text:
Why Shop with Us
Customers also Bought:
Previously known as Suterra WCM Balck Stripe Delta Kit. WCM Black Stripe Delta Kit is an advanced monitoring trap kit specially designed for clothes moth. This package contains black stripe triangular (delta) trap that's made of laminated card and pheromone lure which attracts clothes moths. The inner surfaces of the trap are coated with sticky "dry touch" glue to efficiently catch insects. Use the trap with lure to bait them easily. This trap has a unique black stripe print that is proven to attract more moths and increase catch rate by 15%. The WCM Black Stripe Delta Kit will help eliminate pests that can cause serious damage to natural fibre based fabrics such as drapes, clothes, tapestries, carpets, etc.
Each kit contains 10 traps.
|Active Ingredient:
|Pheromone lure
|Target pests:
|Webbing clothes moth (attracts males only)
|For use in:
|Storage areas or near items at risk such as closets to protect clothes, museums or areas with tapestries, rugs, etc.
|To be placed or hung in areas of potential or known infestations such as closets & bedrooms or fibre processing / storage areas. Hanging ties are included.
|Pet safe:
|Yes, when used as directed on the product label
|Shelf Life:
|Up to 6 weeks
|14.76 x 5.12 x 0.80
|Black and white
|Material / Construction:
|Trap: Card
Lure: Cellulose
|Special Features:
|Contains pest specific 6-week duration pheromone lure. Dry touch glue. Non-toxic
|Time to Kill:
|Detection in 0 - 48 hrs.
|Parts Included:
|10 Traps, 10 Hangers, 10 lures
|Shipping Weight:
|0.49 lbs
|Manufactured By:
|Agrisense Industrial Monitoring (Mfg. Number: 10089)
Product Documents
Remove the cover paper from the glued surface of the trap and remove the pheromone lure from its foil sachet. Place the lure in the center of the glued area. Then fold the two sides of the delta up, so that the glue is on the inner surface and insert the tab in the slot. Fold over the crease at the top of the trap.
The traps should be placed in storage areas or near items at risk throughout the year. The timing of pest appearance will vary with the external climate and other factors such as the use of central heating etc.
This insect is noted as a poor flier and will not normally venture very far from the source of infestation. The traps should be placed on shelving or on the floor where the materials to be monitored are kept. Avoid hanging the traps. If traps must be hung then this should be as low as possible above the monitored items. One trap should be sufficient to monitor a single confirmed spaces e.g. a wardrobe. Where larger areas need to be monitored a minimum of 1 trap per 10m2 should be used.
Disposable Traps and Lures should be changed every 6 weeks. Remove the cover paper from the glue on the trap and lure from its sachet. Place the lure centrally on the base section of the trap. Fold the trap to form its delta cross section and lock the tabs together.
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3 of 3 people found this review helpful:
Excellent so far
By Js in New Mexico on 08/19/2015
If this works as well as the pro pest control I called, I have just saved $150! So far have caught several, but many were caught in the preceding months. Then it slowed way down. So far, this is an excellent product
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text:
Correcting 'Dodd–Frank' to actually end 'Too Big to Fail'
Hearing on "Examining How the Dodd–Frank Act Could Result in More Taxpayer-Funded Bailouts", Washington, D.C., June 26, 2013 ; "I implore the members of this important committee and the Congress to not succumb merely to the illusion of hope. Don't listen to the siren song of the megabanks and their lobbyists. Take action to deal with the unfair advantages that these institutions enjoy… Leveling the playing field is a just cause for 99.8 percent of American banks and for all Americans."
If you experience problems downloading a file, check if you have the proper application to view it first. In case of further problems read the IDEAS help page. Note that these files are not on the IDEAS site. Please be patient as the files may be large.
|Date of creation:
|Contact details of provider:
| Web page: http://www.dallasfed.org/
More information through EDIRC
|Order Information:
| Email:
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subset: commoncrawl


text:
Marijuana: Good or Bad
Javier Gonzalez
May 27, 2010
Dr. Fathiah Inserto
Marijuana is considered an illegal drug yet thousands of the American people consume marijuana for medical purposes. Is marijuana good for you? There has been a lot of controversy concerning the topic of marijuana throughout the last few years. When the topic of marijuana is brought up the majority of the public interpret marijuana and an illegal drug as one. Most of the public may associate marijuana as unhealthy for the human body and even being arrested and going to jail when used. The other half of the public would interpret marijuana as a cure to their illnesses and a way of leading a normal and healthier life because the use of prescribed medical marijuana has helped to tame their illnesses.
Illegal drugs are considered bad for a person because the end result of using illegal drugs could possibly be addicting thus creating a huge health risk and may even be fatal. Marijuana is a drug that is considered illegal, but when prescribed by a doctor marijuana may be used for medical purposes in order to help patients fight illnesses. Medical Marijuana has proven to fight and help with several illnesses not limited to the following; neuropathic pains, migraines headaches, sleep deprivation, appetite loss, and general pain of fibromyalgia and chemotherapy.
A study was conducted in 2003 through 2005 concerning the use of medical marijuana being used for the purpose of suppressing neuropathic pain (Dr. Donald Abrams, 2003.) The study shows that more then four million united states residents suffer from neuropathic pains, and more then 37% of those four million people who are suffering from neuropathic pains use medical marijuana as a source of medication in order to reduce or in some cases stop neuropathic pain. In the same year another study was conducted, (San Francisco hospital) out of 50 patients that were suffering from...


text:
One Inch Wonders
Making an impact on youth art education programs
Client: Inch x Inch
Categories: Illustration, Misc.
Purchase: Buy the Pack
Every month, the guys at Inch x Inch invite a new artist to contribute artwork to become a button pack! That button pack is then sold to "club members" and 40% of all the money made goes directly to Art with A Heart and 826Chi to make a direct impact on young artists.
Sadly, the Legend of Zelda / Tron mashup button didn't make the cut, but it's too cool to not share, right?


text:
665"It's "CLEAR" To Me, How About You?"
Expand Messages
- Apr 10, 2003______________________________________________________________________
"It's "CLEAR" To Me, How About You?"
BY; Jimmy Stix (www.jimmystix.com)
April 10th 2003
As I read the "Wall Street Journal" article on Clear Channel, I could
not help but wonder?
I Wonder if Clear Channel really sees the Big picture? I wonder if
they really know what's going on in COUNTRY? I wonder if they really
care? Why do I say this?
When the Chief Operating Officer Mark Mays, makes this statement
before the U.S. Senate Commitee on Commerce, Science and
Transportation in January, that it's play lists are driven listeners
tastes and desires, largely determined through audience research
performed by it's stations, and that the company (Clear Channel)
takes it's role as a steward of the public airwaves very seriously,
and doesn't want to jeopardize what it considers to be an important
public trust.
Now, I first want to say here, so there is no misunderstanding of
what I am about to say, I do not have anything personal against
anyone in Clear Channel, or any of it's affiliates.
I just have an observation that may help them continue to show a
willingness to put our COUNTRY in a direction that all, Americans who
love country music will be happy with. And I'm sure they will agree,
that's what they want as well. Right?
To get back now to the statement made to our U. S. Senate Committee
in January.
The research they make is limited to mostly audience determination,
and they use this to base, what direction their play list will go
in. Who they will play on the public airwaves, and who they will not
play. Is this a true system gauge to go by, in order to know what the
American Country Music lovers want to hear on Radio, and see on TV?
After all, it's been said by me, many, many times over the past 14
years, that "New Country Radio", (And this is more than just Clear
Channel "New Country Radio" I'm talking about here) Their Audience
is not the majority of Country Music Lovers. Why? Because the
majority of country music lovers are not listening to "New Country
Radio", even though they would love to listen to the FM Country Radio
In other words, "New Country Radio" (And that includes Clear Channel)
is missing a great deal of country music that belongs on radio, and
the majority of country music lovers in America are not getting to
The audience research you make, are not getting to the massive
country people who are upset that radio has forgotten such artist
like all the Legendary Stars. These stars are still on the major
markets with brand new releases. Your audiences make up just about
ten percent of the American Country Music lovers here in the USA.
