lead to false ortholog links in some scenarios. Because our local synteny exploits proximate synteny information we expect lower false positive ratios than those obtained with MSOAR.
Most importantly, when the orthology is defined by codon sequence similarity, testing any hypothesis of selective pressures on orthologous gene presents tautological challenges. Since orthology detection by local synteny is not based on the comparison of coding sequence information between candidate orthologs, testing the selective pressure between orthologs becomes a valid comparison of largely independent variables. However, gene order also degrades over evolutionary time. How well synteny will be able to effectively identify ancient orthologs remains to be seen.
Gene duplications are a major force in genome evolution [27]. Genes are duplicated through two main duplication mechanisms; DNA-mediated and RNA-mediated [27,28]. DNA-mediated duplications can include multiple genes and associated intergenic sequences and introns. On the other hand, RNA-mediated duplication, or retrotransposition (RT), only copies coding sequences in the duplication event. Retrotransposed genes had been considered mostly "dead on arrival", but recent studies [29,30] show that there are many functional RT copies in the human and mouse genomes.
Sometimes the difference between coding sequences of parental genes and RT copies is not large enough to distinguish the RT copy from the parent. RT copies, however, do not share introns or flanking genes with the parental paralog. Therefore, local synteny and gene structure can often separate the RT copy from the original gene. However, when neither has an intron, only local synteny will distinguish parental and RT copies. Based on our random sampling, approximately 8~10% of Pfam orthologs are intronless gene pairs.
Reconstructing phylogenetic trees informs our understanding of the evolutionary history of gene families. Using tree reconciliation between a species tree and gene tree we can identify duplication and lost events on the tree. However by distinguishing two duplication mechanisms, DNA-mediated and RNA-mediated, not just identifying duplication events, we can sometimes place duplication events in an appropriate phylogenetic context. For example, as Figure Figure66 and Figure Figure77 show, when coding sequence does not distinguish paralogs, local synteny can determine whether DNA or RNA-mediated duplication occurred first. Local synteny can also help place RT duplication events before or after speciation (Figure (Figure7).7). Even when the coding sequence of RT duplicates drifts apart, pre-speciation RT genes often retain local synteny. Conversely, when RT duplicates are young enough to be indistinguishable by coding sequence comparison, synteny can discriminate between pre-speciation duplications, and independent RT duplication events in parallel lineages. Finally, local synteny information can often resolve the order of iterative DNA-mediated duplication events in large gene families (see also [9]).
In this paper, we investigate the concordance between coding sequence based orthology (Inparanoid) and local synteny based orthology. In the 5 mammalian genomes studied, 93% of the sampled inter-species pairs were found to be concordant between the two orthology methods, illustrating that local synteny can largely substitute for coding sequence in identifying orthologs. However, 7% of pairs were found to be discordant. Discordance is often associated with evolutionary events like retrotransposition, iterative DNA-mediated duplication, and genome rearrangement. Analysis of discordant cases between local synteny and Inparanoid shows that local synteny can differentiate between true orthologs and recent retrogenes, can split ambiguous many-to-many orthology groups into more precise one-to-one ortholog pairs, and, when employed in a genome-wide screen, might help in highlighting possible cases of non-orthologous gene displacement by retrocopied paralogs in mammalian genomes.
Five species analyzed (human, chimp, mouse, rat and dog) were obtained from Ensembl release 48 [31]. We only used protein coding genes in the Ensembl database. For genes with multiple alternative transcripts we used the longest transcripts as the representative ones. We used Pfam families [13] to choose ortholog candidates. Since there are more than hundreds of millions possible protein pairs among five genomes in Pfam families, we sampled our datasets in the following way. First, we randomly selected 1,000 families from 3,418 Pfam families which have at least two representative proteins from different genomes. One cross-species protein pair was selected from each Pfam family in order to avoid a bias from big families. We used 10 sample datasets for the LCA experiment and one of them was used in the discordance analysis.
Local synteny is measured by homology between the neighboring genes of two genes of interest. In this study the maximum unique homology matches between two sets of six neighboring genes (three upstream and three downstream neighbors) was used. The matches do not need to be co-linear or between genes on the same strand/orientation either (see Figure Figure1),1), which allows for genome micro-rearrangements as well as gene losses and insertions in the flanking region. The homology between neighboring genes is decided by pre-calculated Blastp [11] results in the Ensembl Compara database [14]. To avoid having high local synteny due to proximate tandem array genes, we considered the tandem array genes as one neighboring gene. Within a tandem array, each gene was counted separately.
