text:
Interlocking inflatables combine to create AirX Inflatable Theme Parks
Designers and manufacturers of
quality inflatables, trampoline parks and soft play areas.
Interlocking inflatables combine to create AirX Inflatable Theme Parks
Airspace Solutions.com presents its Atlantis modular aqua inflatable system. A unique and versatile set of sealed inflatable components that combine to offer a multitude of configurations for open water, lidos and commercial pools.
Any adventure is possible with our amazing swimming pool inflatables. Aquaruns, pool slides and sealed pool inflatables. Children can sail the seven seas, explore a ghostly ship, confront a giant crocodile, win gold at the Olympics and even become a pop star!
Inflatable play needs no introduction. If you can run, bounce or slide on it, we can design and manufacture it.
A exciting new phenomenon, but massively popular, trampoline and jump centres are heaps of fun for a wide variety of ages. We offer full consultancy, design and installation of everything from giant foam pits, to freestyle trampolining and dodge ball arenas.
A unique way to make a real impact, you can promote your offering on just about anything inflatable. Whether you want a start or finish arch, a bespoke branded inflatable, or a giant replica of your product, we'll make it happen.
Transforming an unused space into a vibrant, energetic soft play area is one of our specialities. From soft play packs to huge multi-level play structures, we design and build playrooms for the ultimate fun experience.
From the leisure to play industry, from wet to dry, from indoors to out, we design and manufacture inflatables, soft play equipment and trampoline jump arenas.
For incredible active play products – engineered in the UK, to the quality and safety standards you would expect – you've come to the right place.
Send us a question and we'll get back to you.


text:
Rebound Hospitality is dedicated to preserving
and enhancing unique properties that
serve and connect communities.
Rebound Hospitality is dedicated to preserving
and enhancing unique properties that
serve and connect communities.
Rebound Hospitality Core Values: Energy, Integrity, Community, Results Driven, Entrepreneurial and Teamwork. (We do nothing alone)
Rebound Hospitality cares deeply about the communities where we live and work. The term 'Community' is a core value and is also included in our mission statement. We are proud to be a community driven organization. Read More
Fairfield Inn & Suites Northfield – General Manager / Job Northfield, MN – June 11, 2018 – Rebound Hospitality is pleased to announce the hiring of Ken Pankow as General Manager of the Fairfield Inn & Suites Northfield – www.marriott.com/mspnf. The hotel is scheduled to open in early September of this year. The Fairfield by […]
Rebound Hospitality received the Winneshiek County Impact Award at the Annual Chamber Luncheon, held at Luther College in Decorah, Iowa on January 22nd. The award was given by Winneshiek County Development, Inc. to recognize Rebound Hospitality's major impact on the Decorah economy. "Rebound was honored and humbled to accept this award on behalf of our […]
Thanks to everyone who attended the Fairfield Inn & Suites by Marriott Grand Opening Celebration in Decorah, Iowa! More than 250 people enjoyed a tasty lunch catered by Restauration, took self-guided tours of our beautiful new hotel, and heard comments from Arne Sorenson, CEO of Marriott International. What an incredible day! We are proud to […]
You are invited to help us celebrate the Grand Opening of the beautiful new Fairfield Inn & Suites Decorah! Lunch will be served from 12-1 with a short ceremony to follow. Hope you can join us! Facebook Event Page Fairfield Inn & Suites – Decorah 2041 IA-9, Decorah, IA 52101 – 563-382-8800
Exchange Building in St. Paul sold, will become boutique hotel http://www.startribune.com/exchange-building-in-st-paul-changes-hands-will-become-boutique-hotel/449088413/ Star Tribune – By Paul Walsh October 3, 2017 — 9:07pm A century-old office building at a prime downtown St. Paul corner, with a music conservatory as its major tenant, has been sold and will become a boutique hotel, the buyer announced Monday. The […]

subset: commoncrawl


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A family history of one type of cancer increases the risk of not only the same variety but also of cancers at other sites, researchers reported.
The finding, from a large network of case-control studies, suggests there may be "cancer syndromes" among close relatives, according to Eva Negri, ScD, of the Mario Negri Institute for Pharmacological Research in Milan, and colleagues.
Those syndromes in turn might indicate that there are genetic factors that influence several cancer types, Negri and colleagues argued online in Annals of Oncology.
The findings might help researchers "focus on the identification of additional genetic causes of selected cancers and on optimizing screening and diagnosis," Negri said in a statement.
The risk of cancer is known to be increased in people who have a family history of malignancy at a so-called "concordant" site, the researchers noted, but there is little information about a family history of cancer at discordant sites.
To examine the issue, Negri and colleagues looked at participants in a network of case-control studies on 13 cancer sites, conducted between 1991 and 2009, that included 12,647 cancer patients identified at hospitals in Italy and the Canton of Vaud in Switzerland.
The studies also included 11,557 controls, who were patients admitted to the same hospitals for reasons other than malignancy.
The investigators collected data from all participants on history of any cancer in first-degree relatives, as well as information on such things as sociodemographic characteristics, anthropometric measures, lifestyle and dietary habits, and personal medical history.
For each relative with a history of cancer, the participant was asked about his or her vital status at the time of interview, current age or age at death, cancer site, and age at diagnosis.
As expected, a family history of cancer at any site was associated with an increased risk of cancer at the same site, Negri and colleagues reported.
Interestingly, for most cancer sites, the link with family history was stronger when the participant was younger than 60 when he or she was affected.
For example, Negri and colleagues reported, the odds ratio for ovarian cancer, given a family history of the disease, was 20.1 when the participant was younger than 60 and 3.2 when she was 60 or older.
But the researchers also found significantly increased risks for:
- Oral and pharyngeal cancer given a family history of laryngeal cancer, with an odds ratio of 3.3
- Esophageal cancer, given a family history of oral and pharyngeal cancer, where the odds ratio was 4.1
- Breast cancer, given a family history of colorectal cancer or hemolymphopoietic cancers, where the odds ratios were 1.5, and 1.7, respectively
- Ovarian cancer in the light of a family history of breast cancer, with an odds ratio of 2.3
- And prostate cancer, given a first-degree relative with bladder cancer, where the odds ratio was 3.4
As with concordant cancers, the risks were "somewhat stronger" for some sites when the participant was younger than 60 at diagnosis, the investigators reported.
The researchers cautioned that some of the discordant site associations they found might have been chance findings and need confirmation. On the other hand, they said they "cannot exclude" the possibility that other such associations exist but didn't emerge because of the limited statistical power of the study.
They also noted that data on family history was self-reported.
The study was supported by the Italian Association for Cancer Research, the Italian Ministry of Education, and the Swiss League Against Cancer. The journal said the authors did not report any conflicts.
- Reviewed by Robert Jasmer, MD Associate Clinical Professor of Medicine, University of California, San Francisco and Dorothy Caputo, MA, BSN, RN, Nurse Planner
- Download a complimentary source PDF.
last updated


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For my card today I have gone with Pink Christmas!
I used gorgeous sparkly silver ribbon which you can get here and pink seam binding which you can get here.
The papers are from DCWV. I added tree die cuts, silver padded stars, pink gems and lots of glitter. Oh and finished with silver pompom.
Tilda is coloured with promarkers.
I would like to enter this into
Dutch Dare - non traditional Christmas colours
PaperPlay - Christmas is coming
In The Pink - Pink Christmas
Magnolia-licious challenge - pink Christmas
The Dutch Magnolia Chickies - Christmas card
Simply Magnolia - Tis the season
Tilda's Town - celebrate Christmas
Be sure and pop by the Inspirational Blog and enter the draw.