When you say that teens in America do not want to hear the older
stars and their music, even their brand new releases, can you see you
are wrong about that?
All ages of country music people out here want to hear the country
music legendary stars, especailly their brand new country music.
How important is it to "New Country Radio" that you provide the best
listening experience for all country people in America?
Clear Channel Radio CEO John Hogan announced; "Strong relationships
with artists and record labels are a priority for our
business,".....................and provide opportunies for us to
create better ways to market and promote music for all concern."
All concern? Well, we hope so Clear Channel, for everyone's sake,
here in America, where we never want to segregate it's people in any
area of life. United we stand, and divided we fall, isn't that the
American way? Of course it is.
Will Clear Channel, and other companies that own radio stations with
a country format, really reach out to all the country music people to
bring us the best possible listening experience for all Americans,
young, and older?
Only time will tell, right?
After all, nobody wants to see Clear Channel suffer, not even me.
You could truely qoute me on any part of this article gang,
especially the following.
I guarantee if Clear Channel, and other "New Country Radio", FM
companies, would only research beyond their Audiences, they will
find that the majority country music lovers do, they do, want
the "New Country Music" on the "Airwaves" that the Legendary Stars
have on the major markets right now, and these stars have many more
releases planned for the future.
And to add to that, the strong relationships with artists and record
labels are a priority for Clear Channel's business, and we could only
pray, it's with all artists, and record labels, here in America.
Because so far, the only artists and record labels having a strong
relationship with Clear Channel, and other "New Country Music" radio
stations have not, included the Legendary Stars and their label
I stand by my statement gang, that any radio station that calls
themselves COUNTRY, and who do not play the brand new releases from
such legendary stars, are not COUNTRY! They can not be.
Prime time COUNTRY radio without our legendary stars and their brand
new music, is like having the CMA AWARDS SHOW on TV every year,
without our legendary stars as a part of the show. It's just not the
American Country music show without these seasoned stars. The
legendary stars who are still alive, like the great JOHNNY CASH,
deserve the major attention of every American country music Lover
now, and in the future.
And to say that your country music audience do not want to hear the
brand new country music coming from all the legendary stars, is like
saying, I, Jimmy Stix, don't like the young new stars that are on
your stations now. And that's stupid! It's stupidity that
misunderstands the truth. What is truth? The truth is, every person
who says they love country, loves the legendary stars! And we, the
country people want to hear these stars!
So you see, even your present audience, who love country music,
right? Do want to hear the legendary stars' brand new releases, they
must, if they truely love country music here in America they do? Why
wouldn't they? What would be their reason not to?? I'd like to hear
it, and everyone reading my article would like to hear their reasons
too, don't you gang?
Yes! We love the older country music of the legendary stars, even the
ones who have past away. And when your stations play these songs on
weekends, we love it! We are not looking to have these country songs
on prime-time!
It's the brand new releases coming from all the living legendary
stars, like the GREAT JOHNNY CASH, that we want on prime time radio!
Mr Mark Mays said in the WAll Street Journel, that his company takes
its role as a steward of the public airwaves very seriuosly, and
doesn't want to jeopardize what it considers to be an important
public trust.
Well, Mr Mark Mays, we will trust that you will see to it that the
legendary stars and their brand new releases get on the prime time
airwaves soon, for it is to all of our best interest that you do so.
_______________________________end of article.


text:
What was the most Memorable thing...
View Single Post
November 28th, 2006, 11:39 AM
Platinum Supermommy
Join Date: Feb 2005
Location: Louisiana
Posts: 11,318
Yea...right! My son was newly four when he not only witnessed but assisted at my birth with Remy. He was GREAT! He never looked afraid and he would come and sit by the side of the tub and put a little hand on my arm and say, "breathe mommy...you just have to get the baby out!" And then he leaned down when I was on my hands and knees and the baby was crowning and said, "OHHHH, it's a baby!"
He also cut the cord and helped Mattmos clean up after the birth. So...from our experience with this child. We are just in love with having our children share in the birth as much as they feel comfortable.
"I am a midwife. It is not just what I do, it is what I am, and I grow in it."
-Ruth Cobb
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text:
|10 Apr 2014
More than one external USB drive-how to identify?
Had this problem for ages. I've got two sometimes four Western Digital My Passport external drives connected. In Safely Remove, how can I tell which one is which? "WD My Passport 0740" is the name of one in Safely Remove. The other is "...0748". On the back plate of the drives, no such ID # exists, just S/N and P/N. In the past, to get around this, I just shut down and unhook the drive I don't want. How to identify which is which in Safely Remove?
edit: Also, are 0740 and 0748 assignations that stick with the hardware when connected on any PC? Or, is it a system assignation that might be different on another machine? ie are these port numbers, not the drives?
|My System Specs
|10 Apr 2014
I simply set the policy to quick removal and don't bother with the icon on desktop.
|My System Specs
|10 Apr 2014
Safely Remove > right click Open Devices and Printers > My Passport Properties (only General and Hardware tabs) > under Hardware tab, highlight WD My Passport > click bottom right Properties button > WD My Passport 0748 Device Properties > only General, Volumes, Driver, and Details tabs ...no "Policies" like in your screenshot.
|My System Specs
|10 Apr 2014
Go to Dev Mgr and expand dvd drives.
Right click on external hdd and click Properties.
You will see Policies.
|My System Specs
|10 Apr 2014
Vista, Windows7, Mint Mate, Zorin, Windows 8
Another option is to give each disk a different name - in Computer (highlight and right click).
|My System Specs
|11 Apr 2014
Windows 7 Home Premium 32 bit
Apart from a unique drive label as aforesaid by whs, I also stick a label on the external HDDs indicating the unique drive label so that when I plug out , I plug out the right external HDD.
|My System Specs
|11 Apr 2014
Win 7 Ultimate 64 bit
|My System Specs
|Thread Tools
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text:
Sagun Home Care Inc.
About Sagun Home Care Inc.10560 Main St. Ste 209
Fairfax, VA 22030
Phone: (703) 273-8520
Fax: (703) 273-1381
Provides services to: children, adults, seniors
Service Area for Sagun Home Care Inc.
About Sagun Home Care Inc.
We want what is best for those we love... and we know that home, if at all possible, is the best option. But home care can be costly, only provide limited services, and qualified, caring providers can be difficult to find. Plus, in today's world, it can be just about impossible for a family member to dedicate the time needed to be a caregiver. Sagun Home Care, Inc. understands these potential hurdles. That's why we offer expansive personal home care services at affordable hourly rates by fully bonded & insured caregivers who truly and deeply care. Because of our rigorous background checks and thorough hiring process, we believe that you will find Sagun Home Care, Inc. the most loving, flexible, expansive, and reasonably priced home care and companionship in the Arlington, Fairfax, Loudoun and Prince William counties, Alexandria City and surrounding areas.
Sagun Home Care takes our responsibilities to those we care for very seriously. Part of that involves offering more than just the basic necessities, but also the compassion and respect each of our clients deserve. Just as important, however, is earning and maintaining the trust of our clients. Below are important reasons you can trust Sagun Home Care:
1.All our caregivers are fully bonded & insured
2.Our thorough Caregiver Screening Process includes state criminal background checks, motor vehicle record check, reference checks, certification and license verification
3.Screenings conducted on all caregivers before working with a client home
4.Licensed by the Virginia Department of Health
5.Our caregivers receive ongoing training including monthly in-service educational programs and formal training
6.Sagun Home Care, Inc. performs unannounced quality control visits by the company president to ensure continual excellence in service
7.No Long-term contracts. Affordable hourly rates! Hourly or Live-In Caregivers.
8.We Accept Medicaid Waivers, Private Pay, Long-term Care Insurances
9.We tailor your care according to your personal needs
10.Locally owned and operated by Virginia licensed CPA
Overall Rating 5.0 / 5.0 ★★★★★
Alice Ndunguru★★★★★ 7 months ago
Sagun Home Care Inc. Services
- Private Duty Home Care
- Home Care
- Home Health Aid
Cities Served by Sagun Home Care Inc.
