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subset: commoncrawl


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Apple Inc. Chief Executive Tim Cook has said the company plans on entering new product areas this year, and, if the latest reports are any indication, Apple will release multiple versions of what's being called its "iWatch" this fall. (The image here is a photoillustration is not meant to depict an actual Apple product.)
Apple AAPL, -0.13% wouldn't be the first company to release a smartwatch — Samsung released its first Galaxy Gear watch last September — but it will certainly be a big deal if and when it comes out. It will also be the next step forward in how we tell time. So, what with the Summer Solstice occurring this weekend, and the days about to get just a little bit shorter, let's take a look at 10 of the most important timepieces through history.
— Rex Crum
1. The sundial
Probably the oldest method man has used to track the passing of the day, the earliest sundials date back to 3500 B.C. and the ancient Egyptians. Not very practical these days, but if you're ever driving up I-5 at Redding, Calif., you can check out the famous Sundial Bridge at Turtle Bay..
2. Prague's Astronomical Clock
Built in 1410, the Astronomical Clock at Old Town Square in Prague was heavily damaged during the Prague Uprising at the end of World War II but began working again in 1948. If you ever visit Prague and need to meet someone, you won't be alone if you tell them to meet you at the clock's tower at the top of the hour.
3. Big Ben
Without a doubt, London's Big Ben is the most famous clock in the world. The tower that houses the four-faced clockworks (and "Ben," its biggest bell) stands at the north end of the Houses of Parliament. The clock began striking the hours in 1859.
4. The pocketwatch
It's amazing that hipsters everywhere haven't taken to wearing the pocketwatch. The old-school timepiece hearkens back to the Victorian Era and the days prior to Prohibition. Works best if you have a vest on and look like a train conductor and/or Teddy Roosevelt. The one shown belonged to President Abraham Lincoln.
5. Dick Tracy's two-way wrist radio
This is one of the most famous gadgets in pop-cuultural history. Dick Tracy began wearing his two-way wrist radio in 1946 and hasn't taken it off since. Given the appearance of Samsung's smartwatches, that company is at least a little indebted to the comic-book crime fighter.
6. The Timex
The Timex was the subject of the one of the classic ad campaigns of all time, with newsman John Cameron Swayze putting the watch through its paces, often involving motorboats. And, even now, if you hear "Takes a licking and keeps on ticking," no other product comes to mind.
7. Texas Instruments LED watch
If you were a certain type of kid in the 1970s, few things felt cooler than strapping one of TI's TXN, +0.12% LED watches on your wrist. Of course, you had to press a button for the time to show up, but at least you didn't have to deal with learning to read those pesky hour and minute hands, right?
8. The Casio
What if pressing that button on the TI watch was just too much activity for you? Well, Casio had you covered with its quartz digital watch, introduced in 1991. Today, you may know Casio best for its line of G-Shock watches, which are popular in sports and the military.
9. The Swatch
Admit it. In the '80s, you had one of these plastic-bodied timepieces. You probably even put one of the neon "Swatch Guards" on it. You may have even matched your clothes to your Swatch. And then your girlfriend laughed at you.
10. The Rolex
The grandaddy of them all. Fewer things in any product category say "I've arrived" like a Rolex. If you don't have $1.16 million to pick up a 1942 Rolex Chronograph, you can slum it and get a Rolex Oyster Perpetural Cosmograph Daytona ... for just $104,000.
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• 5 reasons you are poor
• How much to tip everyone
workJobs where women make much less than men
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Is there an item I can purchace that allows me to draw a small item, such as a potion, as a free action? I recently discovered consumable items but it sucks to use two actions to be able to use them.
I'll talk to my DM and if he doesn't go for using a single action to draw and use a potion, I'll go for the bandalier. Sadly the quick draw feat is unavailable. Is it a high level feat or is it a racial or class feat?


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research guide
editors & contributors
write the editor
The confrontations between police and demonstrators at the Stonewall Inn in New York City the weekend of June 27-29, 1969 mark the beginning of the modern glbtq movement for equal rights.
Formed soon after the Stonewall Riots of 1969, the short-lived but influential Gay Liberation Front brought a new militancy to the movement that became known as gay liberation.
The sexual revolution of post-World War II America changed sexual and gender roles profoundly.
"Leather" is a blanket term for a large array of sexual preferences, identities, relationship structures, and social organizations loosely tied together by the thread of what is conventionally understood as sadomasochistic sex.
Although best known for her crusade for women's suffrage, Susan B. Anthony spoke out on a range of feminist issues.
With reports from hundreds of sub-Saharan African locales of male-male sexual relations and from about fifty of female-female sexual relations, it is clear that same-sex sexual relations existed in traditional African societies, though varying in forms and in the degree of public acceptance
Androgyny, a psychological blending of gender traits, has long been embraced by strong women, soft men, members of queer communities, and others who do not easily fit into traditionally defined gender categories.
A cultural crossroads between Asia and Europe, Russia has a long, rich, and often violent heritage of varied influences and stark confrontations in regard to its patterns of same-sex love.
Congratulations to Good Morning America weatherman Sam Champion and longtime partner photographer Rubem Robierb, who have announced their engagement. According to a posting on the Good Morning America website on October 5, 2012, the couple plan to marry later this year.
In a blog posting on September 30, 2012 about the wedding of MSNBC news anchor Thomas Roberts and Patrick Abner, I noted that among the celebrities attending the event was Sam Champion. I had recognized Champion in photos of the wedding published on facebook.
Champion and Robierb may have decided to announce their wedding plans on the Good Morning America website today because of the publication this morning of a New York Times article about the Roberts-Abner wedding in which they are quoted.
The New York Times article by Jacob Bernstein is a beautifully written and moving account of the relationship between Roberts and Abner. In describing the wedding, however, Bernstein notes that "Among the 170 or so guests at the reception was Sam Champion, the weather anchor at ABC's 'Good Morning America.' He took a turn on the dance floor with his partner, the photographer Rubem Robierb."
Caught up in the spirit of the moment, Champion told the reporter, "We're getting married New Year's Eve in Miami." Robierb corrected him: "We'll do it here officially, and then have a party in Miami."
Although Champion has widely been perceived as gay and has been seen at various charities that benefit glbtq youth and causes, as far as I know he has not previously publicly declared his sexual orientation.
According to the Good Morning America website, Champion will discuss his announcement on the program on Monday, October 8, 2012.
The Kentucky native is a veteran newscaster in New York. He joined WABC-TV's Eyewitness News team in 1988. He joined the Good Morning America team in 2007. On April 7, 2012, he debuted as host of Sea Rescue, an educational and informational program that focuses on the rescue and rehabilitation of animals.
Champion and Robierb met in Miami, where the latter lives, four years ago. A native of Brazil, Robierb is a fine-arts photographer who exhibits his work in Miami, Atlanta, Santa Monica, and New York.
The humorous video below was made as a tribute when Champion left ABC's New York City affiliate for Good Morning America in 2007 after 18 years.
learn more about glbtq contact us advertise on glbtq.com
glbtq and its logo are trademarks of glbtq, Inc.
This site and its contents Copyright © 2002-2015, glbtq, Inc.


text:
What does DCOR stand for?
What does DCOR mean? This page is about the various possible meanings of the acronym, abbreviation, shorthand or slang term: DCOR.
We've found a total of 3 definitions for DCOR:
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Defense Committee On Research
Defense Committee On Research
Division Central Office of Record
Still can't find the acronym definition you were looking for? Use our Power Search technology to look for more unique definitions from across the web!
"DCOR." Abbreviations.com. STANDS4 LLC, 2015. Web. 2 Jul 2015. <http://www.abbreviations.com/DCOR>.


text:
Hold down Control (PC) or Command (Mac) key + mouseclick to select more than one option
Home >
Fairfax / Tysons Corner >
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Blackfinn Ameripub
Seasoned With Our Signature Blackfinn Seasoning & Seared In A High Temp Broiler. (GF).
Served On A Butter-Toasted Bun With Fries, But Feel Free To Sub With Any Other Side Item, Small Salad Or Cup Of Soup. Go "Naked" And Sub The Bun For A Crisp Lettuce Cup.
All Sandwiches And Wraps Are Served With Fries.
Pick 2.
For Sharing
They're Great For Sharing, Although You May Not Want To.
Choose One Item From All Four Sections.
Flowing With Refreshing Flavors From Our Taps To You.
So Fresh, We Practically Picked 'Em.
They're Classics For A Reason, Plus A Few Of Our Own Creations You'll Crave Too.
Listed From Crisp And Bright To Full, Round And Buttery.
Like A Little Black Dress Or A Pair Of Comfy Khakis: A Perfect Fit For Any Occasion.
There's Nothing Like A Rich And Satisfying Glass Of Fine Red Wine.