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Asturias v. Smith et al
Plaintiff: Eduardo Asturias
Defendant: Gerold Smith, Zaner, Scott Hassell, O'Bryant and Johnson
Miscellaneous: staff attorney
Case Number: 4:2015cv00176
Filed: January 29, 2015
Court: Alabama Northern District Court
Office: Middle Office
County: Etowah
Referring Judge: Staci G Cornelius
Presiding Judge: C Lynwood Smith
Nature of Suit: Civil Rights
Cause of Action: 42:1983
Jury Demanded By: None
Available Case Documents
The following documents for this case are available for you to view or download:
|Date Filed
|Document Text
|August 17, 2016
|MEMORANDUM OPINION as more fully set out therein. Signed by Judge C Lynwood Smith, Jr on 8/17/2016. (AHI)
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Taking a cue from her always stylish mom Kate Moss, 9-year-old Lila Grace made a furry fashion statement as the pair were spotted out and about in London, England yesterday (November 26). Looks cozy!
While Lila definitely takes after her supermodel mama, it's Kate's half-sister Lottie who seems to be following in her famous footsteps. The 13-year-old recently made her modeling debut for Storm Model Management, the same agency that discovered her big sister when she was just 14.
The sisters' dad Peter Moss tells the UK's The Daily Mail that Lottie, who was a bridesmaid at Kate's wedding earlier this year, won't be rushing into a career in modeling, saying, "It's early days really. Storm, Kate's agent, felt that it was worth giving Lottie a test shoot and seeing how she turned out. She had a lovely time and she seemed to come out quite well – so everybody's happy so far…. It will be very strictly controlled because she's not 14 yet so it's very early days. "


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Hello! Each Wednesday I share some of the photos that stopped me in my tracks while I was scrolling Instagram the previous week. Here are this week's picks!
Almost like you're cooking outside! So bright and fresh!
I've never seen that tile used outdoors, but I love it!
That wallpaper. Good heavens! Her caption said it was Farrow & Ball.
Wow! Once our porch is painted, I'll be on the hunt for a swing with ropes like that!
Aaaahhhhh. Looks like such a neat place to start the day!
PS- Speaking of Instagram, I'd love to connect with you over there too. Just click the banner below to swing by!


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Vocabulary Word
Word: approbation
Definition: approval
Definition: approval
Sentences Containing 'approbation'
With those words, and a final snap of his fingers, Mr. Stryver shouldered himself into Fleet street, amidst the general approbation of his hearers.
She liked him too little to care for his approbation.
She could see him instantly before her, in every charm of air and address; but she could remember no more substantial good than the general approbation of the neighborhood, and the regard which his social powers had gained him in the mess.
Mr. Gardiner declared his willingness, and Elizabeth was applied to for her approbation.
In terms of grateful acknowledgment for the kindness of his brother, though expressed most concisely, he then delivered on paper his perfect approbation of all that was done, and his willingness to fulfil the engagements that had been made for him.
This was enough to prove that her approbation need not be doubted: and Elizabeth, rejoicing that such an effusion was heard only by herself, soon went away.
You were disgusted with the women who were always speaking, and looking, and thinking for your approbation alone.
A general murmur of approbation showed that the narrator had faithfully detailed their misfortunes and sufferings.
``Excellency,''said Pastrini,``I am delighted to have your approbation, but it was not for that I came.''
``I assure your excellency,''said he,``that at least it shall be my study to merit your approbation in all things, and I will take M. Ali as my model.''
``This communication,''continued the procureur, in that cold and decisive tone which seemed at once to preclude all discussion,``will, we are sure, meet with your approbation.''
It had been agreed that the old man should express his approbation by closing his eyes, his refusal by winking them several times, and if he had some desire or feeling to express, he raised them to heaven.
``In order to render an act valid, I must be certain of the approbation or disapprobation of my client.
said the count, the approbation of whose eye Villefort had frequently solicited during this speech.
``But one thing I can tell you, of which I do not think you are aware; that is, that I have always been opposed to this marriage, and that the contract was entered into entirely without my consent or approbation.''
A murmur of approbation ran through the assembly; and at this moment, Albert, had nothing more transpired, your father's cause had been gained.
``Nothing shall be done without the full approbation of Albert de Morcerf.
My allegation on the contrary, that it met with such approbation as to leave no doubt of our being able to raise two thousand pounds by voluntary donations, they considered as a most extravagant supposition, and utterly impossible.
The colony settled its own form of government, enacted its own laws, elected its own magistrates, and made peace or war with its neighbours, as an independent state, which had no occasion to wait for the approbation or consent of the mother city.
But if the company were bad stewards and bad sovereigns, when the whole of their neat revenue and profits belonged to themselves, and were at their own disposal, they were surely not likely to be better when three-fourths of them were to belong to other people, and the other fourth, though to be laid out for the benefit of the company, yet to be so under the inspection and with the approbation of other people.
The sovereign, though he might have some indirect influence in those elections, and though it was sometimes usual to ask both his consent to elect, and his approbation of the election, yet had no direct or sufficient means of managing the clergy.
As for them that do not, what manner of men they be at home, or abroad; day or night, how conditioned themselves with what manner of conditions, or with men of what conditions they moil and pass away the time together, he knoweth, and remembers right well, he therefore regards not such praise and approbation, as proceeding from them, who cannot like and approve themselves.
This same Knight of the Lions, who was called not long since the Knight of the Rueful Countenance, sends by me to say may it please your highness to give him leave that, with your permission, approbation, and consent, he may come and carry out his wishes, which are, as he says and I believe, to serve your exalted loftiness and beauty; and if you give it, your ladyship will do a thing which will redound to your honour, and he will receive a most distinguished favour and happiness."
For your highnesses must know that, going a few days back to kiss her hands and receive her benediction, approbation, and permission for this third sally, I found her altogether a different being from the one I sought; I found her enchanted and changed from a princess into a peasant, from fair to foul, from an angel into a devil, from fragrant to pestiferous, from refined to clownish, from a dignified lady into a jumping tomboy, and, in a word, from Dulcinea del Toboso into a coarse Sayago wench."
Though their speculations seem abstract, and even unintelligible to common readers, they aim at the approbation of the learned and the wise; and think themselves sufficiently compensated for the labour of their whole lives, if they can discover some hidden truths, which may contribute to the instruction of posterity.
Moralists have hitherto been accustomed, when they considered the vast multitude and diversity of those actions that excite our approbation or dislike, to search for some common principle, on which this variety of sentiments might depend.
It will be equally easy to prove, and from the same arguments, that _liberty_, according to that definition above mentioned, in which all men agree, is also essential to morality, and that no human actions, where it is wanting, are susceptible of any moral qualities, or can be the objects either of approbation or dislike.
The mind of man is so formed by nature that, upon the appearance of certain characters, dispositions, and actions, it immediately feels the sentiment of approbation or blame; nor are there any emotions more essential to its frame and constitution.
The characters which engage our approbation are chiefly such as contribute to the peace and security of human society; as the characters which excite blame are chiefly such as tend to public detriment and disturbance: Whence it may reasonably be presumed, that the moral sentiments arise, either mediately or immediately, from a reflection of these opposite interests.
One night when Miss Murdstone had been developing certain household plans to her brother, of which he signified his approbation, my mother suddenly began to cry, and said she thought she might have been consulted.
said my aunt, with an air of unqualified approbation; 'and never pretend to be anything else, for I know better!'
He sneaked back into his cell, amidst a little chorus of approbation; and both Traddles and I experienced a great relief when he was locked in.
'Ambition, love of approbation, sympathy, and much more, I suppose?
As my intentions are good, so have they had the good fortune to meet with approbation from the sober and substantial part of mankind; as for the vicious and vagabond, their ill-will is my ambition.
The reason why I did not publish this book till the end of the last sessions of parliament was, because I did not care to interfere with more momentous affairs; but leave it to the consideration of that august body during this recess, against the next sessions, when I shall exhibit another complaint against a growing abuse, for which I doubt not but to receive their approbation and the thanks of all honest men.
(1893-1938) was a poet, novelist and screenwriter whose poems attracted the approbation of H. L. Mencken, whose works were produced on stage and on film, and who had several screenwriting credits for work on properties where he was not the author of the original work.
Henry Vane, and Edmond Ludlow to be Commissioners for the nomination of officers to be presented to the consideration and approbation of the Parliament.
As I see it, the Court of Appeals has already completed that inquiry, and its judgment, trained on this Court's "Bakke" and "Grutter" pathmarkers, merits our approbation".
He wrote: I could not have imagined ... that this childish book be received with universal approbation by the learned world.
We have no wish to mislead; no interest to gratify by unsparing abuse or indiscriminate approbation."
More Vocab Wordsequivocal - (of words or statements) ambiguous; intentionally misleading; (of behavior) questionable; OP. unequivocal
lionize - treat (a person) as a celebrity
clapper - striker (tongue) of a bell
traverse - go through or across
chase - ornament a metal surface by indenting; follow rapidly to catch
mores - conventions; moral standards; moral customs
persevere - continue steadily in spite of difficulties
apropos - with reference to; regarding; Ex. remarks apropos (of) the present situation; ADJ. ADV.
braggart - boaster; bragger
demotic - of or pertaining to the people