Sagun Home Care Inc. serves ZIP codes that encompass all or part of each of the cities listed below.
Burke, VA
Centreville, VA
Clifton, VA
Dulles, VA
Fairfax, VA
Falls Church, VA
Manassas, VA
Mc Lean, VA
Reston, VA
Vienna, VA


text:
Easy Low-Carb Bodybuilding Breakfast: Egg White & Veggie Muffins (Video)
Checkout this great low-carb breakfast recipe with egg whites and veggie muffins from Micheal Kory Fitness.
1 cup broccoli (85 g)
1/4 cup chopped onions (40 g)
1/4 cup low fat shredded cheese (30 g)
1 cup liquid egg whites (240 mL)
Seasonings of your choice
Hot sauce (optional)
Makes 1 serving.
Per serving:
250 calories
5g fat
15g carbs
34g protein
Tags: breakfast, egg whites, muffin, recipe, video


text:
Added to Shopping Bag
Vaneli Di Notte Pointed-toe Rhinestone Detailed Dress Heels Size 7.5n Black Pumps
Listed by Dominic Scacci
Size: 7.5
Price: $140.99
Returnable for site
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Di Notte Pointed-toe Rhinestone Detailed Dress Heels Size 7.5n Black Pumps
Size: 7.5
$140.99 Shipping Included
Vaneli Di Notte Pointed-toe Rhinestone Detailed Dress Heels Size 7.5n Black Pumps
Shipping Included
Pay with Credit Card, PayPal, or Affirm.
3 people saved this item
This item has visible signs of wear
Item Number:
Design Details:
Vaneli Di Notte Black Pointed-toe Rhinestone Detailed Dress Heels Size 7.5N, thin heel, tiny silver rhinestone embellished throughout shoe, pointy toe, vero cuoio, inner foot exposure
Stylist Tips:
Looks great with a black or silver evening gown
Signs of Wear:
Some wear on sole and insole, no major signs of wear
Insole: N/A
Style: Classics
Material: Fabric,Leather
Pattern: Studs
Style Tags:
Fri 11/24/17 - Mon 11/27/17 (5-8 days)
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text:
The 10 Russian spies deported in a sensational spy swap with Washington are being questioned by the special services in a prolonged debriefing at a secret location in Moscow, reports said Tuesday.
The Moskovsky Komsomolets daily said that the 10, including the glamorous Anna Chapman, were not allowed to leave a compound owned by the Foreign Intelligence Service (SVR) and were subjected to tests including lie-detectors.
There has been sharp criticism in the Russian press and by ex-agents of their spycraft, notably their use of social networking sites and apparently archaic techniques such as invisible ink.
"At the current time the agents are working with specialists," the report quoted a source in the Russian special services as saying
"They are trying to clarify how their cover could have been blown in such situations."
The Interfax news agency also quoted a security source as saying that the 10 were in Moscow and were working with the "competent organs".
It said that some of the agents had expressed a desire to change their names "in line with a witness protection programme," the agency's source added.
The SVR has refused to give any comment on the 10 agents but the reports were the first significant claim of details about the spies' fate to have emerged since their swap for four Russian convicts.
Moskovsky Komsomolets said they would be released in the next few weeks, so long as the probe showed that serious errors had not been committed in their work as agents in the United States, the report said
"For clarifying all the details, interviews are being carried out along with different kinds of tests which include lie-detectors," its source said.
"This should not be called an interrogation in the true sense of the word. But if it turns out that serious mistakes were made, spies, employees of the SVR, can be fired."
The report said that mobile phones did not work at the compound and the agents were not allowed to leave. However, they were being supplied with all necessities.
The idea that their cover was blown as a result of "treachery" within the service was also being examined, it added.
Immediately after landing in Moscow on Friday, the 10 were taken to SVR headquarters in Yasenevo outside Moscow but exactly where they were being questioned now was not clear, it said.
The arrest of the agents sparked fears that the espionage scandal could harm improving ties with Washington, but Friday's spy exchange appeared aimed at limiting any damage.
Some of the agents had been working as deep cover "sleepers" for as long as a decade although Chapman -- who has become a tabloid celebrity over the last fortnight, had gone to the United States more recently.
The paper's source meanwhile rubbished US media reports that the arrests of the agents were hastened by a panicked telephone call from Chapman to her father, who is believed to have a background in the Russian special services.
"Agents know what to do in these situations. She would not have turned to her father. At the Russian embassy in the US there is a person in charge of security and other questions," the source said.
Such is her fame that a local newspaper in her native town of Volgograd has launched a contest -- entitled "Let's help our guys with a good song" -- to compose the best song about her.
"Any scandal can end cheerfully, optimistically -- for example with a good song," Andrei Serenko, the editor of the Gorodskiye Vesti newspaper told Interfax.


text:
Admirals rally past Senators 3-2
The Norfolk Admirals scored two goals in the third period to rally past Binghamton 3-2 on Saturday night at Scope.
Sponsored by:
The Norfolk Admirals scored two goals in the third period to rally past Binghamton 3-2 on Saturday night at Scope.
Injuries to Anaheim's top goaltenders mean opportunity for Admirals' netminders.
Sports around Hampton Roads during the weekend of Oct. 31, 2014.
The Admirals managed two goals in a defensive showdown, but it was all they needed.
Norfolk surrenders late goals after managing to tie the game in the third period.
John Gibson had 32 saves and Nic Kerdiles and Antoine Laganiere teamed up for a spectacular goal in the Admirals 3-2 win over Manchester.
Anaheim Ducks goalie John Gibson has been assigned to the Admirals for the weekend.
The Admirals didn't have enough to follow up their thrilling Saturday night win over the Bears.
Sports around Hampton Roads this weekend included the Norfolk Admirals' 15th Season Opener and NSU taking on Deleware State.
Chris Wagner scored two short-handed goals in the first period and added the game winner in the third in the Admirals 5-4 win over Hershey.
New coach Jarrod Skalde and veteran Chris Wagner talk about the Admirals upcoming season which begins Saturday night at Scope.
The Norfolk Admirals introduced Jarrod Skalde as its new head coach on Friday afternoon.
The Admirals lost their top enforcer Zack Stortini to the Philadelphia Flyers organization after he signed a two-year deal with Philadelphia…
NORFOLK, Va. (WAVY) – The hockey season is over in Hampton Roads, and the Norfolk Admirals spent Monday Morning doing the one thing no team …
ST. JOHN's, Newfoundland (WAVY) – The Admirals season came to a close Friday night in Canada. Facing elimination in game 6 of the Eastern Co…


text:
Blazekick doom
July 14th, 2008, 9:07 AM
The game is very addictive. I wanna know what game had you up all night. It was Emerald that had me up all night. The whole Battle Frontier thing was a smart move on Game Freak's part. Also the extras, oh man the extras. I got my Pokemon Diploma (This achievement is only enabled if you have pretty much all of the Pokemon RPG's) and of the other available things for the game.


text:
I have actually called ribbon storage the Holy Grail of Scrapbooking- and it is. Everybody is looking for the perfect solution...someone could make a fortune, I swear! Wanna see mine? See? Ribbon stays vertically stored, no rolling, room for almost a hundred rolls. Want to know what it cost me? Nothing. Zip. Zero. Nada. Gotta love it! You want to know where, right? Big Lots. So, here is the story...I am out on a mission one day last year- ribbon storage or bust! I have gone to literally every store I can think of- nothing. Now, I am willing to pay some bucks, here- it just didn't exist. Finally, I am walking through BL, and what do I spy? Their Christmas ribbon for sale---in this handy little cardboard endcap. I asked the manager- cause it was almost empty- and she was thrilled to give it to me. A little alteration...and we have ribbon storage. I actually have another one in storage- there for when I need it. Just thought I'd mention it...it being the Christmas ribbon selling season, and all.
Scrapping Cents Sightings:
BL: Lots of cool MM Garden Party accessories...for $1!
Scrapbook News and Review: Sept issue is up- and remember: pay for one month, and you get access to EVERY, single issue we have ever published for that month. Now, THAT is a bargain!!
Sunday, September 21, 2008
Scrapping Cents
Posted by Anne House at 1:28 PM
1 comment:
- 15minL8 said...