text:
Sponsored by Cerro Coso Community College Foundation
Dinner & Calcutta Auction
Extra Tickets Available
April 27
Sign-In: 9:30am
Shotgun Start: 10:30am
April 28
Coffee & Donuts: 7:30am
Shotgun Start: 8:30am
$800 per 4 Person Team
Includes Green Fees, Cart Rental, Saturday Night Dinner & Sunday Lunch
Team Makeup
All teams must have an A, B, C, and a D player on their team.
Any player who does not have an established handicap must provide a phone number of their home course for validation. Higher handicap players may be designated to fill a low handicap slot.
Team Play
The team must use three drives from each player each day. If a player's drive is selected they may not hit the next shot unless it is on the green.
All Calcutta proceeds will be paid out (number of pay outs will be determined by the number of teams entered). Pin prize for long drive and wind shirts for the 1st place team.
Donate directly to the Cerro Coso Community College Foundation


text:
Catalogue Number: 100-9765
- Compatible with Kärcher K1-K4 series pressure washers
- Designed with a rotating pencil jet for precise cleaning
- Ideal for moss covered or weathered surfaces
Sorry, there are currently no sellers for this product.
Product Details
The K1-K4 Kärcher dirtblaster lance combines a powerful high-pressure jet with a rotary nozzle, providing up to 50% more cleaning power over conventional high-pressure jets. For use with K1-4 series Kärcher pressure washers.
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Product specifications
Product specifications
Key Information
|Decking brush
|Product Height
|1.0 Centimetre
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|0.15 Kilogram


text:
From Our Editors
Literary-device lovers can spend hours browsing through Nostalgic Books and Comics, which offers the latest in comic books and graphic novels, as well as renowned classics written by the hallowed monsters of plot. Those who mysteriously sprout word bubbles will identify with Nostalgic's selection of new comics (prices vary; around $3 each), such as the 700th issue of Superman, or the newest selection from Jim Henson's Fraggle Rock series. Explore the annals of narrative artwork history with an old comic (average price $2), or finger through the store's action-packed paperback (around $8) and hardcover (around $20) book section, which runs the gamut of sci-fi/fantasy tales such as Neil Gaiman's Stardust, but also stocks mainstream bestsellers. The store also offers T-shirts (around $15) and posters (around $10), in addition to occasional evening screenings of films such as Independence Day and The Sandlot.


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Soft and more subtle than the conventional Synthetic Mink lashes, these are a great lash for those looking for a more natural look and lighter feel.
Posted by Unknown on 10th Apr 2015
Best lashes I have ever used
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I have used many types of synthetics. My Clients and I agree that after applied the result is a very flirty mascara look especially with the C curl. Just keep in mind when purchasing that this brand's C Curl may sometimes be more like a D Curl. Might be a good idea to have B Curl handy.
Posted by Brandi on 2nd Mar 2015
Easy to grab with tweezers, look amazing
Posted by jenny luu on 14th Feb 2015
quality look and feels AMAZING. super soft and black. will def order this when out!
Posted by Amanda P. on 18th Nov 2014
I ordered these on the. 07 diameter to start doing volume lashing. These are the softest lashes I've come across yet. They have an amazing natural feel and look. Love them! And the shipping was awesome, had them within 2 days of ordering!


text: OverviewBack to top Tender - AfterBite Outdoor Insect Bite Relief Combo Pack - 2 Tubes Item# :76313UPC# :044224010988 Brand:After Bite Size/Form:2 Tubes Ship Weight:0.15 Tender - AfterBite Outdoor Insect Bite Relief Combo Pack - 2 Tubes Be doubly prepared for bug season with the AfterBite® Outdoor Combo Pack. Keep AfterBite® at home, in your car, in your tackle box and camper. Don't let insect bites ruin your family fun.The combo pack comes with AfterBite® Outdoor, the extra strength gel that contains ammonia with baking soda and tea tree oil to soothe the skin and stop the itch and pain, and AfterBite® Kids, a gentle, non-stinging cream with baking soda, aloe vera and tea tree oil for instant, soothing itch relief.AfterBite OUTDOOR is a powerful itch treatment that provides immediate relief! Extra strength gel contains baking soda and tea tree oil to soothe the skin and stop the itch and pain.AfterBite KIDS is a gentle non-stinging cream containing baking soda, aloe vera and tea tree oil. Now Mom can treat even the most over-scratched bug bites without fear of stinging and whining.Ideal Uses: Protection from Black Flies Protection from Chiggers Protection from Deer Flies Protection from Fleas Protection from Horse Flies Protection from Mosquitoes Protection from Stable Flies Protection from Ticks


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|231 posts found
Novice Member
Joined: 4/10/09
Eve is the only game where I would have another game (mostly minecraft) running to past time between stuff. If im mining,hauling, ship spinning, flying to missions, I would be playing another game or doing something else to fill in the gaps. Im not the only person that does this either.
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Apprentice Member
Joined: 8/15/08
I have a secondary game/mini game running while playing any mmo to be honest. I'm sure I'm not alone in this. Eve isn't the only game with gaps. And if I take into account that 80% of that time I am either doing research, looking up information or planning what to next, or talking to ingame people (game-based activities), then that 'gap' becomes close to none.
Mashing buttons endlessly is not the only definition of playing a game, sadly too many people refuse to admit that. Part of the fun in eve is knowing how to optimize your time spent playing.
Hard Core Member
Joined: 6/10/13
on the doing other things whilst playing, very few games have 'eve-ability' to do this
currently im working fulltime on the production T2 chain, but also hammering "world of warplanes" now many games allow you to play 2 games lol
This post is all my opinion, but I welcome debate on anything i have put, however, personal slander / name calling belongs in game :) were of course your welcome to call me names im often found lounging about in EvE online.
Novice Member
Joined: 9/30/10
Originally posted by uplink4242
When playing EVE I am known to read a book while mining or doing other things that have that gap that you spoke of. I also know those who watch movies or TV while playing EVE. It is fairly common to multitask while playing EVE.
Currently bored with MMO's.
Novice Member
Joined: 5/12/10
What's sad to me is that EvE is boring and almost everyone here will agree. The majority of people who "play" the game do so semi-afk, completely afk, watching movies, reading books or playing other games. Anyone else see the problem with that? I don't semi-afk when I'm playing Dota2, Planetside2, nor will I be when playing EQNext. And what's worse is that EvE to so many represents the BEST sandbox PvP game we have right now, and for the last 10 years. How did that happen?
Let's be real: EvE's population isn't really growing. People claim it is but it isn't. It's stagnating. The majority of the accounts you see are multi-boxed, and about 30% of the rest are trials. 0.0 is dead in the majority of regions, and unless you blop and can fly ships that cost 100s of millions of ISK and require literally YEARS of skill training, there's not much action for you. The content is stale and boring from top to bottom.
The new players that actually quit are wise. For the rest of you that stick it out, realize that you will never "shape some glorious destiny" and it will be literally YEARS before you will be on par to do any of the high-end blop fleets. And that's if you train perfectly and have ISK available already. If not, well you get to farm and farm and farm and farm, because you won't be making any legitimate money on your own without a lot of work for a very long time. If you think you can step onto a playing field dominated by a time-based skill-queue and go toe-to-toe against toons that are 2-7+ years ahead of you in skills, ISK, and experience... well think again. Not that the actual combat in the game is fun anyway. Oh, and if you do decide to try and PvP you better have some form of a cash-cow because its an ISK-negative experience. PvP is not for money-making, any more than money-making is for fun.
I've tried to like the game, but everytime it's ultimately the same boring crap. Top to bottom I don't get the appeal, never have and despite my efforts never will, and ultimately this saddens me because on basically every front it's the best and only option we have out there.
EQnext don't fail me.
Elite Member
Joined: 4/04/11
Started playing last year more seriously, loving it (all pve so far). Horses for courses impsy.
rpg/mmorg history: Dun Darach>Bloodwych>Bards Tale 1-3>Eye of the beholder > Might and Magic 2,3,5 > FFVII> Baldur's Gate 1, 2 > Planescape Torment >Morrowind > WOW > oblivion > LOTR > Guild Wars (1900hrs elementalist) Vanguard. > GW2(1000 elementalist), Wildstar
Now playing GW2, AOW 3, ESO, LOTR, Elite D
Advanced Member
Joined: 4/29/12
Originally posted by Impsy
I have some bad news for you already. You are overly hyped, like the rest of this weird forum. This is never good. Me? I'm just stocking up popcorns and beer for when EQNext releases and is a major flop xD
Ontopic: EVE is .... weird ... I have 2 year old account and I get back in and out every X months for some reason. But the lack of tasks to do just kills me. Ye ye ye I know EVE is supposed to let you do your own stuff the way you want and all buuut :/ I don't really know how to do stuff and the game is not really helping me. Sure there are missions but they quickly become boring and repetitive. So I'm left with scanning to gain moneyz for that precious skill or next ship class. And what's worse ... there is a vast majority of 3rd party utilities that, yes, help A LOT, but me as a returning/new player have no idea of any of it. There are no decent EVE guides, I'm not stupid >.> I faceroll every and any game I play yet I somehow can't really comprehend EVE. I have 2 kills because some jerks decided to attack my vagabond few months ago :U That was fun but I don't feel ready for a massive fight. The combat doesn't require skill. It requires knowledge and understanding of the game. And we all know it has thousands of attributes that contribute to your survival ._.
I wouldn't say I hate EVE. When I am not playing it I do appreciate it, but when I start playing im like "dafuq am i supposed to do here" and I really try hard to find a reason to stay for more than 2 months but I just get tired and go back to my other games
*Jumps back to Battlefield 3 ^_^ *
Apprentice Member
Joined: 8/15/08
Originally posted by Impsy
And again, that's only your oppinion with little to no facts in there. No, not everyone agrees the game is boring otherwise it wouldn't be the most populated sandbox in the market. There are plenty of people who love the gameplay and still sit there daily for years. How often does this happen in other mmos? Not much. As for the pvp argument that is complete bull, there is action almost everywhere if you know how to look for the right corp. This game is supposed to be all about player interaction; if you don't look for it, don't expect it to magically become interesting for you because it clearly isn't designed for solo play. Also, PVP generates plenty of isk at times (which again, requires prior research...).
But have fun with your future 'eve-killer', or whatever games are hyped to be amazing. Your tears will be noted.
Novice Member
Joined: 8/04/10
I'm getting real tired of hearing the whole EVE is a simulation.
Yeah, a space sim game that has modules that say things like "Reduced weight of the ship...". Oh yeah, real space simmy there. :)
Novice Member
Joined: 8/15/04
Originally posted by Sk1ppeR
What jumps out at me is the lack of mentioning other players in any way, shape or form. This is notable because EVE really does rely on player interaction for pretty much everything. If you are looking to the devs for help, you're looking in the wrong place; you'll never find it. The devs are there to create the world as a whole, not to shape individual experiences. For that latter part, you need to turn to your fellow players. They are the ones who ultimately provide both help and direction in the game, even if you rarely or never directly interact with them.
Advanced Member
Joined: 10/13/05
I'm down to around 3 months a year now. You get to a point where you see all the glass barriers and nothing anyone tells you is going to make you not see them.
I'm pretty sure you have the same issue I have though OP. It isn't EVE it's all games. You found their limit. Either lower your expectations or find a new hobby.