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Nemesis Now 3D Rectangular Keyring featuring Anne Stokes artwork. This double sided keyring has the high quality lenticular printed artwork with a quality metal key chain and edging.
Dimensions: 5.6cm x 3.6 cm approx.
Artwork: Mystic Aura is a purple and blue mystical fairy with purple roses in her hair. Her hand is reaching forward controlling a floating crystal ball like orb. All around the Mystic Aura fairy are butterflies and roses she wears a pentagram, heart, crystal and dragon motif.
About the Artist: Fantasy artist Anne Stokes lives and works in Yorkshire, England. Her art features a wide subject range including fierce dragons, dark fairies, seductive vampires, melancholy angels and many, more.
Additional Information
|Nemesis Now


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When you look around infobarrel, are you jealous of everyone making loads of money whilst you sit on the sidelines, wondering what you did wrong?
What went wrong? NOTHING.
Infobarrel is a great website but it certainly takes a bit of practise to start earning some bucks! If you think you can be earning more than what you are now, then READ ON.
Here's what you can do to MAXIMIZE your infobarrel profits!
1) Make sure you already hooked up your google adsense!
2) Make sure you are already earning money from adsense, even if its pittens!
Have you completed the above so far? Move onto the next section.
1) How many articles have you written?
If the answer is less than 10, then you have some way to go.
If the answer is around 50, then you are well on the way.
2) If you need more articles, set yourself a goal everyday.
It's not realistic to type out 50 articles a day, it's just impossible! So instead, just write maybe 1 or 2 a day. Before you know it, you will have an army of articles!
3) Infobarrel showing your articles?
Make sure they are - are they long enough? They must be 400 words long to make it onto the index. Make sure they're being accepted and not denied too, simple rule!
4) Make relevent articles!
No one wants to read about your life or some weird way to grow corn crops, try picking something popular and hype it up. The more articles you write about a popular topic, the more chance they'll see your articles!
5) Find an obscure niche to exploit
Has no one talked about kettles and pots yet? Go for it! Just search for your own favourite little niche and write up the infobarrel article asap!
Have you done all the above, but still not earning a decent amount from infobarrel?
1) Quantity, baby!
You need perhaps 100 articles, perhaps even up to 500 articles, to start earning a good lump of money from infobarrel. Your best bet is to just keep going at it and dont give up! This is where EVERYONE usually gives up! You can beat this.
2) Quality!
If you're producing bad quality articles, then you're wasting both time and money. You should just write useful articles and publish them on infobarrel, to earn money through adsense.
3) Add friends on Infobarrel
Friends will check out your articles, help you around the website and do alot more for you then you would first imagine. Infobarrel is so far a great, fresh community to be a part of.
Keep on writing!
When you look around infobarrel, are you jealous of everyone making loads of money whilst you sit on the sidelines, wondering what you did wrong?


text:
Explore Homeschooling
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graine cannabis autofloraisonLa marijuana est mise hors-la-loi dans la majorité des pays du monde au cours du XXe siècle: la convention de l'ONU (1961) proscrivant un cannabis dans tous la majorité des pays signataires est indiscutablement une retombée du Marijuana Tax Act de 1937 aux États-Unis d'Amérique. C'est une bonn
The Best Property, Most Luxurious Home in Northern Virginia: 23072 OGLETHORPE COURT, ASHBURN, VA 20148Home of the 2017: An enchanting estate at Brambleton, within Northern Virginia. This stunning modern home makes incredible use of its surroundings, with a casement glass window showcasing wooded nature and a serene placid lake. The great room opens to an interior kitchen, foyer and deck, bringing
Deutsch Sprache Lernen…Wie wir uns seine Sprache aneignen, untersuchen unterschidliche wissenschaftliche Disziplinen zB. Die Linguistik, die Entwicklungspsychologie, die Didaktik und andere. Die Forschung unterscheidet dabei den Sprachwerb und das Sprachenlernen...Wenn wir uns bei Kindern anschauen, über welches spra
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The quantification of alloreactivity, in phrases of balance involving effector cells and Treg, could enable the identification of patients at threat for acute rejectionAt the moment, one-12 months graft survival prices right after renal transplantation are exceeding 90%. Nevertheless, the existence-very long administration of immunosuppressive drugs is accompanied by numerous facet effects. Next to elevated threat of infections and malignancies, the use of the
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Even so, mitophagy was not just an artifact of overexpression of mCherryParkin, as mitochondrial material for every mobile also diminished in nontransfected cells subjected to simulated ischemiaTo determine if Parkin is critical in vivo for IPC-induced p62 recruitment to the mitochondria, we assessed p62 translocation after IPC in wild kind and Parkin knockout mice. To figure out the effects of simulated ischemia on mitochondrial decline, mitochondrial information was as opposed in HL-o
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The Most Important Thing Missing From Google's Announcements
Yesterday, during Google's opening keynote at its I/O developer conference, it seemed that the search giant unveiled everything under the sun. We're talking about a new cross-platform social gaming service, a unified cloud messaging platform, a Samsung Galaxy S4 running stock Android, a new streaming music service, a revamped Google Maps interface, a push into education, and much more.
However, there was one incredibly important announcement that some investors were expecting, but was notably absent as the keynote ended. There was no new Nexus 7.
Just being honest
Rumors have been leaking for months that Google was preparing to launch a second-generation of its flagship 7-inch tablet to follow up the success of the first-generation model. The popularity of that device has helped propel manufacturer ASUS to the No. 3 spot in the tablet market, behind Apple and Samsung but overtaking Amazon.com and its Kindle Fire.
Q1 2013 Units
Q1 2013 Market Share
19.5 million
8.8 million
2.7 million
1.8 million
Source: IDC.
Not all of those 2.7 million units were Nexus 7 devices, but it's safe to say that the vast majority of them are. Before the Nexus 7, ASUS was having trouble topping 1 million in quarterly tablet units. Now it has no problem shipping well over 2 million per quarter.
The Nexus 7 is approaching its first birthday, which means it's about time for a product cycle refresh. That's particularly important considering that Apple just recently got into the small-sized tablet segment with the iPad Mini, and Amazon is likely preparing to launch third-generation Kindle Fires in September if the e-tailer sticks to the same 1-year cycle. Apple is priced at a premium $329 and putting on pressure from above, while Amazon will probably hold its ground at $199.
It appears that Chrome and Android chief Sundar Pichai wasn't just playing coy when he shot down the idea of major product launches this week -- he was just being honest. The Nexus 7 is beginning to age. Google needs to refresh the small tablet within the next couple of months to maintain momentum in that market.
It's more important than ever to understand each piece of Google's sprawling empire. In The Motley Fool's new premium research report on Google, we break down the risks and potential rewards for Google investors. Simply click here now to unlock your copy of this invaluable resource.
The article The Most Important Thing Missing From Google's Announcements originally appeared on Fool.com.Fool contributor Evan Niu, CFA, owns shares of Apple. The Motley Fool recommends Amazon.com, Apple, and Google. The Motley Fool owns shares of Amazon.com, Apple, and Google. Try any of our Foolish newsletter services free for 30 days. We Fools may not all hold the same opinions, but we all believe that considering a diverse range of insights makes us better investors. The Motley Fool has a disclosure policy.
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text:
> Right click on image of choice
> Left click on > save as
> clear box and name as you wish.
> click on > save
> It will now be saved on to your Hard Drive
You will now need to Host the image at a Photo Hosting Site.
I use and recommend Photobucket. It is a great photo hosting site, plus registration is free. Unless of course, you choose the premium service.
> Go to Photobucket
> Sign up > log-in
> Click on the > browse button. Find your image and then click > submit.
> You will then see your pic uploaded
> Three links will show under the image itself.
> Sample Album
> For a small to medium size image. Copy the IMG link.
> IMG Example:
> For a large image. Copy the URL
> This saves on bandwidth. Pages will also load much faster for those on dial-up.
> URL Example: http://img.photobucket.com/albums/v317/sca...18/IMG00057.jpg
> Paste the link into your post
> Note: Actually for images that are quite large, as in desktop screenshots. I use the photo hosting site ImageShack. Which is a free service as well. Since it allows a maximum image size of 1024 kb. Photobucket has a maximum allowance of only 250 kb. They will be re-sized if they are larger. And the image will not be as crisp.
Click on Register in the top right hand corner.
Check your e-mail for your activation link
Click on > Browse
Then click on > Host It
Then click on the link > My Images
You will now see that your image has been uploaded.
> copy eiether the Thumbnail Code for forums. The thumbnail code will create a clickable thumbnail.
Example: Clickable Thumbnail
> Or the Direct code. Just under the thumbnail code.
Example: Direct http://img340.imageshack.us/img340/9320/thimg000573fi.jpg
Now if you wish to insert a signature or a small image, that will show in all of your posts. You must do this.
> Copy the IMG link
> Go to your Control Panel @ Bleeping Computer
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Edited by Scarlett, 04 November 2005 - 10:31 AM.