Have you seen the ribbon ring? If you would like to try it and review it on your website, I would be willing to send you a package of ribbon ring tags at no charge! Just send me an email: email@example.com.
- September 21, 2008 at 4:17 PM
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text:
Origin of a novel protein-coding gene family with similar signal sequence in Schistosoma japonicum
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Evolution of novel protein-coding genes is the bedrock of adaptive evolution. Recently, we identified six protein-coding genes with similar signal sequence from Schistosoma japonicum egg stage mRNA using signal sequence trap (SST). To find the mechanism underlying the origination of these genes with similar core promoter regions and signal sequence, we adopted an integrated approach utilizing whole genome, transcriptome and proteome database BLAST queries, other bioinformatics tools, and molecular analyses.
Our data, in combination with database analyses showed evidences of expression of these genes both at the mRNA and protein levels exclusively in all developmental stages of S. japonicum. The signal sequence motif was identified in 27 distinct S. japonicum UniGene entries with multiple mRNA transcripts, and in 34 genome contigs distributed within 18 scaffolds with evidence of genome-wide dispersion. No homolog of these genes or similar domain was found in deposited data from any other organism. We observed preponderance of flanking repetitive elements (REs), albeit partial copies, especially of the RTE-like and Perere class at either side of the duplication source locus. The role of REs as major mediators of DNA-level recombination leading to dispersive duplication is discussed with evidence from our analyses. We also identified a stepwise pathway towards functional selection in evolving genes by alternative splicing. Equally, the possible transcription models of some protein-coding representatives of the duplicons are presented with evidence of expression in vitro.
Our findings contribute to the accumulating evidence of the role of REs in the generation of evolutionary novelties in organisms' genomes.
KeywordsSignal sequence trap Schistosoma japonicum Repetitive elements Gene duplication Secreted proteins Non-allelic homologous recombination
Signal sequence trap
Non-allelic homologous recombination
DNA level recombination
Non-homologous end joining
Illegitimate recombination
Repetitive element
Open reading frame
Whole genome shotgun.
Evolutionary novelties generated as an upshot of the "nascence" of new protein-coding genes are the bedrock of adaptive evolution and acquisition of novel molecular functions. The ever-growing vast and diverse protein repertoire in organisms can be ascribed to these events, and may explain the increasing heterogeneity among organisms of otherwise common ancestry [1, 2, 3, 4, 5]. Since the pioneering definitive treatise on gene duplication by Ohno about four decades ago [6], geneticists and evolutionary biologists have advanced this traditional notion; creating remarkable insights into the composite patterns and underlying mechanisms of genetic innovations. Some of these mechanisms are illustrated in a supplementary figure (Additional file 1). The advent of the genomics era has most importantly armed scientists with a valuable tool to enhance discovery of the rather intriguing mechanisms underlying the "birth" of new genes [5].
Apart from the canonical gene duplication model as proposed by Ohno [6]; extensive studies in various organisms have not only elucidated other models of gene duplication, including "dispersed" duplication in addition to the more definitive "tandem" duplication [7, 8, 9, 10, 11, 12, 13]; but has also revealed multiple mechanisms leading to the emergence of new functional genes. These include but not limited to: recombination by exon shuffling or exon "scrambling" [4, 14, 15, 16, 17, 18]; retrotransposition by retrotransposons yielding intronless chimeric genes [18, 19, 20, 21, 22, 23, 24, 25]; transduction of genomic segments by transposable elements by skipping the characteristic weak polyadenylation signal in retrotransposons leading to the mobilization of adjacent genomic sequence; or may involve a repetitive element (RE) mediated DNA level recombination (DLR) by a non-allelic homologous recombination (NAHR) mechanism, in which the REs provide the requisite homologous sequences for the recombination of genomic sequences in a non-allelic manner [7, 20, 26, 27, 28, 29, 30]. Horizontal gene transfer between organisms although infrequent, can give rise to new genes in the recipient organism [31, 32, 33]. De novo origination of protein coding genes from previously non-coding genomic sequences is a very important mechanism previously underrated, but accumulating data in many organism show that this event occur more often than previously thought [2, 3, 34, 35, 36, 37, 38, 39, 40]. Equally, a new gene can arise from the fusion of two genes [1, 3, 22] or fission of a "parent" gene [41]. These mechanisms seldom operate singly as they frequently overlap, collaborating in the creation of nascent genes as depicted in the famous origins of Jingwei and Sphinx in Drosophila species [14, 19].
Schistosoma japonicum along with S. mansoni and S. haematobium are the principal schistosome species causing human schistosomiasis. Uncharacteristic of other human invading schistosomes, S. japonicum is also able to infect several non-human mammalian hosts. While S. japonicum and S. mansoni inhabit the periportal veins and cause an intestinal form of the disease, characterized by liver granulomatous fibrosis as a consequence of host immune response to the eggs lodged in the hepatic sinusoids [42, 43]; S. haematobium causes urinary schistosomiasis at the vesical bladder plexus. Although S. japonicum produces similar lesions like S. mansoni, the fibrotic lesions and hepatosplenomegaly, the most severe outcome of schistosomiasis, is relatively more frequent and severe in S. japonicum[44]. Also, in contrast to S. mansoni and S. haematobium, acute disease due to S. japonicum is common in endemic foci and is associated with severe and persistent manifestations that may rapidly progress the host mediated immunopathogenesis, terminating in a network of fibrotic lesions [45]. Secreted proteins from the parasite ova embolized in the liver of the host are accessible to the host immune cells being located at the host-parasite interface and thus constantly exposed to the host liver tissues. Such interactions play critical role in the initiation and progression of granuloma and fibrosis formation by mediating inflammation [42, 43, 44, 45]. Secreted protein candidates thus, possess great potentials for application in interventions aimed at preventing severe hepatic pathogenesis [46, 47] among other applications.
Nascent genes confer extra functional capacities for the organisms to confront the challenges of the ever dynamic environment, and may equally, albeit rarely, inflict some functional constraints. In any case, recently evolved characteristics could best be attributed to either: protein family or domains expansion, gene loss events [48], or more likely, evolution of new genes. S. japonicum relatively exhibit a higher degree of parasitism and dependence on host derived molecules and signals as inferred from genomic and transcriptomic studies [49, 50, 51]; it is able to infect a wide range of hosts, and produces relatively more severe pathogenesis [45]. While these could be attributed to a number of other factors including: selective pressure of parasite-host interactions, the extensive gene loss and protein domain elimination or expansion events observed in its genome and transcriptome [49]; the evolution of novel functional protein coding genes before and after the divergence from other members of the genus Schistosoma could account for these extra characteristics.
Here, we report putative evolutionary novel gene family of Asian schistosomes, S. japonicum on the premise that no homologs of the genes were found in the genome of its evolutionary close relatives in the genus Schistosoma, or in any other organism with a complete sequenced genome. The genes first caught our attention as genes bearing similar or same signal sequence from our previous work that identified some secreted protein coding genes from the eggs of S. japonicum using a signal sequence trap (SST) [47]. Given the available tools prior to the publication of the S. japonicum genome sequence, we had attributed this observation to some alternative or trans-splicing models. The present analysis was inspired by the availability of the invaluable tool presented by the recently published partially assembled genome of this parasite [49]. We adopted an integrated approach utilizing extensive BLAST queries and other bioinformatics tools, transcription and expression analyses, southern hybridization of genomic DNA and evolutionary analyses. We describe evidence of "genome-wide" dispersed duplication of a protein coding gene locus, which may have arisen recently from previously non-coding genomic sequence. The role of repetitive elements as major mediators of the dispersive duplication is analyzed and discussed. Detailed evidence of the potential transcription models of some protein-coding representatives of the duplicons with similar signal sequence is presented and supported by our observations. Finally, based on the identification of non-coding mRNA transcripts as alternatively spliced variants of protein coding mRNAs, we propose that the new genes could be under significant functional selection.