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Writer-lyricist Javed Akhtar, who has been part of the Indian film industry for more than four decades, says so far only Bollywood has not suffered at the hands of Hollywood.
"Hollywood is such a powerful film industry that it has ruined film industries of the world. Wherever Hollywood films reach, the original film industry of that nation got ruined, except India. Our film industry is flourishing be it Tamil and Telugu films," the 67-year-old said here at the music launch of "Ye Khula Aasman".
The Indian film industry turns 100 next year and Akhtar is happy that it is spreading roots across the world.
"Our films are showcased in around one 30 nations abroad. The number of nations that view Hindi films is increasing and this is not an ordinary thing," said Akhtar.
"We have made some good and bad films. We have made some excellent films as well. I think Indian cinema is at such a point that the future can only be bright and in another 10 years it will reach great heights," said Akhtar, who has penned songs for films like "Mr. India", "Tezaab", "1942: A Love Story", "Dil Chahta Hai" and "Refugee".
Akhtar forayed into Bollywood as a scriptwriter with "Yakeen". Later he joined hands with Salim Khan and the duo together wrote blockbusters like "Zanjeer", "Sholay", "Yaadon Ki Baraat", "Deewar", "Kranti" and "Don".
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Actually its Hollywood which is in the dumps now. In the last 2-3 years very few blockbusters have come out of Hollywood! They dont make movies like Titanic anymore. Most of the recent releases are insignificant in their domestic collections here in the US. Only news from Hollywood is which star got married or divorced and who had how many kids.
ahahahahahaha Yeah Right! *Rolls eyes*, Wake up hun.
No more exists family friendly organic indian themed movies like back in the 90's. So dont feed us BS of Hollywood not ruining bollywood. In most ways, the story telling has bettered but the stories now days suck and are not indian. So blaaah on you B'wood.
Read on to know more... 7
Let's see what our Telly stars have to say?
Actor Arjun Kapoor says he wanted to become a director, but ideas ... 1
What made Salim Khan ask the makers to remove Bajrangi Bhaijaan song ...


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BY Brent Dirks on Fri December 12th, 2014
In our latest gift guide, we offer 10 perfect gifts that any Mac owner would love to receive this holiday season.
Continue reading →
BY Brent Dirks on Fri December 14th, 2012
We're wrapping up our Gift Guide series with seven accessories perfect for any Mac user.
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D. Orlando Ledbetter of the Atlanta Journal-Constitution writes that impending free agent Steven Jackson "appears" to be the Falcons' top target in free agency. There's been a lot of speculation in recent weeks that Jackson could make his way to Atlanta now that he's expected to void the final year of his contract and test the free agent market. The Falcons released Michael Turner a few days ago, but signing Jackson seems to make a lot of sense in terms of the fit. Jackson would not only be able to take on a similar role in the team's offense, but would almost certainly be an upgrade for them. Jackson has said that he would like to finish out the remainder of his career with the Rams, so there's at least a decent chance he could re-sign with them in the next few weeks. The post Falcons Prepared To Target Steven Jackson In Free Agency? appeared first on NFL Trade Rumors -.
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On Nearly Getting Struck By A Car
By 1337W422102 34 Comments
While I'm no stranger to being hit by a motor vehicle under controlled conditions, today was somewhat different. While crossing a downtown Montreal street, some dude pulled the corner without slowing down and almost hit me head on.
My first instinct was to block rather than dodge.
Maybe I have been doing too much gaming after all. It wouldn't be fair to blame this on videogames when really it was just me being an idiot.