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So work-wise I am still reasonably on top of everything (well it is only 1 week into the year). I still need catch up on the emails a bit, order more supplies for the shop, and I have a looming deadline for the Sewing World Magazine bag, but otherwise most things are in order and under control, that is until this came though the letter box for me - DAMN!
This book is going to save my short ass. We crafters have access to so much yummy fabric, but short folks do not have access to yummy skirts. Ohhhhh if I start thinking about whipping up some skirts now I just might have some to wear for spring/summer.
Some of the skirts you can make with this book.
The book doesn't have patterns, instead it teaches how to make your own - no wait, come back! The instructions are clearly written and they shouldn't freak a beginner out which is great because I'm not too hot at dressmaking. The first part of the book teaches you how to measure yourself, how to transform those measurements into types of skirt, and it teaches you stuff like how to do hems, zips, and darts etc.
How to attach a waistband.
I think I'll try this one first...
Here are the instructions for the skirt above. It looks reasonably achievable and only moderately swear-worthy...I'll let you know how I get on.
Have any of you bought this skirt book yet? Which skirt/s did you make?
I'll announce the Amy Butler Pattern monthly sewing contest winners in my next post.


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This house, located in one of the best residential areas of Badalona, enjoys a peaceful setting and it is too well connected to Barcelona.
The gentle slope of the land has conditioned the layout of the house and outdoor areas with a perfect distribution in different plants averages.
We find a first level of access with garage, hallway, a bathroom and a bedroom that provide access to the rest of the home.
Walk down a small flight of stairs we have the great living area that opens to a generous outdoor area with access from both: the living room and the kitchen. This corner invites you to enjoy all year the mild Mediterranean climate. A barbecue complements this fabulous highlight of the house.
At the upper level is the night area, with 4 bedrooms and a bathroom.
Complete this property a convenient and spacious sun terrace overlooking the wonderful environment in which the home is located.
The large garden is also organized in terraced levels. We have a large pool and ample space with some trees, ideal for recreation of children.
10 minutes from Barcelona, very well connected to the B-20 highway and in a quiet setting overlooking the mountains and the city of Barcelona, we find this ideal for first or second residence residential enclave.
The abundance of pine trees and typical Mediterranean vegetation and proximity to Serralada Marina, make this area an ideal place for nature lovers who do not want to renounce the convenience of proximity to the city.
Availability upon agreement. This material is based upon information, which we consider reliable, but because third parties have supplied it, we cannot represent that it is accurate or complete, and it should not be relied upon as such. This offering is subject to errors, omissions, and change of price or withdrawal without notice. Taxes, notary and register fees must be paid by purchaser, the commission by the seller as stipulated by Spanish regulations.


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Hearts Crossing Ranch Series, Book 3
June 2011
White Rose Publishing
E Book
$ 3.99
155 pages
Inspirational Romance
Rating: 5 Cups
Hooper Martin is busy helping with his brother's wedding. As someone who has been undergoing cancer treatments, he hopes he will be around for plenty more weddings and able to care for his young daughter, Ella.
Malia Cameron, Mallie, is invited to the wedding by her brother, one of the groomsmen. She is a cancer survivor, even though she knows at any time things could be reversed for her. One glimpse of Hooper and she is utterly smitten.
Hooper was getting ready for his brother's wedding when the guests began arriving. He had no idea after meeting Mallie things would change in his life. He was certain no one would ever want him, especially since his wife left him even when he was healthy. She did not even wish to be around their daughter. Then Mallie enters his life, a woman who loves the cowboy life and is a cancer survivor. She is bubbling inside when she meets Hooper and very glad she is able to attend the wedding. Mallie and Ella even make a connection. Has Hooper met the love of his life who can share the best of days with him and Ella, or is this just a dream?
I have to admit Sanctuary was a spellbinding story. It captivated this reader completely. Just when I thought it could not get any better, Ms. Hanson throws in a curve with the ex-wife. This remarkable read was extremely hard to put down. The refreshing characters of Mallie and Hooper really connect with the reader. They indeed are unforgettable, as well as the secondary players in this marvelous story. It felt like being surrounded by a huge loving family. This extraordinary read is one that I truly recommend to everyone. Tanya Hanson gives the reader promise in her characters.
Reviewer for Coffee Time Romance & More
Awards | Author Services | Contact Information | New Author Information


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There has been much talk on the intertubes recently about the "death" of WPF, or the possible subsumption of WPF by the Silverlight platform. As well as the possibility that the Windows team at Microsoft are not buying into XAML and WPF/Silverlight, or more fundamentally, the whole Managed code revolution. Apparently, the Windows team—from what I read—would rather take a bet on compiled JavaScript and HTML5, which is surprisingly similar to Silverlight in goals but not in technology or implementation.
While some of this talk may be alarmist or just rumour, for which people are talking solace in posts like this, and tweets like this. Anyone who has worked in the technology industry for any period of time will have to admit to this immutable truth: Everything changes. All the time.
Today's WPF was yesterday's WinForms, today's ASP.NET MVC was yesterday's WebForms, and so on. There have been dozens of UI technologies developed over the years for the Windows platform, both managed and unmanaged alike, but here's the fundamental point: technology does not die, it's abandoned, it cannot be killed (just look at IE6 and I can still download Visual Basic 3.0 from MSDN today!)—what this means is that actually YOU have a choice, YOU decide when a technology is dead to YOU; not the Windows team, not anyone else, but simply YOU. The decision is all yours.
Here's just a few UI technologies that I've worked with over the last 15+ years:
- Access (v1.0 and v2.0)
- Excel (v4.0 macros and v5.0 VBA)
- Word (v2.0 Macros and v5.0 VBA)
- Visual Basic (3, 4, 5 and 6)
- IDC and HTX Web Templates
- HTML/DHTML/CSS and JavaScript (from the late 90's to today)
- Classic ASP (from V1 until it was replaced with Managed Code and ASP.NET in 2002)
- ASP.NET (1.0, 2.0 and 4.0)
- ASP.NET MVC (1.0 and now 2.0)
- Silverlight (1, 2 and now 4)
- Windows Forms (all versions)
- WPF (All versions)
Interestingly, you can use just about any one of these technologies, in one form or another, still today to build your applications for the Windows platform.
I think of it this way: Even if Microsoft announced that WPF will not be actively developed from today—this very day, even though there are a reported 200+ developers working on Silverlight and WPF at this very moment—we'll still have five years of patches and maintenance. Five years of you being able to choose to use it in a production environment, even then the technology won't be "dead". You may simply choose to abandon it long before then, but that does not mean it's dead, far from it.
So to hear that WPF is "dead" or that I'm going to have to brush up on my JavaScript and learn HTML5 in the near future it's like: "Well, like duh! Dude. News just in: water is wet…". Things change, deal with it. Learning something new comes with the territory; if the Windows team want to abandon managed code, WPF and Silverlight, for whatever reason (and remember, this might not even be true, this is what's being spouted on the intertubes) then so be it. But *I* don't have to, I get to choose, and today I choose WPF and Silverlight and probably will for at least the next 5 years… unless something more shiny comes along ;)
Think deeply, code well. If you have any comments or opinions that you'd like to share on this topic or any other, I'd love to hear from your. [-PJ]


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Senior Management Appointments: President Stresses Competence, Youth and Diversity
Tunis, 1 March 2005 – The President of the African Development Bank (AfDB) Group, Mr. Donald Kaberuka, has announced the appointments of four new Vice Presidents and the Secretary General of the institution. The appointments came at the end of a competitive, global search process that involved some 500 candidates from around the world.
The new appointees are: Mr. Joseph B. Eichenberger as Vice President, Planning Policy and Research (PRVP); Mrs. Zeinab Bashir El Bakri as Vice President, Operations, West and Central Africa Region (OCVP); Mr. Mandla Sizwe Vulindlela Gantsho as Vice President, Operations, North, East and South Africa Region (ONVP); Ms. Arunma Oteh as Vice President, Corporate Management (CMVP); and Mr. Modibo Toure as Secretary General. Mr. Thierry de Longuemar, stays on in his position as Vice President for Finance.
In announcing the appointments, the President of the Bank Group pointed to qualities of competence, youth and to the geographical and gender diversity of the new management team. All of the appointees are under 50 and include, for the first time in the history of the ADB, two women as Vice Presidents.
Mr. Kaberuka also noted that the men and women he had selected, after recommendations from an eminent interview panel of highly respected international personalities, came with impeccable academic qualifications and had held high-level positions in global and regional institutions such as the, the Asian Development Bank, the Development Bank of Southern Africa, the United Nations and the AfDB itself.
The team will join the President in leading implementation of a reform agenda that seeks to transform the Bank into a more effective development institution that is also a centre of knowledge on African development issues.
The New Leadership Team
- Mr. Eichenberger, 48, comes from the Asian Development Bank where he served as Senior Vice President and Deputy CEO;
- Mrs, El Bakri, 49, currently Director for Social Development, was Acting Vice President in the AfDB in 2005;
- Mr. Gantsho, 43, is Managing Director of the Development Bank of Southern Africa ;
- Ms. Arunma Oteh, 41, has been AfDB Director of Treasury since 2001;
- Mr. Modibo Toure, 47, is a member of the UNDP Transition Team. He was UNDP Resident and Humanitarian Coordinator in Ethiopia from 2004 to 2005.