Results and discussion
Sequence characteristics of a novel protein-coding gene family with similar signal sequences in S. japonicum
*SST isolatedS. japonicumegg cDNAs with similar signal peptide
Gene Products
GenBank cDNA Accession
GenBank Protein Accession
Signal Peptide
AY570737 (1027 bp)
AAS68242 (271aa)
AY570744 (983 bp)
AAS68249 (260aa)
AY570753 (1038 bp)
AAS68258 (174aa)
AY570748 (854 bp)
AAS68253 (203aa)
AY570756 (848 bp)
AAS68261 (124aa)
AY570742 (1037 bp)
AAS68247 (274aa)
*UniGene entries forS. japonicummRNAs and ESTs bearing the similar signal sequence (n = 195)
UniGene Name
UniGene ID (UID)
Set of likely mRNA transcripts (GenBank)
Gene Products (Database annotation)
AY814448, BU780442est
Egg protein SjCP3611
FN317637, BU772954est
Hypothetical protein
Egg protein SjCP1531
AY570748SST, AY223245, AY222916, AY813542, EF127834, EF140742, FN323799, FN323800, FN323801, FN323803, FN323793, FN323792, FN323791, FN323790, FN323788, FN323785, FN323782, FN323781, FN323779, FN323778, FN323777, FN323776, FN323773, FN323772, FN323771, FN323770, FN323769, FN323768, FN323767, FN323766, FN323765, FN323764, FN323763, FN323762, BU772060est, BU766145est, CX862012est
Egg protein SjCP3842
Egg protein SjCP1084
AY570753SST, AY570744SST, AY814685, FN327232, FN327137, FN318042, FN321065, FN321060, FN321059, FN321058, FN321057, FN321056, FN321055, FN329815nc, BU768978est, BU780021est
Egg protein SjCP3611, Egg protein SjCP501, Hypothetical proteins
Egg protein SjCP1731
FN326953, FN330298nc
Hypothetical protein
AY570756SST, FN327121, FN327254, FN327253, FN327241, FN327233, FN327229, FN327224, FN327222, FN327216, FN327196, FN327185, FN327163, FN327158, FN327154, FN327129, FN327125, FN327115, FN327089, FN327083, FN327073, FN327057, FN327050, FN327049, FN327045, FN327042, FN327035, FN327022, FN327018, FN327014, FN327000, FN326998, FN326978, FN326973, FN326961, FN326960, FN326959, FN326930, FN326905, FN326883, FN326882, FN326881, FN326859, FN326857, FN326852, FN326851, FN326841, FN326831, FN326829, FN326822, FN326808, FN326801, FN326790, FN326770, FN326740, FN330540nc
Egg protein SjCP400, Somula protein
Egg protein SjCP3842, Hypothetical protein
AY813755, FN320057, FN320056, FN320514, FN329566nc, BU768160est, BU774105est, BU770186est, BU779051est
Egg protein SjCP3842, Hypothetical protein
FN327242, FN327131, FN327087, FN326854, BU776301est
Hypothetical protein
AY813975, FN329814nc, BU769048est
Egg protein SjCP1084
Hypothetical protein
AY570737SST, FN328299nc
Egg protein SjCP1084
Egg protein SjCP3842
Egg protein SjCP1084
Egg protein SjCP1084
Hypothetical protein
Hypothetical protein
Hypothetical protein
FN326786, FN318043, CX861530est
Hypothetical protein
Hypothetical protein
Schistosoma japonicumgenome contigs containing the similar signal sequence (n = 34)
*Contig [GenBank Accession No.]
Transcription Strand
Size, kb
Signal Sequence coordinates
4848 – 4779
276 – 205
999 – 928
2413 – 2342
3856 – 3785
3284 – 3217
19023 – 19094
6502 – 6431
42511 – 42440
9335 – 9264
493 – 564
10498 – 10427
7860 – 7789
433 – 504
5768 – 5697
12367 – 12438
383 – 322
4663 – 4602
2484 – 2544
337 – 408
4411 – 4342
388 – 459
925 – 854
271 – 200
1646 – 1717
446 – 517
2389 – 2319
5231 – 5160
1295 – 1366
656 – 585
1094 – 1165
1918 – 1847
1131 – 1060
4249 – 4320
Schistosoma japonicumScaffolds containing the similar signal sequence (n = 18)
Scaffolds [GenBank Accession]
Contigs within the scaffolds
To assess whether some homologs or at least some similar domains exist in other species, BLASTN and BLASTP searches using both the signal sequence and the entire coding sequences of the mRNAs and protein sequences as queries showed that these genes have no homologs in any other organism, but their expression in S. japonicum is supported by evidence from transcriptome and proteomic data. A search on several protein domain databases showed that although our candidates were classified in the same protein family with similar domains and assigned to a domain ID (ProDom:PD884968), no related domain or protein family was found in any other organism. The absence of these genes in the genome of S. mansoni, S. haematobium and other published genomes cannot possibly be attributed to sequencing gaps or annotation errors since the WGS sequencing approach is considerably reliable [49], and the fact that we adopted a multiple species approach covering the entire available sequenced genomes of all species makes this even more improbable [37, 40]. Given the accumulating evidences of de novo origin of new genes from previously non-coding DNA sequences [2, 34, 35, 36, 37, 38, 39, 40], we propose that the coding sequence of these genes may have recently originated de novo from previously non-coding DNA sequences in the ancestral forms, and subsequently duplicated and dispersed in the genome. This represents a more plausible interpretation than the improbable alternative hypothesis of concurrent gene deletion or inactivation in multiple ancestral lineages.
Species and strain specific expression
Nevertheless, while it is completely normal to verify this exclusive evolution and dispersed duplication hypothesis by confirming the physical localization of the gene loci in the genome and chromosomes by performing synteny analysis, we are unable to achieve this because we do not have access to a fully mapped chromosome information of the genome of S. japonicum. However, the distribution of the contigs and scaffolds bearing the similar signal sequence apparently suggests a dispersed distribution. To confirm this hypothesis and to exclude the possibility of overlapping among the loci, we generated the restriction map of six of the genome scaffolds bearing duplicated loci based on the information on the genome map, performed southern hybridization using restriction endonuclease digested genomic DNA from S. japonicum species and strains; and were able to match the expected probe binding fragment sizes with the observed bands on the hybridization blots (Additional file 4). Also an ancestral homolog is required for synteny analysis, however, we could not find a homolog in S. mansoni, another member of the genus with sequenced genome; and the genome of other more closely related species like S. mekongi and S. malayensis are not yet sequenced. Unless new evidences emerge from future updates in the sequenced genomes, we hold true that these genes have newly evolved, probably from modifications on previously non-coding ancestral DNA sequences and subsequently disperse duplicated. As opined in previous studies, the short length of our identified genes is an expected property of nascent genes because of improbability of evolution of long open reading frames (ORFs) and the complexity of intron splicing signal [38]. We expect these novel genes to be of functional significance since new genes tend to display accelerated sequence and structural changes towards neo-functionalization [1], and most newly characterized genes from other species have been shown to be characteristically functional [35, 56]. Other workers showed that the common pathway for de novo protein-coding gene evolution involves a piece of DNA sequence to be transcribed via recruitment of all transcription core promoters, other elements and machines; followed by the acquisition of a translatable ORF through mutations or other sequence alteration mechanisms [2, 35]. Together, our findings support the presence of these intrinsic features of novel genes in the identified candidates, including the gradual model of novel protein coding gene origination.