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In the last years more than 20 vertebrate genomes have been sequenced, and the rate at which genomic DNA information becomes available is rapidly accelerating. Gene duplication and gene loss events inherently limit the accuracy of orthology detection based on sequence similarity alone. Fully automated methods for orthology annotation do exist but often fail to identify individual members in cases of large gene families, or to distinguish missing data from traceable gene losses. This situation can be improved in many cases by including conserved synteny information.
Here we present the SynBlast pipeline that is designed to construct and evaluate local synteny information. SynBlast uses the genomic region around a focal reference gene to retrieve candidates for homologous regions from a collection of target genomes and ranks them in accord with the available evidence for homology. The pipeline is intended as a tool to aid high quality manual annotation in particular in those cases where automatic procedures fail. We demonstrate how SynBlast is applied to retrieving orthologous and paralogous clusters using the vertebrate Hox and ParaHox clusters as examples.
The SynBlast package written in Perl is available under the GNU General Public License at http://www.bioinf.uni-leipzig.de/Software/SynBlast/ webcite.
Conserved synteny is the (local) maintenance of gene content and order in certain chromosomal regions of related species. Several studies on chromosome evolution [1-5] demonstrated that conserved synteny exists not only between closely-related species but also over very long evolutionary timescales. Long-range conserved synteny is a particularly frequent feature around developmentally important genes [5], demonstrating that rearrangements are not an unbiased random process in genome evolution.
Conserved synteny is, however, not only of interest as a phenomenon in genome evolution, but provides valuable practical information for the analysis of families of homologous genes. It is a long-standing problem in comparative genomics to identify orthologs, i.e. pairs of genes from two organisms that are separated from each other by a speciation event. In general, the task to distinguish true orthologs from paralogs cannot be solved based on pairwise comparisons. Gene loss, differences in evolutionary rates [6], and convergent evolution often distort the sequence similarities to an extent that makes it impossible to determine orthology from the gene tree. Genomic linkage with genes whose orthology relationships are clearer (i.e. which are more conserved across species and have fewer in-species paralogs) than others can be exploited because linked genes likely share their duplication history. Local/tandem duplications place new copies into a new genomic context, large-scale duplications coordinately duplicate the genomic context and gene loss becomes obvious if it leaves large parts of the genomic context intact while erasing the gene of interest. Therefore, conserved synteny information may demonstrate the loss of a particular copy of a gene and hence put a restriction on which extant gene copies are potential orthologs. If the genomic context of duplicated genes has sufficiently diverged prior to a speciation event, synteny can even provide direct evidence for or against orthology.
There are three basic approaches towards automated orthology identification.
1. Similarity-based clustering methods. This group includes the popular reciprocal pairwise best hit approach and refinements (such as Inparanoid[7-9]), as well as more sophisticated methods that initially represent homology as many-to-many relations. In [10], for instance, the "homology graph" graph of initial blast hits is refined by iteratively removing sub-optimal edges.
2. Phylogenomics-based methods (such as the tree-based Ensembl Compara[11] pipeline). These approaches first cluster homologous genes, then construct a gene phylogeny, attempt to reconcile it with a prescribed species tree and use the resulting mapping between gene-tree and species tree to assign orthology and paralogy relations. An alternative use of phylogenetic information is made by PhyOP, synonymous rate estimates to distinguish between orthologous and paralogous segments in closely related genomes [12].
3. Methods utilizing conserved synteny to infer true orthology between relatively recently diverged species. Methods range from whole genome alignments to combinations with similarity- and phylogenomics-based approaches. Examples are the commercial "syntenic-anchor" approach from Celera [13], the former Ensembl Compara pipeline (prior the June 2006 release). Algorithms that are primarily designed to determine syntenic regions and break points between them also fall into this category [14-18].
Despite substantial improvements in this area, the automatically generated results are still far from being perfect, and the annotation provided by databases such as Ensembl Compara[11] or OrthoDB[19] are neither sufficiently complete nor sufficiently accurate for many applications. For instance, an in-depth study of lineage-specific differences in a family of transcription factors requires not only the complete complement of family members in each species, but also a flawless gene phylogeny (which implies a correct orthology assignment).
The SynBlast tool is designed to assist the manual curation of such data and to focus on individual loci of interest. In contrast to most approaches to genome-wide orthology annotation, it does not operate on pre-determined gene (proteome) sets but it searches the nucleotide sequence of the entire target genome. Hence it does not exclude possible homologs only because they are missing from annotation tracks. Instead of attempting to automatically extract an assignment of orthologs and paralogs, SynBlast instead provides the user with detailed information on all plausible homologs and their genomic context. To this end, web-based graphical overviews that can easily be compared with one another are generated. While heuristic rules are employed by the software to propose a plausible rank-ordering of the homologs with the aim of determining the correct orthologs, its primary purpose is to present conflicting information to the researcher in such way that it facilitates the decision of a human curator.
Algorithms and Implementation
Overview of the SynBlast pipeline
SynBlast is a "semi-automatic" pipeline that is implemented as a suite of Perl scripts (SynBlast package). In order to allow automatic retrieval of proteins from syntenic regions and comparison of assignments with existing annotations the Ensembl system and databases [11] were chosen as standard reference sources. Therefore, the pipeline scripts make use of the Ensembl Perl API to retrieve reference annotation and sequences from the Ensembl Core databases as well as homology annotations (for comparison only) from the Ensembl Compara database.
The workflow is summarized in Figure 1. It starts from a focal protein coding gene of interest whose homologs are to be detected.
Figure 1. SynBlast pipeline steps. (1) A focal protein coding gene of interest and its surrounding genes (within a certain flanking size) are selected as reference set. Protein sequences and genomic positional information are either compiled manually or retrieved from Ensembl using the tool getEnsemblProteins.pl. (2) tblastn searches of all reference proteins are performed against selected target genome databases. (3) The resulting blast hit tables are scanned for regions of possibly conserved gene content. These regions are stored in separate blast hit tables. (4) The resulting sets of possibly syntenically conserved blast hits are evaluated based on their sum of blast bit-scores obtained by means of a gene loci order alignment to the reference set. The final decision on orthology or paralogy of ranked syntenic blocks is left to the user.
Step 1, in order to include synteny information, the adjacent protein coding genes within a certain genomic distance (flanking size, e.g. 1 Mb up- and downstream) are added to the reference set. The system requires both the sequences and positional information (orientation and (relative) start and end positions) of the reference genes. This information can be provided manually by the user in form of a text file containing tab-separated entries of gene identifiers and their genomic coordinates. Details are given in the SynBlast Tutorial, which is included in the Online Supplemental Material [20]. Alternatively, the corresponding files can be generated using the getEnsemblProteins.pl script which retrieves sequences and annotation information from Ensembl databases.
Step 2 consists of translated-blast searches using all reference proteins as queries on a selection of genome databases as targets. Resulting blast hits are expected to be in tabular (NCBI BLAST) format for further processing. Again this step can be performed manually using any program that creates blast-like tabular output, including NCBI BLAST[21], WU-BLAST (http://blast.wustl.edu webcite), or BLAT[22]. When the genome data is available locally in NCBI BLAST format, the script doBlastJobs.pl automatizes this step using local NCBI BLAST. It is needed to include the reference species as target genome as well in order to enable the subsequent normalization of blast scores and to detect possible paralogous clusters that the user should be aware of when interpreting final pipeline results. Those paralogous regions of the reference set should be used as reference in a subsequent pipeline run as well to avoid false positive orthology assignments.
In step 3, we search for potential regions of conserved synteny (syntenic target blocks). To this end we collect blast hits that are located within regions of limited size on the target genome. The purpose of this filtering step is to extract candidate subsets of blast hits (or HSPs, high scoring pairs) that can be treated separately in the following. At this stage we do not consider gene order, but gene content information, i.e. a user-specified number of query-specific hits must be contained at minimum in each candidate subset. The procedure is implemented in the script doSyntenyFilter.pl, and is described in detail in the following section.
In step 4, all detected candidate regions of conserved synteny from step 3 are evaluated w.r.t. their agreement with the reference gene order. The technical details of the doSyntenyAlignment.pl procedure are described below. The candidate syntenic regions are sorted according to a scoring scheme that combines sequence similarity, synteny information, and orthology versus paralogy information. The results are presented as HTML files in a web browser together with graphical representations of gene order alignment matrices and paths. Graphics such as those shown in Figure 2 allow the user to readily identify small-scale rearrangements such as translocations or inversions.
Figure 2. Dotplot graphic and detailed alignment graphic. The dotplot (top) visualizes the content of the alignment scoring matrix used to calculate the global alignment with free endgaps where the reference loci (p) and target loci (q) are arranged in rows and columns, respectively. Brightness of green color indicates the relative score value (rscore) of sequence similarity for a particular locus match. The dotted squares correspond to the optimal alignment path, which is shown in the alignment drawing (black box). Swapped orientation of a single target locus (w.r.t. a matching reference locus and the cluster orientation) is indicated by shaded boxes and opposite arrow orientation in the dotplot and alignment drawing, respectively. (Note that in this example the alignment path contains only aligned loci which share the same orientation.) The focal reference gene is highlighted by a light frame and orange color in the dotplot and alignment drawing, respectively.
Extraction of syntenic target blocks – doSyntenyFilter.pl
A region of one of the target genomes is considered as a candidate for a syntenic target block if it contains blast hits from at least N different proteins of the query set within an interval of length at most L. The parameters N and L are set by the user, both either directly or indirectly (relative to the reference set). They reflect the expected rate of gene loss and the expected structural similarity. The maximal regions of contiguous blast hits fulfilling these criteria are extracted separately for each target sequence. Depending on the status of the genome assembly, the sequence can be on a chromosome, a scaffold, or a contig. Small scaffolds or contigs pose a problem for this step as a target block syntenic to the query region may be mapped to several different scaffolds. In this case, SynBlast reports two or more separate syntenic regions and/or misses parts of the regions if less than N query proteins map to some of the scaffold regions. Note that for some genomes allelic variants are assembled into different scaffolds. SynBlast then reports all these scaffolds and it is left to the user to recognize this.
In its current implementation, the candidate subsets of blast hits are found by a sliding window approach. In addition to the number of query proteins ≥ N that have blast hits within a sequence window of size ≤ L also the sum of all maximal HSP bit-scores for these proteins is recorded. This yields a convenient measure to prefer the higher-scoring subsets when there are overlapping intervals (in particular if L and N are too small). We currently use a greedy approach that selects a specified number of target block intervals in decreasing order of the score sum and skips all intervals overlapping a previously selected interval by more than a specified threshold. For a detailed description of the various effects following changes to the parameter settings of L and N we refer to the SynBlast tutorial, which is provided as part of the Supplemental Material [20].