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Witnesses told Israel's Yedioth Internet Web site the first explosion triggered several other blasts in nearby vehicles that were also carrying rockets and ammunition.
Estimates of the number of dead ranged from seven to 19.
Hamas blamed an Israeli helicopter, but the Israeli military denied involvement and the Palestinian Interior Ministry said the blast was set off by the mishandling of explosives.
The rally was held in the Jebaliya refugee camp, one of the last military-style parades before a ban on flaunting weapons in public — agreed to by all militant groups — takes effect Saturday evening.
Witnesses said participants, including children, crowded around the pickup truck just before the explosion. The witnesses said the truck carried two homemade rockets.
One man, who only gave his first name, Hussam, said he helped pull three men out of the pickup, two of them dead and one still alive. The side of the pickup was charred.
The witness said he saw five dead children nearby. Dozens more were wounded. The Hamas military wing, Izzedine al Qassam, is popular with youngsters and when the pickup with the gunmen arrived at the rally, many crowded around the vehicle.
After the blast, men carried bodies wrapped in blankets and body parts to nearby cars.
At Shifa Hospital in Gaza, doctors treated patients on the emergency room floor because they ran out of beds. Masked Hamas men wheeled in casualties, including children.
The truck was not heavily damaged by the blast.
One witness, Hazem Abu Rashad, 18, said the truck had two rockets in its bed. Three militants rode in the back and at least three more were inside, he said.
"There was smoke all over, and then we saw people in pieces, but we couldn't make out what really happened," he said.
Also Friday, Palestinians temporarily opened the crossing between Gaza and Egypt, taking control of a border for the first time in their history. Palestinians hoped the two-day opening would set a precedent and pressure Israel to reach a permanent border agreement with them.
It was the first time Palestinians crossed the Gaza border without Israeli supervision in 38 years, reports CBS News Correspondent Robert Berger. Israel still wants supervision of the border, fearing that weapons and terrorists could be smuggled across the border. Israeli and Palestinian negotiators are seeking a compromise.
Earlier, Israeli forces killed three Palestinian gunmen in a West Bank raid.