Evidence of dispersed duplication from a source gene locus
Additional file 5: Simulations using our raw data to show DNA-Level recombination mediated by REs by NAHR mechanism. The movie created from a Powerpoint presentation (Additional file 10) represents the basic approach we utilized in our analysis to show evidence of DNA level recombination by a non-allelic homologous recombination mechanism. The raw data obtained from BLAST searches and RepBase repetitive element prediction report was used to present a simulation that demonstrates that the duplicated locus is flanked on 5` and 3` ends by retrotransposons of the classes RTE_SJ and Perere respectively. We proposed that these repetitive elements could have provided the requisite homologous stretch of DNA that is required for such DNA level recombination. NAHR can be inter-chromosomal, intra-chromosomal, inter-sister chromatid, or intra-chromatid to give rise to disperse duplicates of the intervening genomic locus. This movie was created from an original Powerpoint presentation (Additional file 10) (MOV 6 MB)
The role of repetitive elements (REs) in dispersed duplication of genomic sequences is fairly documented from previous studies in model organisms [15, 20, 27, 28, 30, 58, 59]. The precise mechanism of this retrotransposon mediated dispersed duplication is not clear but may likely involve RE-mediated DNA level recombination, most likely by non-allelic homologous recombination (NAHR), alternatively called ectopic recombination (see illustration in Additional file 6). Due to their extremely high copy numbers, REs create structural modifications in the genome by providing the requisite highly similar DNA sequences, initiating recombination between non-allelic elements [20, 25, 60], the result of which could be deletion, shuffling, duplication or transduction of a genomic DNA segment. Structural modifications introduced in the genome by NAHR mechanism can progress between non-homologous chromosomes (inter-chromosomal), between homologous chromosomes (inter-homologous or intra-chromosomal), between sister chromatids (inter-sister chromatid) or within a chromatid (intra-chromatid); giving rise to dispersed duplication of genomic segments, several forms of deletions or may create isodicentric chromosome by forming a mirrored segment in the chromosome by inversion. See detailed cartoon in Additional file 6. [60].
Many studies in other organisms have elucidated the role of REs in mediating sequence duplication, transduction and other structural variations by ectopic recombination mechanism. Notable among these is the human Alu element for which several reports suggest a role in mediating NAHR and other structural modifications in the human genome [7, 20, 61]. Yang et al found an excess of repetitive sequences proximate to the breakpoints of duplicated gene loci in the genome of the fruit fly Drosophyla melanogaster, and have suggested that a NAHR mechanism, mediated by REs accounted for the birth of the new duplicons [1, 27]. Another study performed on human individuals concluded that NAHR accounted for over 40 % of detected genomic sequence duplications in the human genome [30]. Illegitimate recombination (IR), incomplete crossing over and non-homologous end joining (NHEJ) are other possible mechanisms of gene duplication by DNA-level recombination, but NAHR play a more significant role in producing typical dispersed duplications [1] while the other mechanisms in addition to NAHR are more likely to produce tandem duplicates. Although we could not clearly identify the exact breakpoints of the duplications at both ends still for lack of a reference ancestral homolog and partly due to sequencing gaps, the fact that homology among all the scaffolds examined uniformly terminated at the same point with Perere on the 3` end (Figure 3 and Additional file 5), and traces of the observed predominant retrotransposons (SjR2) was found at the exact positions as they occur in the putative source locus (Figure 4) confirm that these gene loci could be products of dispersed duplication from a single genomic source locus.
In addition to RE-mediated DNA-level recombination by NAHR, gene duplication events are also attributable to RE-mediated retrotransduction mechanism either on the 5` or 3` directions [27]. Xing et al and other groups have demonstrated the role of retrotransposons in the duplication of entire genes and creation of previously un-described genes by analyzing SVA (SINE, VNTR and Alu)-mediated retrotransduction events in the human genome [20, 29]. However, we did not specifically identify any chimeric duplicon originating via a retrotransduction mechanism among our datasets. Furthermore, retrotransposons including SjR2 characteristically encode reverse transcriptase and endonuclease, and can therefore transcribe and 'paste' a gene sequence into new locations in the genome [3, 22, 62]. However, retrotransposed genes are characteristically intronless since the introns are usually spliced out during the process of retrotransposition. Our duplicons retained their introns, although in some case some portion of the introns may have either degenerated or deleted during duplication and subsequent sequence modifications [3, 22, 63]. A further evidence that a retrotransposition mechanism is unlikely in our observed cases was that while retrotransposons would not duplicate the promoter regions of duplicated gene based on the process of transcription and insertion of retrocopies [1, 57] which leaves the newly retrotransposed sequences to acquire new regulatory sequences from adjacent genes or through mutations in order to be functional [14, 19, 24]; the protein coding duplicons observed among our duplicated gene loci retained the same or similar core regulatory region and signal sequence as the source locus, suggesting that they may not have been products of retroposition and may equally explain the parallel assumption of coding potential at their new duplication loci without the need to form chimeric structures with adjacent genes.
Evolution of translatable ORF and evidence of expression of duplicated genes
Some of the duplicons appear degenerative in homology and are relatively shorter than the source locus (Figure 3, also see Additional file 5) thus are consequently redundant and non-coding at the new locations as opined in the canonical view on the fate of new duplicons [6, 9]; which assumes a tendency to be lost because of genetic drift under natural evolution [29, 57]. However, our data provide evidence that some of the duplicons have evolved into protein coding genes with distinct products at their new loci, the fate of which could tend to either sub-functionalization to the source gene [8, 64] or neo-functionalization by acquiring new distinct functions [9, 65]. In addition to the two duplicons with alternative splicing variants, which we further explored in the next section, some representatives of the protein coding duplicons were depicted in a supplementary figure (Additional file 7). The nucleotide sequences of these genes are still appreciably similar but accumulation of mutations and other sequence modifications have given rise to novel protein coding ORFs, encoding putatively distinct products. We identified and mapped each cDNA sequence to the genomic contigs using information we generated from GeneMark and GeneQuest gene predictions [66] and confirmed by alignment of the cDNAs to the genomic sequences using NCBI Spling program. This approach was necessitated because the fully mapped and annotated genome of S. japonicum is not presently available in the public databases. Intriguingly, our results corroborate the available UniGene and GenBank entries. Nevertheless, it is notable that we only assessed the duplicated copies on the basis of possessing the similar signal sequence. There is possibility that some other duplicons from this source locus could be involved in initiating other forms of structural modifications at other loci when incorporated into the coding region of other genes, but this was not investigated here.
Functional selection by alternative splicing
The precise recognition of exon-intron junction in a precursor mRNA (pre-mRNA) by the splicing machinery is central for the production of functional translatable mRNAs. However, there is often uncertainties in the choice of recognizable splice signals, resulting in a process termed alternative splicing [17], which enables the origination of multiple mRNA transcript variants from a single gene locus [67, 68, 69]. Alternative splicing mechanism could result in 'intronization' of an exon or 'exonization' of an intronic sequence. Ideally, the creation of an intron from a previously exonic sequence could lead to the loss of an ORF in coding genes. In evolving genes however, functional selection possibly by mutations may evolve the required splice signals and induce the intronization of an exon in a transcribed but non-coding mRNA gene sequence to create a translatable ORF encoding a functional protein. Conversely, while exonization of an intron could disrupt a translatable ORF in a coding gene, selective pressure may also evolve new splice signals within an intron to yield exons that could create a translatable ORF from a previously non-coding gene locus or a chimeric ORF from a protein-coding gene.
On the other hand, exons 5 and 6 of a coding mRNA variant [GenBank:AY570742] predictably transcribed from one of the progeny loci [GenBank:CABF01023364] were skipped in a non-coding shorter variant [GenBank:FN329677] without a translatable ORF (Figure 6 and Additional file 9). We observed that the sequences of exons 5 and 6 were similar and was repeated five times in tandem within this locus, but only two copies of the tandemly duplicated potential exons were incorporated into the coding sequence of the mRNA to create exons 5 and 6 of a protein-coding ORF of 274 codons (SjCP1531). These results represent typical models of alternative splicing by intronization and exonization respectively.
Although in evolutionary perspective, intron retention that creates a translatable ORF is considered more plausible than the reverse process; our data show that both mechanisms are potentially possible. Other groups have also identified intron gains recently in mammalian and rodent retrogenes [68, 69]. The identification of non-coding mRNA variant alternatively transcribed from a single gene locus with a protein coding mRNA (Figure 6) is evidence that a novel protein-coding gene can originate from previously transcribed regions that contain the necessary transcription elements and provide RNA material for a protein translation machine [2, 39, 68]. Exon repetition has also been observed from our data to exist in this organism and could be instrumental in expanding the organism`s coding potential. The 'parallel' expression of the non-coding variant alongside the protein-coding transcripts is of significance and could suggest further that the gene may have been recently evolved. Non-coding RNAs have also been shown to perform some regulatory roles at various levels during gene expression [2, 68, 70]. This could be further explored with our data set. In the two described cases in our analyses, we have treated the non-coding isoforms as evolutionally preceding the coding variants; nevertheless, the reverse could also be the case. In addition to these two cases, we also identified a two-nucleotide insertion into a non-coding mRNA sequence [GenBank: FN330540] that yielded the coding mRNA of schistosomula protein with the similar signal peptide, with many similar transcripts in the database. However, this last observation could be an artifact from sequencing error since the existence of the non-coding transcript was not traceable to the genomic sequence.