Evaluation of syntenic target blocks via gene order alignment – doSyntenyAlignment.pl
In the fourth step of the pipeline, each of the syntenic target blocks (subsets of blast hits), resulting from step 3, is analyzed separately in comparison to the query region. This part of the pipeline consists of several sub-components, which we discuss separately, see also Figure 3.
Figure 3. Evaluation of syntenic blocks. Panel (A) summarizes the mappings from query to target, panel (B) elaborates on particular cases. The query region (panel (A), middle) contains a sequence of syntenic query loci (green), each representing one or more possibly overlapping query proteins
- (i)
- (ii)
- (iii)
- (iv)
(i) The set of reference proteins is linearly ordered (by start/end or mean positions) into so-called "query loci". If the query region contains overlapping proteins, these are combined into a single query locus. Thus, a query locus may comprise more than one query protein (an example is the second query locus in Figure 3A).
(ii) For each query protein we chain all corresponding HSPs into models of target loci. Each HSP consists of an interval α = [a', a"] on the query sequence and a corresponding interval β = [b', b"] on the target sequence. These intervals are linearly ordered for both query and target based on their coordinates.
Intervals on the two strands of a target are treated separately. We furthermore take into account that HSPs must not be too far away from each other, i.e. the maximal genomic extension is restricted to either an absolute or query gene dependent length (locus size limit; see also Figure 3-B1). Again, groups of intervals that are too far apart from each other are treated separately.
For each group of HSPs with common orientation we compute an optimal "alignment" of these lists of intervals using a variant of the Needleman-Wunsch algorithm, similar to Ref. [23]. The query and target intervals of the HSPs, respectively, are considered as characters within the alignment in which a match occurs if both intervals belong to the same HSP. The score of the match is its bit-score. Pairs of (query/target) intervals that do not correspond to the same HSP are considered as mismatches with score -∞. Small negative scores are given to insertions and deletions, endgaps are treated as free. The resulting alignment then defines a consistent chaining of collinear HSPs, here called an "HSP chain" for short. It represents an overall hit for the respective query protein (approximate gene model) with a group score equal to the sum of bit-scores of all HSPs of the chain. A score threshold can be specified to eliminate spurious hits, which otherwise might lead to incorrect groupings of adjacent loci in the next sub step, see also Figure 3-B5. Each HSP chain is furthermore characterized by a start, end, and mean position.
(iii) As in sub-step (i), we now define a linear order of HSP chains by grouping them according to start/end or mean positions into so-called target loci, see Figure 3. If there are HSP chains that reside within the same target locus, (i.e. have overlapping intervals with some HSP chain) while representing hits for the same query protein, only the top-scoring chain is kept, see Figure 3-B6. Thus, we finally end up with linearly ordered blocks of target loci each containing one or more (overlapping) query-specific HSP chains.
(iv) As in sub-step (ii), we use a variant of the Needleman-Wunsch algorithm to obtain a maximum weight sequence of collinear pairs of query and target loci. As before, mismatches are prohibited. Matches are scored as the arithmetic mean of the scores of all matching individual query proteins that belong to the same query locus. Formally, for each pair (q, t) of query and target locus let νqt be the number of matching query proteins between the pair of loci. The corresponding similarity score is
where b(s, ts) is the bit-score of query protein s with its match ts on the target genome as determined in sub-step (ii). In contrast to step (ii), we do not exclude matches between items of different orientation. Instead, we use only a fraction of the score b(s, ts) (adjustable parameter) to penalize those matches. Thus, swapped orientation of a target locus w.r.t. the orientation of its matching query locus within the reference set is generally allowed, but the match score of such a locus is reduced to a user-defined fraction (e.g. 90%). This parameter can also be set to 0, in which case matches with reversed direction are considered as not informative at all. The gene order alignment score is consequently calculated for both orientations of the target block relative to the reference set, and only the higher-scoring alignment is retained in subsequent steps.
In addition to this absolute scoring we also compute relative weights b(s, ts)/b(s, qs) where the absolute bit-score is scaled by the score obtained by matching the query protein s back to its genomic locus qs within the reference genome. The value b(s, qs) is a good approximation for the maximal tblastn score of a given query protein. The relative score is then used as match score during the gene order alignment. This ensures that the matches to each reference locus are scored relative to the information content of the locus.
Since match scores are defined directly between loci we can conveniently combine the visualization of the alignment path and the scoring matrix, see Figure 2.
(v) Finally, all evaluated target regions are compiled in a ranked list in browsable HTML format including graphical overviews of loci scoring matrices (dotplot) and alignments as well as an alignment table displaying additional information for assigned loci. Swapped orientation of a single target locus is indicated in the dotplot by a shaded dot. In the alignment graphics, an arrow with reversed orientation w.r.t. the arrow of its assigned query locus is used.
The ranking can be based on the gene order alignment score (roughly the sum of (weighted) bit-scores for assigned target loci) or the (log)RatioSum score, which is calculated as the sum of (the logarithms of) intra-inter-score ratios of assigned target loci. This score ratio, which is described in detail in the "Methods" section, measures the ambiguity of orthology between two loci based on the existence of close paralogs within the reference. It has proven useful in the process of identifying true orthologs. In combination with the gene order alignment score this score makes it easier to distinguish between putative orthologous and paralogous hits or clusters.
If reference and genome data are taken from Ensembl databases, SynBlast optionally retrieves the EnsemblCompara homology annotations and the Ensembl Core protein coding gene annotations overlapping the target locus interval of the matching HSP chain identified by SynBlast, for comparison.
As a real-life test of SynBlast, we consider here the genomic clusters of vertebrate Hox and ParaHox genes. These genes code for homeodomain transcription factors that regulate the anterior/posterior patterning in most bilaterian animals [24,25]. Hox and ParaHox genes arose early in metazoan history from a single ancestral "UrHox gene" [26]. After a few tandem duplication events, a large scale duplication lead to ancestral Hox and ParaHox clusters. While the ancestral ParaHox cluster remained largely unchanged, the evolution of its Hox counterpart was dominated by a series of tandem duplications. As a consequence, most bilaterians share at least eight distinct paralogous groups (8 in arthropods, and 13 or 14 in chordates) which retained high sequence similarity at the homeobox. Both Hox and ParaHox genes are usually organized in tightly linked clusters [27], with syntenic conservation extending even beyond the core clusters themselves. For instance, an additional homeobox gene, Evx, located at the 5' end of the Hox cluster can be seen as part of an extended Hox cluster, see [28] for more details.
The modern vertebrate genome arose from an ancestral chordate by means of two rounds of whole genome duplication [29,30]. Teleost fishes have undergone an additional round of genome duplication [31,32].
Substantial loss of duplicated genes was the consequence of these duplication events. In the case of Hox clusters there is little doubt about the orthology relationships among the Hox genes of tetrapoda. In teleost fishes, however, the relationships of the duplicated Hox clusters between zebrafish and fugu have long been controversial, see [33] for a discussion, and have only recently been resolved using a dense taxon sampling [30]. It is well known that the relative order and orientation of Hox genes in their clusters have been highly conserved in vertebrate evolution, albeit there is substantial gene loss. The Hox clusters thus are an excellent test case to demonstrate the gene order alignment functionality of SynBlast.
Vertebrate Hox clusters
We used the four human Hox clusters as reference and searched the vertebrate target species with SynBlast. We consider here a diverse set of vertebrate genomes which contains both tetrapods (with 4 paralogous Hox clusters) and teleosts (with 8 paralogons). The cluster locations, gene inventories, and SynBlast scores are listed in Figure 4. In case of genomes with complete assemblies, the correct assignment of cluster orthology and the correct assignment of Hox gene identity is straightforward by visual inspection of the SynBlast cluster alignments, see Table 1 for an example. Here, both the gene order alignment score and the logRatioSum score is suitable to assign cluster identity to the target loci in the zebrafish genome. However, the logRatioSum score clearly out-performs the gene order alignment score in case of the Danio Bb cluster. In combination, the two scores provide the best means to rank orthologous loci at the top. The zebrafish Zv7 assembly contains two inparalog copies DrCa1 and DrCa2 of the zebrafish HoxCa cluster. This is, however, certainly an assembly artifact and contradicts all of the existing literature, see e.g. [34] and the references therein. SynBlast correctly retrieves both copies with comparable scores.
Table 1. SynBlast results for Danio rerio, Ensembl release 46 (Zv7), with the human Hox clusters as query.
Figure 4. Overview on pipeline results for vertebrate Hox clusters. SynBlast results and manually extracted orthologous cluster positions and identities for selected vertebrate species are listed. Unless otherwise indicated, positions correspond to assigned blast hits' intervals from Hox1 to Hox13/Evx hits in gene order alignment. Cluster orientation is w.r.t. the human reference clusters, which are HOXA9_ENSG00000078399_5e5; HOXB9_ENSG00000170689_2e5; HOXC9_ENSG00000180806_3e5; HOXD9_ENSG00000128709_5e5. Unassigned loci from the reference may be due to overlaps of chained HSPs. A '*' indicates loci that are absent in agreement with the literature [45]. Data for Ensembl release 42 (Dec 2006).
In the case of incomplete assemblies, only partial clusters can be obtained. For instance, individual Hox loci of oppossum and chicken are located on small separate scaffolds. For the duplicated genomes of the five teleosts in our data set, we obtained all 7 Hox genes-containing paralogous clusters in agreement with the literature, see [33,35-38]. Since our query consisted of the Hox cluster only, we could of course not retrieve the 8th zebrafish paralogon, which is completely devoid of homeobox genes [39].
Several artifacts of preliminary genome assemblies further complicate the analysis: In the fugu HoxAa cluster we readily detected the artifactual breakage of the cluster into two fragments on scaf.12 and scaf.346. In the older Zv6 assembly of the zebrafish genome, some Hox clusters contained local rearrangements and obviously duplicated gene loci, in particular the HoxBb and HoxCb clusters. Most of these problems have been resolved in the most recent Zv7 assembly, while the Ca artifact has been newly introduced. Table 2 summarizes the discrepancies of the Ensembl Compara annotation for the orthology assignments obtained using SynBlast, the latter conforming to the recent literature [33].
Table 2. Incomplete and erroneous Ensembl Compara orthology annotations for vertebrate Hox cluster loci.
The very well-understood example of the Hox gene cluster demonstrates that the true orthology and paralogy relationships can be determined rather quickly and easily by means of a manual analysis with the assistance of SynBlast. Automatic orthology annotation pipelines, on the other hand, still produce unsatisfactory results despite recent progress.
Teleost ParaHox clusters
The ParaHox clusters of teleost fishes have long been used to contradict the whole genome duplication scenario because of a mainly unduplicated repertoire of ParaHox genes compared to other vertebrates. Even after the teleost-specific genome duplication had been broadly accepted, the small number of ParaHox genes in each cluster and the large amount of gene loss at this locus complicated attempts to decipher their duplication history. Knowledge about the number of paralogous Cdx genes and their assignment to paralogous groups is a good starting point for such a reconstruction. Two studies based on publicly available genome sequences arrived at different scenarios for the history of this particular ParaHox gene in teleost fishes [40,41]. While Prohaska et al. [40] proposed the existence of a Cdx2 gene copy (at least for fugu and tetraodon), Mulley et al. [41] concluded that both copies of Cdx2 were lost and suggested that this loss was compensated by two copies of Cdx1. A more recent analysis that uses additional sequence data [42] settles the discrepancy in favor of [41], supporting the retention of two Cdx1 genes in cichlids. The analysis of [42] in part excludes zebrafish because of problems with the available genome assemblies. Here we demonstrate how SynBlast can be used to facilitate retrieval of candidate Cdx loci and cluster assignments in the zebrafish genome.