We used five orthology detection methods and Ensembl orthology in comparison with our local synteny based orthology. For each of the five orthology detection methods -- namely Inparanoid [1], OrthoMCL [2], SBH (single or one-way best hits), RBH (reciprocal best hits) and BLASTP (one-way Blastp hits with the threshold) -- we used the pre-computed Blastp outputs in Ensembl database as input data. Parameters and thresholds used for each method are as follows:
1) BLASTP: homology detection using E-value cutoff (= 1e-5)
2) SBH: Single-way or One-way Best Hit. 'Best-hit' is defined as the hit (or multiple hits tied) with the highest E-value (E-value cutoff = 1e-5)
3) RBH: Reciprocal Best Hit. 'Best-hit' is defined as same as SBH (E-value cutoff = 1e-5)
4) Inparanoid (v2.0): bit score cutoff = 50 bits and sequence overlap cutoff = 0.5
5) OrthoMCL (v1.4): E-value cutoff = 1e-5 and MCL inflation index = 1.5; MCL package (v02-063) was used.
The frequency table of agreements and disagreements between orthology detection methods was calculated and used for LCA. LCA was performed using the LEM package [32] with default parameters to estimate the false positive and false negative rates. We used a basic latent model to produce Figure Figure33 assuming independence between various orthology detection methods. However, all methods we considered are solely or partially based on protein sequences. In order to account for these dependencies, we applied another latent model with an extra latent variable. With such a model, called latent class model with random effects or a continuous factor (CFactor) model, the responses of different tests are assumed to be independent [15,33]. Although the estimated error rates from the CFactor model (Additional file 6) are less tightly distributed than ones from the basic model, the relative values are not changed significantly compared with Figure Figure3.3. The looser distribution of values in the CFactor model is likely due to a lack of convergence in these runs. For detailed description of two LCA models and the FP/FN graph by the CFactor model, see Additional file 6.
Gene structure similarity is measured by the intron conservation ratio (ICR) between two intron-bearing genes [34]. For genes with multiple alternative transcripts we developed a collapsed gene model that incorporates all potential exons of that gene. Resulting exon coordinates were used to obtain the protein alignments and also to align the positions of introns. ICR between two homologous genes was calculated as the ratio of the number of positionally homologous introns divided by the total number of intron positions from the protein/intron alignment, similar to the method in Rogozin et al. [34]. Introns with less than 40BP were ignored in ICR calculation.
For the discordant cases between Inparanoid, local synteny, and/or ICR based orthology (Panels b and c of Figure Figure44 and part of data in Figure Figure4d),4d), we investigated:
1) any significant Blastp hits other than the sampled pairs (with ICR = 0 in Figure Figure4c4c or with ICR ≥ 0.5 in Figure Figure4b)4b) in order to find RT miscalls by Inparanoid (e.g., Figure Figure55)
2) the other Inparanoid orthologs in all 5 species in order to confirm rearrangement history in those families (with ICR > 0 in Figure Figure4c),4c), and
3) Inparanoid orthologous counterparts of non-Inparanoid sampled pairs in Figure Figure4b4b and Figure Figure4d4d to confirm these sampled pairs from distant paralogs.
Mouse-rat many-to-many Inparanoid ortholog groups were collected and analyzed to reconstruct their evolutionary history by considering the genomic location and intron content of their member genes. Two example ortholog groups (Figure (Figure6666 and Figure Figure7)7) were chosen due to their unambiguous evolutionary history: for most groups it is difficult to unambiguously reconstruct the full evolution due to the presence of intermingled events.
JJ performed the programming and the data analysis. JJ, IIM and CEN participated in the design of the study and drafted the manuscript. All authors read and approved the final manuscript.
Various numbers of neighbors. False positive (FP) and false negative rates (FN) of local synteny measures to the Inparanoid orthologs and Ensembl orthologs, with using different number of neighbors.
Tabular format of Figure Figure5.5. One example of RT miscall cases by Inparanoid confirmed with local synteny and ICR also shown in tabular format.
Example of loss of local synteny case. One example of loss of local synteny along the evolutionary distance.
Tabular format of Figure Figure6.6. An example of many-to-many Inparanoid ortholog groups where a SD event proceeded mouse-rat speciation.
Tabular format of Figure Figure7.7. An example of many-to-many Inparanoid ortholog groups where RT events followed the mouse-rat speciation.
LCA with a CFactor model. Another LCA model to account for the dependency between orthology detection methods and a corresponding FP/FN graph.
We thank Asav Dharia for critical reading of the manuscript and valuable suggestions.
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