We have passably delineated the possible mechanism that led to the identification of several protein coding genes with similar signal sequence, following lead from our work that isolated secreted proteins candidate genes using SST. A trend was described in the genome of S. japonicum whereby a 'newly evolved' gene served as a source locus for dispersed duplication events leading to the formation of several expressed genes with similar transcription core promoter region and signal sequence. We further found that the duplicated gene locus was flanked by non-long terminal repetitive elements (REs), especially of the RTE- like and Perere class. We therefore inferred that REs may have played an important role in this dispersed gene duplication by creating the requisite homologous DNA sequence that mediate a DNA-level recombination, most probably by a non-allelic homologous recombination (NAHR) mechanism. Our findings also provide evidence of logical sequential process of novel gene origination by evolution of transcription core elements followed by translatable ORF. While similar RE mediated phenomena had been observed in other organisms, unlike our dataset, most analyses have centered on the model organisms. Our data contribute to the accumulating evidence that REs mediate diverse recombination events leading to novel gene origination and other evolutionary novelties.
BLAST search
We had earlier identified a particular 81 nucleotides (27 amino acids) sequence, which was commonly utilized as signal sequence by several of our signal sequence trap (SST) isolated S. japonicum cDNAs (Table 1) [47]. The sequence of this signal sequence was employed as query to search for matches in the GenBank non-redundant nucleotide sequence database and expressed sequence tags (ESTs) database for all organisms using BLASTN program in National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) [71]. A search on the NCBI UniGene database [72] that provides information on sets of transcript sequences that appear to come from the same transcription locus was performed to ascertain redundancy and group identified transcripts. For genome-wide searches, the same query sequence and program were used to search the WGS reads from all organisms with sequenced genomes deposited in the NCBI genome databases. In a similar search in the protein database, the amino acid and nucleotide sequence of the same signal sequence was used as query for BLASTP and BLASTX searches respectively. Conserved domain architecture searches on all translation products of the SST identified candidate genes were performed using the conserved domain architecture retrieval tool on NCBI website [73] and compared with same analyses on the ProDom database of protein domain families available online at [74].
Multiple alignments
All multiple sequence alignments of DNA and protein sequences were performed in parallel with ClustalW on MegAlign program in Lasergene 7 DNASTAR software, NCBI bl2seq, COBALT multiple alignment programs, and Multialin interface software [75]. cDNA-to-genome sequence alignments were computed using the free NCBI Spling program [75]. The latest update of the S. japonicum genome map is accessible at [52]. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 5 [76].
Gene prediction
Gene predictions were performed using the GeneQuest program (Lasergene 7 DNASTAR) to predict potential coding regions, starts, stops, acceptors and donor sites using Borodovsky matrix files for Caenorhabditis elegans; and the results compared with that of the Eukaryotic GeneMark.hmm [66] gene prediction server provided freely on the website of Georgia Institute of Technology, Atlanta, USA.
Repeat masking
The whole sequences of all the genome contigs bearing the similar signal sequence were screened against a reference collection of repetitive DNA elements in the RepBase database available at the Genomic Information Research Institute website, using the CENSOR repeat masking software [77]. Sequence analysis figures were generated using real DNA sequences on Vector NTI Advanced 11.0 (Invitrogen).
Designation of putative duplication source locus and probable breakpoint
Reference to a parent gene is required for accurate determination of duplication breakpoint. However, in absence of a reference homolog, we putatively selected the most prominent contig [GenBank:CABF01020060], the longest among the identified dataset (43.7 kb), which significantly covered the length of the other contigs (Figure 3, also see Additional file 5) as the putative duplication source locus and utilized it as such for most of the analyses performed in this study. When the contigs were aligned with the putative source locus, homology was not lost till the 3` end of the aligned sequences. We therefore recruited two contigs [GenBank:CABF01020061 and GenBank:CABF01020062] downstream of the source locus based on genome assembly information, thereby generating at least 5 kb flanking sequences on either side of the duplication source locus. This sequence was then aligned with the genome contigs and scaffolds to identify the exact point where sequence identity disappeared. This point was arguably chosen as the possible duplication breakpoint and utilized as such in our discussions. We further attempted to identify a recurrent consensus sequence at the breakpoints but this was hampered by several sequencing gaps in the partially assembled scaffolds.
Parasites, genomic DNA and developmental stage mRNA samples
Chinese strain of S. japonicum (hereafter abbreviated as Sj) was obtained from Jiangsu Provincial Institute of Parasitic Diseases Wuxi, Jiangsu Province, PR China, while the Philippine and Japanese strains of S. japonicum in addition to S. mekongi (Smk) samples, were maintained in the Laboratory of Tropical Medicine and Parasitology, Dokkyo Medical University, Tochigi, Japan. S. mansoni (Sm) adult worms were maintained by, and kindly provided by the Department of Parasitology, Institute of Tropical Medicine, Nagasaki University, Japan. S. haematobium (Sh) sample was from Department of Immunology and Parasitology, University of Occupational and Environmental Health, Kitakyushu, Japan. Total genomic DNA was purified from cut tissues of mixed sex adult worms from different species of Schistosoma using QIAamp DNA Mini Kit (QIAGEN) according to the manufacturer's instructions. Qualification and quantification of genomic DNA extract was assessed by gel electrophoresis and ND-1000 spectrophotometer (NanoDrop, USA). To obtain sufficient amount of genomic DNA for southern hybridization experiments, the whole genome of each sample was amplified using the GenomePhi DNA Amplification Kit (GE Healthcare) according to the manufacturer's instructions. Equally, total RNA was extracted from parasite eggs, cercariae, 24 h cultured schistosomulae and adult worms of S. japonicum according to the instruction manual of PureLink Micro-to-midi total RNA Purification System Kit (Invitrogen).
Reverse transcription polymerase chain reaction (RT-PCR)
mRNA from eggs, cercariae, 24 h culture schistosomulae and adult worms of the Chinese strain of S. japonicum was used for RT-PCR. The first strand cDNA was synthesized from the total RNA of each developmental stage by using oligo (dT) primer according to the instruction manual of High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and the resulting cDNA was used as template for RT-PCR. The S. japonicum actin gene was used for internal quality assurance. The cDNA sequences of some selected SST identified secreted candidate genes were amplified using pairs of sequence specific primers designed according to the S. japonicum transcriptome data [49] in the NCBI public database. All RT-PCR amplicons were analyzed using gel electrophoresis and confirmed by sequencing using the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystem).