In an intact ParaHox cluster, the Cdx gene is flanked by two ParaHox genes, i.e. Gsh and Pdx, and a number of genes of other gene families. According to [42], the ancestral gnathostome ParaHox genes are organized in four clusters, designated A, B, C, and D in analogy to the Hox clusters (see Figure 5). The Cdx gene of the C cluster has been lost soon after the 1R/2R duplications [41]. No organism with a fourth Cdx paralog resulting from this duplication event has yet been found. Therefore, only three of the four ancestral clusters each retained a Cdx gene: Cdx2 (cluster A), Cdx4 (cluster B), and Cdx1 (cluster D). As a consequence of the teleost genome duplication we would expect to find 8 ParaHox clusters, two A clusters (A1, A2), two B clusters (B1, B2) etc. and up to 6 Cdx genes in the 6 clusters A1/2, B1/2, and D1/2. We start our SynBlast search in the zebrafish genome with the four human ParaHox cluster regions.
Figure 5. Schematic representation of genes flanking the Cdx gene locus in the human ParaHox clusters. Only linked genes relevant for the interpretation of the SynBlast output are shown.
Table 3 shows the scores for all pairs of the four human query loci with the 11 high-scoring target loci of zebrafish. The assignment of true orthologs is more challenging than in the case of Hox clusters but still revealing.
Table 3. SynBlast results for Danio rerio, Ensembl release 46 (Aug 2007), Zv7 assembly with the human ParaHox clusters as query.
One copy of ParaHoxA retained 13 of 24 genes flanking the Cdx2 locus even though Cdx2 itself was obviously lost. This is a case where gene loss can reliably be distinguished from missing data based on well-conserved synteny information (see Figure 6). The second copy retained only 5 of the 24 flanking genes. In line with the analysis of [41,42], we observe that the Cdx2 gene has been lost from both copies. We also observe that one of the two ParaHoxA contains the only copy of Gsh1 which is located at DrA2 (Chr.5), while the only copy of Pdx1 is located at DrA1 (Chr.24). Note that this information independently confirms the assignment of the two zebrafish ParaHoxA paralogs to the ancestral A cluster. SynBlast reports additional syntenic regions in the zebrafish genome that contain homologs of some of the genes of the HsA query. These are located on chromosomes 7, 14, 20, and 21, and can be assumed to be orthologs of the ParaHox B, C, and D clusters. In order to confirm this assumption, we also consider the remaining three human ParaHox regions as queries.
Figure 6. ParaHox example application. SynBlast was used to determine the four pairs of paralogous regions generated by the fish-specific genome duplication from the four gnathostome ParaHox regions. We show alignment dot-plots for the high-ranking hits (according to the gene order alignment score and logRatioSum score (in brackets)) of the four query regions against the zebrafish genome (Zv7, Ensembl release 46, Aug 2007). Parameters for the synteny filtering step were N = 1, L = 2. See text for more details.
The query with human ParaHoxB yields only poorly conserved synteny information. This can be due to the reorganization of this locus when it got translocated to the mammalian sex chromosome X, see [40,43] for details. Nevertheless, we obtain sufficient information from the linkage of the Cdx loci with chic1 to see that zebrafish has one Cdx4 locus, DrB1 (Chr.14). With the human ParaHoxC cluster, which contains the Gsh-2 gene as a query, two paralogous regions in the zebrafish genome can be identified. DrC1 on Chr.20 containing the only surviving copy of Gsh2, while a putative DrC2 locus on Chr.1 contains three high-scoring reference loci (fip1l1, chic2, and clock) and both neighbors of Gsh2, i.e. chic2 and pdgfrA, but is devoid of homeobox genes.
Note that "empty" parahox clusters are not exceptional. Teleost fishes have also lost all homeobox genes in one of the HoxD paralogs (zebrafish, [39]) or one of the HoxC paralogs (pufferfish, [40]), respectively. Loss of a gene of interest can nevertheless be identified due to the retention of neighboring genes given sufficient conserved synteny.
The assignment of orthologs to cluster HsParaHoxD is difficult. Conserved synteny information is relatively rare and only locally given, i.e. the orthologous hits for those query regions are scattered more or less across the target chromosome or even genome, which is probably due to extensive rearrangements. Nevertheless, one Cdx locus is linked to pdgfrB, a D cluster gene. SynBlast detects multiple fragments that map to two distinct zebrafish chromosomes. A plausible hypothesis is to interpret the three hits of Chr.14 at 22 M, 25 M, and 53 M as remnants of one dissolving cluster DrD1, while the two fragments of Chr.21 at 36 M and 43 M constitute the other DrD2 paralog.
In summary, we have located the three retained Cdx genes in the highly fragmented zebrafish genome assembly, and we conclude that three Cdx genes were lost in the aftermath of the fish-specific genome duplication. Due to synteny information, the three Cdx genes can unambiguously be assigned to the paralog groups Cdx4 (one copy, B cluster) and Cdx1 (two copies, D clusters).
Further Examples
In addition to the difficult Hox and Parahox loci we have investigated several examples of human loci with extensive synteny in other vertebrates, some of which are included in the Online Supplemental Material for comparison. In these rather straightforward cases we encountered a rather common annotation problem. Homology-based protein annotation sometimes produces two (or even more) disconnected annotated fragments, in particular when evidence from different sources is used. Since these fragments are not recognized as parts of the same protein, they are subsequently interpreted as distinct homologs of the same protein, resulting in an erroneous "within-species-paralog" assignment. We observed that SynBlast correctly recognizes such disconnected fragments as belonging to the same query item in the HSP chaining step, and hence avoids these spurious "paralogs".
Discussion and Conclusion
The SynBlast tool was developed to assist in the interactive preparation of high-quality orthology annotations. It uses synteny in addition to sequence similarity. A major difference to most other tools is that it does not operate on a "proteome set". Instead, it uses tblastn and a two-level alignment procedure to retrieve the homologs of a set of reference proteins. As a consequence, it is independent of gene predictions and annotations of the target genomes. Known or predicted protein sequences are required only for the query genome. This avoids in particular many of the problems with misannotations in the target genome that may confuse automatic pipelines.
A major advantage of the synteny-based approach is that we also find fairly diverged homologs in a conserved context that would otherwise be discarded due to insufficient sequence similarity, see also [44]. This allows the user to find supporting information for highly diverged genes or gene loss and to distinguish it from the failure to detect sequence similarity. As a consequence, we find that SynBlast is particularly useful to retrieve homologous regions in the presence of high rates of gene loss, such as after the teleost-specific genome duplication. Syntenic regions are found and gene losses can be identified even when the focal genes are lost from one or more paralogons. As demonstrated in the ParaHox cluster example, information on such loci is readily accessible using SynBlast and can be instrumental in deciphering complex duplication/loss scenarios. This is the case in particular when homologous genes that arose through several distinct duplication events are of interest, as in the case of homeobox clusters. Of course, in cases where synteny is not preserved, SynBlast cannot do better than a simple blast search. In such a case, the output of the program at least makes it easy for the user to identify cases of disintegrated synteny. To distinguish orthologous and paralogous regions, SynBlast provides two scoring schemes: one that attempts to evaluate the overall similarity of two syntenic regions (gene order alignment score), and alternatively the relative similarity in comparison to the most similar within-reference paralog ((log)RatioSum score). However, the SynBlast system was designed to aid a careful manual evaluation rather than to provide an automatic pipeline. Hence, it produces extensive graphical and tabular output of all regions in the target genomes that are potentially syntenic to the query region in the form of HTML pages, which also integrates the existing Ensembl Compara homology annotation for comparison. This renders the tool most useful when orthology annotation is not obvious and expert knowledge is required to reach a definitive conclusion.
The vertebrate genomes were taken from Ensembl (release 42, Dec 2006). In case of the ParaHox application and also the Danio Hox example the new assembly version for zebrafish (Zv7, Apr 2007 from Ensembl release 46, Aug 2007) was used. The new Danio assembly was scanned with local WU-BLAST (tblastn, version 2.0 MP-WashU, 04-May-2006). All other blast searches were performed with local tblastn (blastall version 2.2.15 of the NCBI BLAST suite). Genome databases were used in repeat-masked form, and the minimum E-value was set to E = 10-5 or E = 10-4. The maximal size of the target cluster was restricted to twice the size of the reference cluster for all applications (parameter L). The number of different proteins to be contained in a valid synteny region at minimum (parameter N) was set to 1 (Parahox application) or 4 (Hox application). The cutoff for the HSP chain score was set to 100. The cutoff for the maximal overlap (w.r.t. query coordinates) of neighboring consistent HSPs was set to 40 amino acid positions. The fraction of the score used for matches of loci with different orientation was set to 90 percent while the gap penalties were set to 10 (gap in reference sequence) and 2 (gap in target sequence). All scripts were written in Perl (v5.8.8) and executed on PC hardware running Linux.
The intra-score is calculated once for each query protein s, and describes the relative difference of the best and the second-best hit onto the reference genome (i.e. for the closest-related paralogs). This is approximated by their bit-score differences, i.e.
where qs1 and qs2 are the two top-scoring target loci (i.e., HSP chains) within the reference genome. The more distant the closest paralogs in the reference, the more reliable is the assignment of orthologs from the target species.
The inter-score is calculated for each assigned target locus ts within a target genome, defined as its relative bit-score difference to the best reference hit locus qs1:
Hence, the inter-score expresses how "bad" a putative ortholog hit to the target genome is w.r.t. the maximally expected score b(s, qs1).
The ratio of intra-score and inter-score, Sintra/Sinter, quantifies the quality of an inter-species (potentially orthologous) hit in relation to the similarity between paralogs in the reference genome. Therefore, it serves as a measure for the confidence in the orthology of the query and target locus.
The (log)RatioSum score is defined as the sum of the (logarithms of the) intra-inter-score ratios of all target loci assigned within the gene order alignment.
Availability and requirements
The SynBlast package written in Perl is available under the GNU General Public License at http://www.bioinf.uni-leipzig.de/Software/SynBlast/ webcite. It requires a Unix-like environment and several add-on perl modules (DBI, GD) installed, as well as an installation of the Ensembl Core and Ensembl Compara APIs of the appropriate release version, see also the SynBlast tutorial [20] for installation issues. A local version of the NCBI BLAST suite, as well as the genome sequence databases of selected target species is needed to generate the genome-wide similarity search results as part of the pipeline.
Project name: SynBlast
Project home page: http://www.bioinf.uni-leipzig.de/Software/SynBlast/ webcite
Operating System: Unix/GNU Linux
Programming languages: Perl, bash
Other requirements: several add-on perl modules (DBI, GD), Ensembl Core/Compara API, NCBI BLAST (or similar).
License: GNU GPL version 2 or any later version
Authors' contributions
SJP and PFS designed the study, JL developed the SynBlast pipeline and tool, all authors closely collaborated in preparing the manuscript.
We thank Jutta J. Roth, Sven Findeiß, and Stephan Bernhart for valuable comments and discussions. This work was supported in part by the Deutsche Forschungsgemeinschaft (DFG), Proj. No. STA850/6-1 and by the European Community FP-6 project "EMBIO" http://www-embio.ch.cam.ac.uk/.
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- Price: $139.00
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Footwear Shoes Zen Cart Template 28389
Zen Cart 1.3.8 Template that was composed by our webmaster artist Di.
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Description, Sources, Software & Requirements
Foot wear store template with a black and white color scheme. There are two columns with various styles of shoes on display such as heels, platforms, and more. Three is a tall banner in the header with various styles of high heel shoes. The logo is black and says Footwear, which can easily be customized. Footwear Shoes Zen Cart Template 28389 has a width of 1200 pixels and is available in the following sources: .PSD, and .PHP. Software required includes Winzip 9+ (Windows) or Stuffit Expander 10+ (Mac) for uncompressing .zip files, Adobe Photoshop CS+, Adobe Dreamweaver 8+ (or any php-editor), PHP 4.3.2 or higher (PHP 4.4.x for optimal performance), Apache 1.3.x and MySQL 3.2.x or higher, and Zen Cart 1.3.8 only. It has had a total of total of 4 downloads since it first appeared.