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BioMed Research International
Volume 2013, Article ID 697051, 13 pages
In Silico Screening and Molecular Dynamics Simulation of Disease-Associated nsSNP in TYRP1 Gene and Its Structural Consequences in OCA3
1School of Bio Sciences and Technology (SBST), Bioinformatics Division, Vellore Institute of Technology University, Vellore, Tamil Nadu 632014, India
2Human Genetics Foundation, Torino, Via Nizza 52, 10126 Torino, Italy
Received 20 April 2013; Revised 23 May 2013; Accepted 23 May 2013
Academic Editor: Claudio M. Soares
Copyright © 2013 Balu Kamaraj and Rituraj Purohit. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Oculocutaneous albinism type III (OCA3), caused by mutations of TYRP1 gene, is an autosomal recessive disorder characterized by reduced biosynthesis of melanin pigment in the hair, skin, and eyes. The TYRP1 gene encodes a protein called tyrosinase-related protein-1 (Tyrp1). Tyrp1 is involved in maintaining the stability of tyrosinase protein and modulating its catalytic activity in eumelanin synthesis. Tyrp1 is also involved in maintenance of melanosome structure and affects melanocyte proliferation and cell death. In this work we implemented computational analysis to filter the most probable mutation that might be associated with OCA3. We found R326H and R356Q as most deleterious and disease associated by using PolyPhen 2.0, SIFT, PANTHER, I-mutant 3.0, PhD-SNP, SNP&GO, Pmut, and Mutpred tools. To understand the atomic arrangement in 3D space, the native and mutant (R326H and R356Q) structures were modelled. Finally the structural analyses of native and mutant Tyrp1 proteins were investigated using molecular dynamics simulation (MDS) approach. MDS results showed more flexibility in native Tyrp1 structure. Due to mutation in Tyrp1 protein, it became more rigid and might disturb the structural conformation and catalytic function of the structure and might also play a significant role in inducing OCA3. The results obtained from this study would facilitate wet-lab researches to develop a potent drug therapies against OCA3.
1. Introduction
Oculocutaneous albinism type 3 (OCA3) is an autosomal recessive disorder characterized by reduced biosynthesis of melanin pigment in the hair, skin, and eyes [MIM 203290]. This disorder is mostly caused by the genetic mutation in TYRP1 gene. OCA3 is also known as Rufous oculocutaneous albinism. The human TYRP1 gene consists of 8 exons and 7 introns, spanning almost 15–18 kb of genomic DNA in the region of 9p23 [1–4]. This gene encodes a protein called Tyrosinase-related protein 1 (Tyrp1), has a molecular weight of ~75 kDa, and appears to be the most abundant melanosomal protein of the melanocyte [5, 6]. Tyrp1 is comprising of 537 amino acid residues and shares 40–52% of amino acid homology to tyrosinase. The tyrosinase-related family includes tyrosinase, tyrosinase-related protein 1 (Tyrp1), and tyrosinase-related protein 2 (Tyrp2) involved in this enzymatic process that converts tyrosine to melanin pigments. Certainly, two types of melanin are produced by melanocytes, which are pheomelanins (red or yellow) and eumelanins (brown or black) [7]. The first two steps of both eumelanin and pheomelanin production involve tyrosinase catalysing the conversion of tyrosine to 3,4-dihydroxy-L-phenylalanine (DOPA) and of DOPA to DOPA quinone [8, 9]. Then pheomelanogenesis seems to be the default pathway in the absence of MC1R signalling, with a low tyrosinase activity and a high concentration of thiolic compounds, such as cysteine. In another way, eumelanin synthesis requires α-MSH binding to MC1R [10, 11], which transcriptionally activates tyrosinase and upregulates Tyrp1 and Tyrp2 [12–14]. In addition to their roles in pigmentation, tyrosinase family proteins also influence the biology of melanocyte and melanoma. There is evidence that Tyrp1 is involved in the maintenance of melanosome structure and affects melanocyte proliferation and cell death [15–18].
Based on its homology to tyrosinase, Tyrp1 has also been speculated to be another tyrosinase or reveal the tyrosinase-like activity. Tyrp1 shows tyrosine hydroxylase activity, albeit under low substrate (L-tyrosine) concentration, but no DOPA oxidase activity [19, 20]. Based on that human Tyrp1 is involved in conversion of L-tyrosine to DOPA with low turnover rates, sufficient to prime the system by the generation of low amounts of DOPA, a necessary co-factor for tyrosinase activity [21]. Tyrp1 has also been attributed with various other catalytic functions including dopachrome tautomerase (Dct), dihydroxyindole (DHI) oxidase [22] and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) [23]. Mutagenesis studies have recently confirmed that Tyrp1 is actively involved in inactivation of the catalytic activity of tyrosinase [24]. Observing the more number of pathological genetic variants and their structural and functional aspects of OCA3 will aid in development of personalized medicine.
Several computational algorithms used for the accurate prediction of OCA3 uncharacterized alleles for their disease related property. Mutations involved in OCA3 disorder are hard to scrutinize using in vivo examinations. Hence, an efficient experimental design specific to these diseases are mandatory to observe the disease associated mutation of respective SNPs. Several research articles have stated is effectiveness in identifying the deleterious and disease-associated mutations, thus predicting the pathogenic nsSNPs in correlation to their functional and structural damaging properties [25–28]. Computational studies have previously provided an efficient platform for evaluation and analysis of genetic mutations for their pathological consequences and in determining their underlying molecular mechanism [27–33]. Moreover the conformational changes in the 3D structure of the protein account for the changes in its time dependent physiological affinities and various biochemical pathway alterations [34–37]. Here we used set of computational platforms that utilizes sequence-based conservation profile, homology-based structure profile information, and support vector algorithm used to examine the disease associated nsSNPs. In this study we have applied a set of tools like PolyPhen 2.0 [38], SIFT [39], I-mutant 3.0 [40], PANTHER [41], PhD-SNP [42], SNP&GO [43], Pmut [44], and MutPred [45] to show greater accuracy for the prediction of most disease-associated mutations in OCA3 gene and their structural consequence. Further, we carried out molecular dynamic simulations (MDS) to analyse the molecular and structural basis of predicted disease associated nsSNPs. MDS were applied to observe the motion trajectory and atomic interaction of native and mutant (R326H and R356Q) Tyrp1 protein. The overall strategy implemented in this work is shown in Figure 1.
2. Materials and Methods
2.1. Dataset
The data on human TYRP1 genes were collected from OMIM [46] and Entrez gene on National Center for Biotechnology Information (NCBI) Website. The SNP information of TYRP1 gene was obtained from dbSNP (http://www.ncbi.nlm.nih.gov/snp/) [47] and Swissprot databases [48–50]. The amino acid sequence of Tyrp1 protein was retrieved from the Uniprot database (Uniprot ID: P17643). In order to build the mutant structures, we induced the point mutations in the position of 326 and 356 of Tyrp1 protein using SPDB viewer package [51]. These structures were energetically optimized by applying the all atom OPLS force field available in GROMACS package 4.5.3 [52].
2.2. Disease Related SNP Prediction
The single nucleotide polymorphism occurring in the protein coding region may lead to the deleterious consequences and might affect its 3D structure. Here we applied PolyPhen 2.0 [38], SIFT [39], I-Mutant 3.0 [40], PANTHER [41], PhD-SNP [42], SNP&GO [43], Pmut [44], and MutPred [45] tools in order to examine the disease-associated nsSNP occurring in the Tyrp1 protein coding region. PolyPhen 2.0 is based on combination of sequence and structure based attributes and uses naive Bayesian classifier for the identification of an amino acid substitution and the impact of mutation. The output levels of probably damaging and possibly damaging were classified as functionally significant (≤0.5) and the benign level being classified as tolerated (≥0.51) [38]. SIFT prediction is based on the sequence homology and the physicochemical properties of amino acids which are dictated by the substituted amino acid. SIFT score ≥0.05 indicates the amino acid substitution is intolerant or deleterious, whereas the score ≤0.05 predicted it as tolerant [39]. I-Mutant 3.0 is a support vector machine (SVM) based tool. We used the sequence based version of I-Mutant 3.0 that classifies the prediction into three classes: neutral mutation (−0.5 ≤ DDG ≥ 0.5 kcal/moL), large decrease (<−0.5 kcal/moL), and a large increase (>0.5 kcal/moL). The free energy change (DDG) predicted by I-Mutant 3.0 is based on the difference between unfolding Gibbs free energy change of mutant and native protein (kcal/moL) [40]. PANTHER program is a protein family and subfamily database which predicts the frequency of occurrence of amino acid at a particular position in evolutionary related protein sequences. The threshold subPSEC score of −3 has been assigned below which the predictions are considered as deleterious [41]. We filtered the nsSNPs that were combinedly predicted to be deleterious and damaging from these four servers. Further we used PhD-SNP, SNP&GO, Pmut, and MutPred tools to examine the disease-associated nsSNPs. PhD-SNP is SVM based classifier, trained over the million amino acid polymorphism datasets using supervised training algorithm [42]. It predicts whether the given amino acid substitution leads to disease associated or neutral along with the reliability index score [42]. SNP&GO retrieves data from protein sequence, evolutionary information, and functions as encoded in the gene ontology terms [43]. Pmut is a neural network based program which is trained on large database of neutral and pathological mutations [44]. Pmut uses 3 parameters including mutation descriptors, solvent accessibility, and residue and sequence properties to calculate the pathogenicity indexes of given input mutation data ranging from 0 to 1. The mutations with index score greater than 0.5 are predicted to be pathologically significant [44]. MutPred is a web based tool, used to predict the molecular changes associated with amino acid variants [45]. It uses SIFT, PSI-BLAST, and Pfam profiles along with some structural disorder prediction algorithms, including TMHMM, MARCOIL, I-Mutant 2.0, B-factor prediction, and DisProt [45]. Functional analysis includes the prediction of DNA-binding site, catalytic domains, calmodulin-binding targets, and posttranslational modification sites [45]. Combining the scores of all four servers, the accuracy of prediction rises to a greater extent and finally we filtered the most disease-associated mutation.
2.3. Modelling of Native and Mutant TYRP1 Proteins