Southern hybridization
Southern hybridization was performed following standard procedures [78] using the DIG nonradioactive labeling and detection system (Roche, Germany). Briefly, the hybridization probe labeled with DIG-dUTP was synthesized using PCR DIG synthesis kit (Roche, Germany) according to the manufacturer's instructions, and labeling was confirmed by size disparity with unlabeled amplicon as a result of slower migration in agarose gel due to digoxigenin labeling. Genomic DNA from different species of Schistosoma (Sh, Sm, Smk, Sj Japanese (Yamanashi), Sj Chinese (Jiangsu) and Sj Philippines (Leyte, Mindanao and Mondoro isolates) were double digested with three different pairs of restriction enzymes (Eco RI + Eco RV, Eco RI + Hind III and Bam HI + Hind III) to achieve the best possible fragmentation of the genomic DNA. The digested genomic DNA fragments were electrophoresed through 1 % (w/v) agarose gel, depurinated in 250 mM HCl, and denatured by incubating in two changes of denaturing solution for 15 min each (0.5 M NaOH, 1.5 M NaCl). The gels were then neutralized by incubation in two changes of neutralizing solution (0.5 M Tris-HCl at pH7.5, 1.5 M NaCl) for 15 min each, and DNA was transferred to a positively charged nylon membrane (Roche, Germany) by capillary action overnight using 20x SSC solution (3 M NaCl, 300 mM sodium citrate at pH 7.0). The transferred DNA was fixed to the membrane by baking in an oven at 80 °C for 2 hours after rinsing briefly in 2x SSC. After prehybridizing the membrane in 10 ml hybridization buffer (5x SSC, 0.1 % N-lauroylsarcosine (w/v), 0.02 % SDS (w/v), 1 % blocking solution (Roche, Germany)) for 30mins in a hybridization bag, 5μl of the PCR generated hybridization probe was mixed in 50μl of double deionized water, denatured by boiling for 5mins and introduced into the hybridization bag and incubated overnight with shaking at 50 °C. The membrane was washed in two changes of low stringent wash buffer (2x SSC, 0.1 % SDS) for 5mins each at RT, and twice in high stringent wash buffers (0.5x SSC, 0.1 % SDS) for 15 min each at 65 °C. The hybridized probe was then detected using anti-Digoxigenin antibody (Roche, Germany) using CSPD as the chemiluminiscent substrate according to the manufacturer's instructions. The blot was then visualized by exposing to chemiluminiscence for 10 min in a LAS-4000 mini image reader (Fujifilm).
Author contributions
ECM participated in the conception and design of the study, in-silico analyses, molecular experiments, data analysis and interpretation and drafted the manuscript. YC carried out the signal sequence trap (SST) and participated in in-silico analyses. MK participated in the design of the study, SST, in-silico analyses, molecular experiments and data interpretation. MNS participated in in-silico analyses, molecular experiments, data interpretation and revised the manuscript. DB participated in molecular experiments and data analyses. MK2, NH, YC2 and YO maintained parasite life cycle and participated in molecular experiments. SH participated in data interpretation, supervision and revised manuscript for intellectual content. KH participated in the conception and design of the study, SST, in-silico analyses, data interpretation, revised the manuscript and general coordination. All authors approved final version of the manuscript.
We would like to thank Prof. Kenji Hirayama's lab members (Department of Immunogenetics, Institute of Tropical Medicine, Nagasaki University) for insightful discussions. ECM is a recipient of the Japanese Government Ministry of Education, Culture, Sports, Science and Technology (MEXT) PhD fellowship. This study was supported in part by the Global Center of Excellence (GCOE) Program (2008-2011); Grant-in-Aid for 21st century COE program (2003-2008), Nagasaki University; and Grant-in-Aid for Scientific Research B (22406009) and C (23590489) from the Japanese Government Ministry of Education, Culture, Sports, Science and Technology (MEXT). The funding agency played no role in conducting the study, drafting the manuscript and the decision to publish.
Supplementary material
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text:
However, some IDiots clearly should know better. They have advanced degrees in relevant fields and they have received considerable feedback on the claims they post or the books they write. We know they have read the critiques so when they persist in repeating falsehoods, there must be another explanation. They must be lying.
So what do we make of his recent post on Evolution News & Views) (sic) entitled Why All the Fuss Over Some Junk?. We are forced to conclude that Wells is dishonest. Perhaps with the caveat expressed by Peter Medawar many years ago in his review of Père Teihard's The Phenomenon of Man.
Yet the greatest part of it, I shall show, is nonsense, tricked out with a variety of metaphysical conceits, and its author can be excused of dishonesty only on the grounds that he has taken great pains to deceive himself.Read more »


text:
How to Get Better Skin by Using The Quality Skin Cosmetic
Taking the proper care of skin is a very important task for a woman. There are many ways available to get an attractive and gorgeous looking skin. At first, you always look about this thing that you are using the natural ways to get the best skin for your own. If you maintain some rules properly and follow some proper instructions, then it is not too much hard work for you. But if you didn't get your expected result, then you can use makeup items or other products to be a beautiful person. While you are applying beauty products on your skin, you must be aware about the product quality. Low quality products can be harmful to you and your skin. So it is not a wise thinking to use the bad quality products. Now I want to describe some points for you that how quality cosmetics products are valuable for you.
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Being a beautiful person is always liked by every person, everybody needs to be a perfect person in her life. So good quality products will too much helpful for a person. Try to find out the best quality beauty products and use those on your skin. But again don't compromise with the quality.


text:
Posted on 23 April 2005
- 12:00
We travel to Highbury for a crucial Barclays Premiership encounter televised live on Sky as we continue our pursuit of European football.
So while the focus might fall on whether we can hammer the final nail in Arsenal's championship coffin (anything less than victory against us and it's Chelsea's title), midfield maestro Michael is firmly focused on our goals.
"I'm sure the fans will be looking at the title situation but as players, we want to get into Europe and we've now put ourselves in a good position to do just that," said Michael.
"We've probably played it down in the last few months but now, with four games to go, we're in seventh place and we have to be realistic — if we don't get it now, we'll all be disappointed.
"We're going to Highbury to get a result for ourselves and if that means anything regarding the title, so be it. We're all determined to go there and do the business for our own targets and to give our fans the win that they want.
"We've done well away from home over the last few weeks and if we go there with the same attitude and work ethic, we capable of getting a result. We're good enough."
Anyway, Michael has plenty to think about himself — a battle with Patrick Vieira, his first derby for us at Highbury and a determination to put right the 5-4 reverse at the Lane earlier in the season.
Noureddine Naybet put us ahead in that incredible Lane clash back in November, Arsenal recovered to lead 3-1 but we wouldn't give it up as the scoring went 3-2, 4-2, 4-3, 5-3 and finally 5-4 with Ledley King, Jermain Defoe and Fredi Kanoute also on target.
"That will be one everyone will remember for quite a while," reflected Michael. "It was my first taste of the North London derby and certainly one I'll never forget. Hopefully we'll put things right on Monday."
Michael has been in fine form of late with big performances in big games — Newcastle at the Lane, Liverpool at Anfield — but it doesn't get bigger than Vieira and Arsenal at Highbury.
"You live for these games," Michael insisted. "When you are young you see games like this on television and you want to play in them and we're no different.
"I've always enjoyed playing against the best players and you want to test yourself against the best. To be honest, we always seem to do well against the top teams and it's the so-called lesser teams we tend to struggle against. This is a local derby and it's massive for everyone involved."
So is he in the form of his life? "I don't know really," he smiled. "I just try to play my game, try to learn from everyone around me, particularly the coaching staff , and hopefully I'll keep on improving. I'm enjoying my football and have done since I got here and if that's the case, you play freely and to your best. On the other hand, if you are not enjoying it, you tend not to get the best out of yourself.
"It's easy to produce a one-off performance. To get more recognition you need to do it week in, week out. You want to see consistency and we've had that in recent months."
As for Arsenal, Michael doesn't think much has changed in N5 — unfortunately for them, Chelsea have arrived.
"To be fair, they are as good as any other Arsenal team over the years but Chelsea have come on so much this season," he added. "Arsenal and Manchester United haven't exactly changed from great teams into lesser teams, it's just that Chelsea have improved so much and taken the game to another level. That is now the benchmark. For me, Arsenal are just as good as they have been — but we're all confident and looking forward to Monday."
1 July 2017
Heading out to pick up one of our new Nike kits this weekend? Here's a guide to show you where to find your nearest Spurs Shop branch...
21 June 2017
It's 50 years since defensive lynchpin Maurice Norman was forced to retire – and in this second part of our exclusive interview with one of our all-time greats, we hear the 83-year-old's memories of White Hart Lane.
24 May 2017
He might not have been there in person, but Jermain Defoe was very much with us at the Lane's finale.
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text:
Too Close To Home Trailer (1997)
29 April 1997 115 mins
A mother is so consumed with a possessive love for her son that she may have committed murder in order to keep him by her side. When the son's new wife is found murdered, the mother is arrested for the crime. In denial about his mother's guilt, the son, a successful lawyer, agrees to defend her. Based on a true story.
Watch the official Too Close To Home 1997 trailer in HD below or find more Too Close To Home videos on Vidimovie.