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Fashion Fails: Rita Ora, Tilda Swinton, Stacy Kiebler, More
Casey Johnson
June 1st, 2013 4:07pm EDT
June 1st, 2013 4:07pm EDT
Supermodel Laetitia Casta tried on this fashion experiment and it blew up in her face. She seems adamant on holding the feathers in place with her hand and we don't like the odd pink trailer at all. Just all over the place.


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Fish and Crab Melange
|8 Ounce
|Lemon juice
|2 Tablespoon
|1⁄2 Ounce
|Red pepper
|1 Small
|3 Tablespoon
|Crab meat
|4 Ounce (meat from 1 small fresh crab or frozen or canned)
|Asparagus tips
|2 (for garnishing)
1. In a bowl take cod. Season it with salt, pepper and lemon juice.
2. Arrange it on a piece of greaseproof paper, dot it with butter.
3. Fold the edges of greaseproof paper so that it can be shaped like an envelope.
4. Cook it in a steamer, which is held over a saucepan of boiling water.
5. Let it cook for 10-12 minutes or until the flesh can be easily separated from the bone.
6. Transfer the contents from the envelope into a basin. Remove skin and centre bone. Keep it aside to cool.
7. Flake the fish with its own cooking liquor.
8. In a chopping board, cut the pepper into half. Remove seeds. Core and dice the flesh and mix it with flaked cod.
9. On a shallow dish, prepare a bed of lettuce. Decorate the edges with a border of flaked cod and pepper.
10. Mix a rounded dessertspoon of mayonnaise into the crab meat. Arrange it in a smaller circle inside the cod.
11. To serve Fish And Crab Melange, garnish with asparagus tips.