According to the annotated information available in UNIPROT entry-P17643, the predicted deleterious mutation sites of Tyrp1 protein were observed in the topological domain which comprised between the regions 190–385. Hence, we modeled Tyrp1 protein segment which consists of 196 amino acid residues by I-TASSER server [53]. This program works by combining the folds and secondary structure by profile-profile alignment threading techniques for non-aligned regions. For the submitted sequences, five 3D models were obtained and the best model was selected based on the lowest energy. Further the native structure was mutated with the most deleterious substitution predicted in this study. In order to build the mutant structures, we made a point mutation in native Tyrp1 protein at R326H (arginine to histidine) and R356Q (arginine to glutamine) using SPDB viewer [51]. The native and mutant structures were energetically optimized by applying the all atom OPLS force field available under the GROMACS 4.5.3 package [52]. The quality of model structures was verified using the PROCHECK [54] and PROSA [55] programs.
2.4. Molecular Dynamics Simulation
Molecular dynamics simulation was performed by using gromacs 4.5.3 package [52] running on a single Intel Core2Duo machine with 3 GB RAM and running Ubuntu 11.10 Linux package. Structure of native and mutant Tyrp1 protein was used as starting point for MD simulations. Systems were solvated in a cubic box with simple point charge (SPC) water molecules at 10 Å marginal radius. At physiological pH the structures were found to be negatively charged; thus in order to make the simulation system electrically neutral, we added 10 sodium ions (Na+) to the simulation box using the "genion" tool that accompanies with gromacs package. Initially the solvent molecules were relaxed while all the solute atoms were harmonically restrained to their original positions with a force constant of 100 kcal/moL for 5000 steps. After this, whole molecular system was subjected to energy minimization for 5000 iterations by steepest descent algorithm implementing GROMOS96 43a1 force field. Berendsen temperature coupling method [56] was used to regulate the temperature inside the box. Electrostatic interactions were computed using the Particle Mesh Ewald method [57]. The ionization states of the residues were set appropriate to pH 7 with all histidines assumed neutral. The pressure was maintained at 1 atm with the allowed compressibility range of atm. SHAKE algorithm was used to constrain bond lengths involving hydrogen, permitting a time step of 2 fs. Van der Waals and coulomb interactions were truncated at 1.0 nm. The nonbonded pair list was updated every 10 steps and conformations were stored every 0.5 ps. Position restraint simulation for 500 ps was implemented to allow solvent molecules to enter the cavity region of structure. Finally, systems were subjected to MD simulation for 20 ns. We then computed the comparative analysis of structural deviations in native and mutant structure. RMSD, RMSF, SASA, Rg, DSSP, and density plot analysis were carried out by using g_rms, g_rmsf, g_sas, g_gyrate, do_dssp, and g_density tool, respectively. Number of distinct hydrogen bonds formed by specific residues to other amino acids within the protein during the simulation (NH bond) was calculated using g_hbond. NH bond determined on the basis of donor-acceptor distance smaller than 0.35 nm and of donor-hydrogen-acceptor. All the graphs were plotted using XMGRACE [58] program.
2.5. Principal Component Analysis
The calculation of the eigenvectors and eigenvalues, and their projection along the first two principal components, was carried out using essential dynamics (ED) method according to protocol [59] within the GROMACS software package. The principle component analysis or ED is a technique that reduces the complexity of the data and extracts the concerted motion in simulations that are essentially correlated and presumably meaningful for biological function [59]. In the ED analysis, a variance/covariance matrix was constructed from the trajectories after removal of the rotational and translational movements. A set of eigenvectors and eigenvalues was identified by diagonalizing the matrix. The eigenvalues represents the amplitude of the eigenvector along the multidimensional space, and the displacement of atoms along each eigenvector shows the concerted motions of protein along each direction. The movements of structures in the essential subspace were identified by projecting the Cartesian trajectory coordinates along the most important eigenvectors from the analysis. Backbone C-alpha bonds trajectories were obtained using g_covar and g_anaeig of gromacs utilities.
3. Results and Discussion
To determine the deleterious nonsynonymous single nucleotide polymorphisms (nsSNPs), which might be involved in inducing disease associated phenomena, is now among the most important field of computational genomic research. The disease associated mutations can be identified with the help of genome sequencing and its analysis. The advanced method in computational biology has now enabled us to determine the deleterious nsSNPs in the target candidate genes. Computational methods were applied to study the protein structural and functional effect on point mutation at molecular level. In this investigation we implemented multiple computational methods to identify the most likely pathogenic mutations in TYRP1 gene. Our results also revealed that implementations of different algorithms often serve as powerful tools for prioritizing candidate functional nsSNPs. Here we used SIFT, PolyPhen, I-Mutant 3.0, PANTHER, PhD-SNP, SNP&GO, Pmut, and MutPred tools to examine the most deleterious and disease associated nsSNPs from the SNP dataset. The combination of methods based on evolutionary information and protein structure and/or functional parameters were used in order to increase the prediction accuracy.
3.1. Screening of Deleterious nsSNPs by PolyPhen 2.0, SIFT, I-Mutant 3.0, and PANTHER Program
Out of 63 input polymorphic dataset, 42 nsSNPs were found to be "damaging" (0.5 to 1.000) to protein structure and function and the remaining 21 nsSNPs were characterized as benign by PolyPhen 2.0. Among these 42 deleterious nsSNPs, 15 SNPs G63S, W249G, C303G, Y522C, G300E, A380S, V189L, R153C, N132I, N435H, R374G, D343V, T262M, R326H, and R356Q were reported to be highly deleterious with PolyPhen score of 1.000 (Table 1). In SIFT, 34 mutations (G63S, M266T, R146W, W249G, A486T, R73W, T366M, C303G, Y522C, A380S, S305R, G309E, V189L, A409V, L7P, D123V, A31G, N132I, F383L, N96Y, N435H, R114C, G309R, R471W, D343V, G174L, A24T, T262M, R93H, R505C, V319G, R326H, R93C, and R356Q) were predicted to be deleterious with tolerance index ≥0.05 (Table 1). Among these, 17 mutations G63S, M266T, W249G, C303G, G309E, V189L, N132I, N96Y, N435H, R114C, G309R, R471W, D343V, G174L, T262M, R326H, and R356Q were reported to be highly deleterious with SIFT score of 0.00 (Table 1). Furthermore, 29 mutations were identified as deleterious and damaging in SIFT and PolyPhen 2.0 server (Table 1) which also shows a strong correlation between the prediction methodologies implemented by these two servers. SIFT and PolyPhen were shown to have better performance in identifying functional nsSNPs among other in silico tools [60]. The accuracy of SIFT and PolyPhen was further validated through our results, which makes these tools more suitable for the prediction [61]. All the nsSNPs submitted to PolyPhen 2.0 and SIFT were also submitted as input to the I-Mutant 3.0 server. 45 mutations were predicted to affect the stability of the protein structure by I-Mutant 3.0. Remaining out of 18 mutations, 16 mutations showed the neutral effect on protein structure and 2 mutations showed increased stability of the structure. To further validate these results we implemented HMM based statistical prediction method to identify the functionally significant point mutations using PANTHER server. The mutations with subPSEC score less than −3 have been reported to be probably deleterious. 39 mutations with subPSEC score less than or equal to −3 were characterized to be deleterious. We filtered 19 mutations (M266T, R146W, W249G, C303G, Y522C, G309E, V189L, A409V, F383L, N435H, G309R, D343V, G174L, T262M, R93H, V319G, R326H, R93C, and R356Q) which were commonly predicted to be deleterious and damaging by SIFT, PolyPhen 2.0, I-Mutant 3.0, and PANTHER servers (Table 1).
3.2. Prediction of Disease-Associated nsSNPs
We applied PhD-SNP which is based on support vector machine tool to further classify the predicted deleterious nsSNPs as disease associated. Total 19 nsSNPs which were commonly predicted in SIFT, PolyPhen 2.0, I-Mutant 3.0, and PANTHER tools were further used in PhD-SNP server. Out of 19 mutations, 16 of them (R146W, C303G, G309E, V189L, A409V, F383L, N435H, G309R, D343V, G174L, T262M, R93H, V319G, R326H, R93C, and R356Q) were predicted to be disease associated (Table 2). In SNP&GO, 19 nsSNPs were predicted to be disease associated. To verify this prediction, we further employed artificial neural network (ANN) based Pmut tool. Out of 19 nsSNPs, 10 mutations showed pathogenecity and remaining 9 nsSNP showed as neutral (Table 2). Particularly, R326H showed higher pathogenecity level with pathogenicity index of 0.9314 (Table 2). 8 mutations (R146W, G309E, G309R, D343V, T262M, R93H, R326H, and R356Q) were predicted as most disease associated by PhD-SNP, SNP&GO, and Pmut servers. These 8 mutations were further analysed by MutPred tool to predict the SNP disease-association probability and probable change in the molecular mechanism in the mutant. We found R326H to be highly deleterious with general probability () scores of 0.938 and was predicted to induce the loss of stability with () score of 0.0202, showing confident hypothesis. R356Q was found to be highly deleterious with general probability () scores of 0.801 and was predicted to induce the loss of catalytic residue at R356 with () score of 0.0446, showing confident hypothesis. At the end of so many mutations considered, we screened R326H and R356Q as the most deleterious and disease associated mutation in TYRP1 gene (Table 3). This prediction could be endorsed with the observed experimental data [62].
3.3. Modelling of Protein
The human Tyrp1 protein (between domain regions 190–385) was modelled by automated protein structure prediction program, I-TASSER [53]. The program used more than ten templates to model the protein. The top most template (PDB ID: 3nm8A) has covered 86% of the Tyrp1 protein query sequence. The best structure with high confidence score was collected and used for further investigations. The disease associated mutations of R326H and R356Q can probably alter the native conformation of the Tyrp1 protein structure. Hence we made a point mutation in native Tyrp1 protein at the position of 326 (arginine to histidine) and 356 (arginine to glutamine) to build the mutant structures. The quality of the modeled structure of native and mutant Tyrp1 protein was evaluated independently by the PROCHECK [54] and PROSA [55] programs, which showed good stereochemical properties of the modeled proteins. Native Tyrp1 protein showed 91.7% of residues in most favoured and allowed region and -score value of −3.4. Mutant of R326H showed 92.3% of residues in most favoured and allowed region and -score value of −2.13. Mutant of R356Q showed 92.3% of residues in most favoured and allowed region and -score value of −2.81. Native and mutant (R326H and R356Q) Tyrp1 structures showed the -factor in the ranges of 0.40, 0.45, and 0.46, respectively. The overall -factors of native and mutant Tyrp1 protein structures (acceptable between 0 and 0.5) were produced by PROCHECK in the range of 0.40–0.46. These scores implicate high confidence level and hence the structures were selected for further MD analysis.
3.4. Molecular Dynamics Simulation
To understand the structural and functional behaviour of the prioritized disease associated mutations, we performed molecular dynamics simulation for native and mutant Tyrp1 proteins. The following seven factors, namely, tolerance index, PSIC score, DDG value, subPSEC score, disease-association study, pathogenecity index, general score (), and property score (), which correspond to conformational changes in protein residues due to the mutation, may lead to affect the functional behaviour of Tyrp1 protein. The results obtained from above analysis further inspired us to study the dynamic behaviour of native and mutant (R326H and R356Q) Tyrp1 proteins. We studied RMSD, RMSF, Rg, SASA, and NH bond variations, DSSP, density plot, and ED analysis between the native and mutant (R326H and R356Q) Tyrp1 protein structures. Further, the RMSD for all Cα atoms from the initial structure was examined to study the convergence of the protein system. In Figure 2(a), native and both mutant (R326H and R356Q) structures showed similar way of deviation till 3050 ps from their starting structure, resulting in a backbone RMSD of ~0.14 to 0.52 nm during the simulations. After this, native structure retained the maximum deviation till the end of the simulation resulting in the backbone RMSD of ~0.51 to ~0.66 nm, respectively. R326H and R356Q mutant structures showed the minimum deviation till the end of the simulation, resulting in the backbone RMSD of ~0.37 to ~0.51 nm and ~0.38 to ~0.54 nm, respectively.
This magnitude of fluctuations, together with very small difference between the average RMSD values after the relaxation period (~0.52 nm), leads to produce stable trajectories in simulation, and it provided an appropriate basis for further analysis. The average value of RMSD during the simulation time period both native and mutant (R326H and R356Q) structures is signified in Table 4. Through the aim of determining RMSF we predicted whether the mutation disturbs the dynamic behaviour of residues. The RMSF values of native and mutant (R325H and R356Q) structures were collected and shown in Figure 2(b). Analysis of fluctuation score depicted that the higher degree of flexibility was observed in native structure than mutant (R326H and R356Q) Tyrp1 protein structures. The radius of gyration (Rg) is defined as the mass-weight root-mean-square distance of collection of atoms from their common center of mass. Therefore it provides an insight into the overall dimension of the protein. Radius of gyration plot for Cα atoms of protein versus time at 300 K is shown in Figure 2(c).
In Figure 2(c), at the end of the simulation native structures showed greater Rg value than mutant (R326H and R356Q) structures. The native structure showed Rg value of ~1.76 nm at 0 ps, ~1.82 nm at 4000 ps, ~1.82 nm at 9500 ps, ~1.85 nm at 11800 ps, ~1.86 nm at 14600 ps, ~1.86 nm at 17500 ps, and ~1.87 nm at 20000 ps. R326H mutant structure showed Rg value of ~1.76 nm at 0 ps, ~1.78 nm at 4000 ps, ~1.86 nm at 9500 ps, ~1.84 nm at 11800 ps, ~1.82 nm at 14600 ps, ~1.81 nm at 17500 ps, and ~1.83 nm at 20000 ps. R356Q mutant structure showed Rg value of ~1.77 nm at 0 ps, ~1.84 nm at 4000 ps, ~1.79 nm at 9500 ps, ~1.79 nm at 11800 ps, ~1.80 nm at 14600 ps, ~1.77 nm at 17500 ps, and 1.78 nm at 20000 ps, respectively. The average Rg value was 1.84 nm in native, whereas the mutant R326H and R356Q structures showed average Rg value of 1.81 and 1.79 nm, respectively, signified in Table 4. A notable change was observed in both mutant (R326H and R356Q) structures as compared to the native. The change of SASA for native and mutant (R326H and R356Q) proteins with time is shown in Figure 2(d). Solvent accessibility surface area accounts for bimolecular surface area that is assessable to solvent molecules. Decreased value of SASA in mutant structures denotes its relatively shrunken nature as compared to the native structure. The change of SASA of native and mutant proteins with time is shown in Figure 2(d). Native and mutant (R326H and R356Q) structures showed similar fashion of deviation till 12000 ps from the initial structure, but after this native structure showed greater value of SASA than mutant (R326H and R356Q) structures. The average SASA value was 74.5 in native, whereas the mutant (R326H and R356Q) structures showed average SASA value of 71.2 and 69.1, respectively, as depicted in Table 4. We also observed notable differences in NH bond pattern during simulation, whereas the native structure showed less participation in NH bonds formation with other amino acid, while in mutant (R326H and R356Q) structures there was greater number of NH bonds (Figure 2(e)). The average value of NH bond was signified in Table 4.
The NH bond results of native and mutant Tyrp1 structure according to the RMSD, RMSF, Rg, and SASA plot results depicted that the mutant (R326H and R356Q) structural conformation became rigid upon mutation which may lead to disturb the functional behaviour of the protein. This was further supported by the atomic density plot and PCA analysis. The consequences of these molecular changes were clearly observed in the atomic density distribution plot. There was a significant change in density distribution in mutant as compared to the native and it was depicted. Moreover the native structure shows highest atomic density distribution of 41.9 nm−3 but mutant (R326H and R356Q) structures showed 52.2 and 52.3 nm−3, respectively, (Table 4) (Figure S1a-c available online at: http://dx.doi.org/10.1155/2013/697051). It was further indicated that native structure has more flexibility than mutant (R326H and R356Q) structures.
The spectrum of the corresponding eigenvalues indicated the level of fluctuation and dynamic behaviour of protein molecule in the system and was basically confined within the first two eigenvectors. Both mutant (R326H and R356Q) structures covered a small region of phase space particularly along PC1 plane than native (Figure 3(a)). Overall flexibility of two proteins was further examined by the trace of the diagonalized covariance matrix of the Cα atomic positional fluctuations. We obtained the following values for native and mutant (R326H and R356Q) structures: 11.56 nm2, 10.74 nm2, and 8.16 nm2, respectively (Table 4), and again it was confirming the overall increased flexibility in native than mutant (R326H and R356Q) structures at 300 K.
We applied the DSSP algorithm [63] to monitor changes in secondary structure during the simulations. As shown in Figures 4(a)–4(c), a difference is observed only at the level of Helix from the amino acid residual position of 150 to 175 and the level of sheet from the amino acid residual position of 5 to 20. In DSSP, the most significant structural change was an increase in helical content and absence in -sheet, which was observed in both mutant (R326H and R356Q) structures.
To examine how the structure got damaged and leads to affect the functions upon mutation, we analysed the native and mutant (R326H and R356Q) structures at different time scales (Figure 5). It was clearly observed that there is continuous loss of helix in native structure than mutant structures till the end of the simulation. This was well supported by the DSSP analysis. It indicates that both mutant (R326H and R356Q) structures showed an increase in helical content and total absence in -sheet in the amino acid residual position from 150 to 170 and 5 to 20, respectively. In general, helices are mostly rigid, whereas spanning loop regions are mostly flexible. [64–66]. Based on that here, both mutant structures (R326H and R356Q) showed more helical content which leads to more rigidity in the conformation. On the basis of DSSP analysis, it was confirmed that due to mutation the Tyrp1 structure became rigid. Therefore, it seems evident that both mutations (R326H and R356Q) have cruel damaging impact on protein structural orientation and its function. This prediction is also endorsed with the observed experimental data [62, 67]. This study provides an essential insight into the underlying molecular mechanism of Tyrp1 protein upon mutation and in future it may help to develop a personalized medicine for OCA3.
4. Conclusion
Computational analysis has now become a roadmap to define a standard disease specific SNP at molecular level. In this study we screened two most disease associated mutations (R326H and R356Q) which are related to OCA3. Molecular dynamics simulation approaches have also been extensively used to report the structural consequences of the deleterious predicted point mutations. The flexibility loss is observed in RMSD, RMSF, Rg plot which is further supported by a decrease in SASA value in both mutant structures. This may produce a major impact on the structural conformation of Tyrp1 protein, which also affects the function of Tyrp1 protein. Due to mutation, the structure became more rigid which is also supported by NH bond, density plot, PCA, and DSSP analysis. Our result suggests a significant computational roadmap to detect the OCA3 associated SNPs from the large SNP dataset and reduce the expenses in experimental depiction of pathological nsSNPs. Further the predicted R326H and R356Q mutations can be further studied by wet lab scientist to investigate the evidence of Tyrp1 protein mutation in association to OCA3 and develop a potent drug target for OCA3.
|Non-synonymous single nucleotide polymorphism
|Oculocutaneous albinism type3
|Tyrosinase-related protein-1
|Molecular dynamics simulation
|Root-mean-square deviation
|Root-mean-square fluctuation
|Radius of gyration
|Solvent-accessible surface area
|NH bonds:
|Number of hydrogen bonds
|Principal component analysis
|Definition of secondary structure of proteins.
The authors gratefully acknowledge the management of Vellore Institute of Technology University for providing the facilities to carry out this work. They thank the anonymous reviewers for their helpful comments and critical reading of the paper.
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text:
Vaakkoi forest is in southern Finland in Vihti and partly in Espoo, about 30 kilometres from the center of Helsinki. If arriving by a car, there's a parking place at the start of the trails. There are no marked trails, so you should know the routes or have a map and some short of orienteering skills.
On the 6th of December, on the Independence Day of Finland, the first snow had arrived covering trails quite totally. Snow gave some light and sun was shining for a moment but still the day was quite grey and the length of the day was only few hours. There were quite many streams to pass since I lost the trail occasionally. I was wearing my true companions of 4 years, Lowaboots but at this trip I noticed they lost their water resistance.
The sun was going down very quickly and because of trail finding operation the trip was shorter than planned. The sausages reserved for the fire at the lean-to at pond Kämmenlampi were saved for another time. There are many ponds and lots of trails in the area so I'll have to visit it again when there's no snow.