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Bridget grew up next door to none other than Harry Styles. She always encouraged His singing career and him her art. But after spending 2 years without each others company, Never missing one night time phone call, they start to realize it may be more than a friendship, Harry Attempts to set Bridget up with Louis, though the two have never met in person. There is no doubt it is Love at first sight.
ItIsWhatItIsLarry 2 years ago
I found this story because I was writing "Drunk One Direction" on google and you're cover is i there and I clicked it and... here I am! Really like it :) PS- I was searhing drunk one direction on google because niall was drunk last night and it was really funny :D
@CesisLoveNH ME TOO!!! everyone i was with jumped at that part.... and i wasn't paying attention to the screen because my cousin was making a lot of noice with her candy rapper and i was getting ready to tell her to stop and out of nowhere they mutts came!
hahaha!! :D I love Effie too.... Peeta, Katniss, Gale, and Prim!! :D lol I cried lots after Rue died!!
Effie??? are you reading The Hunger Games??? :) lol... that was a FANTASTIC movie!!!!!!!!!


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on ArtFire
on ArtFire
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Welcome to ayannasuniquetreasures
Welcome to Ayanna's unique treasures ! I am having a sale ! Every item in my shop is 50 % off ! Now is the time to pick out that perfect gift for that loved one ! I'm clearing out my inventory to make room for new items .Come check out what i have before they're all gone ! I want to thank you for your business and for looking at my treasures ! * I now accept paypal express as my payment method! Quicker check out time and you don't need a paypal account to use it ! More »


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Dont have one, but very interested on wat you think and how it shoots? Id love to have one for my ccw, but Im scared its to heavy. Let me know. Tnx, HG
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31 year old woman | North Hollywood, CA, USA | Seeking men 27-35 within 50 miles
Easygoing and optimistic
I havn't updated my profile in a while and in that time period, a lot has changed so I'm going to take this moment to do so :) Here's a little bit "about me"...I love to find new, beautiful, interesting, places to go to locally...I never mind driving distances and actually find it very attractive when a man doesnt make a big deal of it as well...I love being introduced or finding random hole-in-the-walls, those always tend to be the best..I read up on yelp and various blogs on new interesting things to do, see, taste ..I use to run an excursion program where I would take 50 Russian g'mas to various destinations, I loved it!..One of my favorite places is the Terranea Resort and the Lighthouse in Palos Verdes..breathtaking...I really would love to take a road trip cross country and experience small towns..that and camping...never been...I'm very laid back and dont do well in clubs...I like lounges, hookah bars, house get-togethers, movie nights..and anywhere with good company and great conversation...I recently switched jobs and am working in downtown for a major retailer..experiencing new uncharted (well at least for me) territory down over on this side of town...have an MA in Business Management and will be my own boss one day :) In a man, I love a nice smile, good sense of humor, a romantic side (but not too clingy), and most importantly good communication skills..I'm very upfront and honest, dont play games, and expect the same from the other person. I can be a little sarcastic at times so a match for me would have to be quick and witty (but not obnoxious). At this point in my life, I'm a relationship-one man- type of gal...I'm known for telling great stories and buying interesting Groupons :) ... If you feel like we might have some things in common, feel free to say hi.
Alumni connections , Coffee and conversation , Dining out , Hobbies and crafts , Movies/Videos , Museums and art , Music and concerts , Nightclubs/Dancing , Performing arts , Shopping/Antiques , Travel/Sightseeing , Volunteering , Wine tasting
Billiards / Pool, Bowling, Dancing, Inline skating, Skiing, Swimming, Tennis / Racquet sports, Walking / Hiking
I exercise 3-4 times per week.
I have Other
Middle of the Road
On the weekends i usually like to go to movies, nice restaurants, hookah bars, lounges, house parties, anywhere really as long is it's with good company
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What she is looking for
|5'4" (162cms)
|5'9" (175cms) to 6'6" (198cms)
|Body type:
|About average, Athletic and toned, A few extra pounds
|Blue, Grey, Green, Hazel
|Dark brown
|Black, Light brown, Dark brown
|No Way
|No Way, Occasionally
|I'll tell you later
|No preference
|Other profession
|No preference
|I'll tell you later
|No preference
|Never Married
|Never Married
|Have kids:
|Wants kids:
|No preference
|White / Caucasian
|Middle Eastern, White / Caucasian, Other
I was born in Ukraine and speak Russian fluently.
|Agnostic, Atheist, Christian / Catholic, Christian / Protestant, Jewish, Spiritual but not religious, Other, Christian / Other
|English, Russian
|Arabic, Dutch, English, French, German, Hebrew, Italian, Norwegian, Portuguese, Russian, Spanish, Swedish, Other
|Graduate degree
|Some college, Bachelors degree, Graduate degree, PhD / Post Doctoral


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Vintage Clothing Selling Groups on Facebook
Below are links to Facebook groups where sellers post items for sale. Most groups focus on certain eras or styles. Each group has their own rules for showing interest in buying an item and for posting items for sale - some allow outside links to items for sale on other venues and some groups do not allow links. Be sure to read each group's rules before you post.
You have to request membership to join most of the groups. There are many others groups out, these are just a selection of some that I'm familiar with.
*** Antique Textile & Clothing Exhange 1800s-1930s
*** 1920s 30s and 40s market place
*** The 30s and 40s clothing exchange
*** Retro Flamingo Vintage Clothing/Accesories/Kitch Buy & Sell - 1960s & Earlier
*** Retro Flamingo 70's 80's & Reproductions - 1970s and later
*** Your Lingerie Delight! vintage and modern lingerie for sale
*** Novelty And Border Print B/S/T
*** Positively Haight Street - buy sell and trade
*** Golden Age Men's Vintage Buy & Sell
*** Vintage Weddings
*** Just love it Vintage Boutique
*** THE HEFTY HIDEAWAY plus size vintage sell and swap
*** Vintage Patterns Bazaar
*** Ooh La La! Vintage Swap and Sell Shop - Mid-Century 1940-50s, rockabilly, pin-up vintage.
*** Ooh La La Frocking 20s 30s 40s 60s 70s 80s Vintage Swap & Sell
A new Swirl Dress for me, in a slightly different style
We all love a classic vintage Swirl dress, and this week I added another to my wardrobe. It's a very cool black print on dark vivid turquoise with small white flower buds.
This one is a little different style from my others. It still has the single Swirl button on the back of the neck and it wraps on the back as usual. But this one ties on each side to a set of ties that are attached on the front instead of wrapping and tying to itself.
This one also has a v-neck with a collar trimmed in solid piping and piping right under the bust, which also makes it different from my other Swirls.
Love it! Washed and ready to wear this Sunday, I already have a pair of stockings that are nearly the same turquoise as the dress.
I love that it's a dark color, because most of my Swirls are kind of Summer-y colors and I don't get to wear them much in cold weather.
- 1/17/2015 08:09:00 PM Labels: Swirl, vintage for me 2 comments:
Vintage bookcase, filled with everything except books!
We managed to get it into the truck with the cedar chest and boxes and bags of vintage clothing and linens that we cleaned out of the house. It sat in our garage for a while until I decided where to put it. I finally got it cleaned and set up today. It still needs a little oil to freshen the wood, but it's looking good.
I devoted each section to some of my favorite things - Kewpies, Oriental stuff and Fairy stuff. Pretty.
Vintage 1940s Home Perm (Torture) Kit
This week I was invited to go to a house clean-out. The property was a 1930s twin house, the woman's grandparents (married in 1913) had lived on one side, her parents (married in the 1940s) lived in the other. The grandparents died in the 1970s and her parents just basically closed off that side of the house, left the grandparent's items in there and just used that side for storage and part of it as an art studio.
The woman's mother passed several years ago and the father just passed last year, so now she is having both sides cleaned out to sell the house.
We went through the grandparent's side which was mostly 1940-50s items, furniture, clothing, linens etc. Nothing really older than 1930s, which was probably when they moved in, and not much after 1960s. I got a nice load of clothing, dresses and lingerie, some linens and wedding photos from the 1940s. A lot of what I left behind was just all faded and moth eaten from having been neglected for so long.
We just glanced through the parent's side of the house, the woman is still removing things that she wants. The decor is still 1950-60s, but the clothing looks 1980-90s, so I don't think I'll get much clothes-wise from there.
From the grandparent's side, I found this cute little suitcase. It turns out that it is an old at-home perm kit, probably from the 1940s. Actually 2 sets of clips, spacers, rollers etc. squeezed into one case.
I just though that it was pretty cool.
There are even 3 partial bottles of old perm lotions in there.
Sears Approved.
Some of the lotions and booklets.
It took all of the items below to do one curl/wave.
The 1st piece is felt and rubber with a slit in the middle that you pull the section of hair through down to your roots.
Next is a metal and rubber spacer that you clip onto the hair at the roots.
Then you roll the hair in the paper onto the metal curler.
Then you clip the whole thing.
All of this would have been so heavy on each curl.
Each wave/curl on your head would look like this -
The Sears Booklet and the Ann Barton Booklet that came with each set of clips.
- 10/31/2014 08:29:00 PM Labels: hair, vintage purchases No comments:
Yeah! May's Barkbox is here!
Yeah! May's Barkbox is here! Gus & Harlow are signed up for the Barkbox 6 month subscription which costs $19.00 per month, I had a discount code from their Facebook page so I received 10% off.
Each month we get a box with 4-5 goodies and fun stuff to play with, rip up and gobble up. This is our second month and it's a lot of fun, I'm as excited to open the box as they are.
Barkbox has plans from 1 month to 1 year.
Use this link to sign up and you'll receive $5.00 off - https://barkbox.com/r/QKJIRSNKFY
Here are some poorly lit cellphone picks of Gus and Harlow with this month's box.
Each month we get a box with 4-5 goodies and fun stuff to play with, rip up and gobble up. This is our second month and it's a lot of fun, I'm as excited to open the box as they are.
Barkbox has plans from 1 month to 1 year.
Use this link to sign up and you'll receive $5.00 off - https://barkbox.com/r/QKJIRSNKFY
Here are some poorly lit cellphone picks of Gus and Harlow with this month's box.
mmmmm, something smells yummy!
Harlow grabbed the stuff squeaky toy, it's days are now numbered.
No toy lasts very long when my dogs play with it
Good stuff this month - a rubber Megalast Ball, Rozcoe the corduroy kitten by Loopies, Apple Cobbler treats by Snicky Snaks (those seem to be a big hit and they smell good) and No Grainers BBQ chicken jerky treats. Looking up the prices of the items online, the box value is at least $32.00
Yeah May Barkbox!
- 5/19/2014 04:37:00 PM Labels: barkbox, Gus, Harlow No comments:
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