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Addressing the Daily Mail and James Delingpoles crazy climate change obsession article « Met Office News Blog
Addressing the Daily Mail and James Delingpoles crazy climate change obsession article « Met Office News Blog
Could it be that the billions spent on computer models were wasted because all the variables were not considered?
Variables such as;
Alterations in the earths magnetosphere.
Increases in phytoplankton due to damage to the food chain from over fishing and pollution of the environment with heavy metals.
Solar activity
And so on.......................
They choose the version they like without ever actually reading and considering the alternative one. And sadly, the choice is often based on peer pressure, politics or what's easier to follow.
We see this time and time again. The science is linked and posted and it is very very clear that it is not even read. It's pure denial and doesn't even deserve to be referred to as scepticism.
If it is cold they will sell a lot of warm stuff.
Capitalist: A man who has gained an unhealthy love of his money - the root of all evil according to the Bible.
Social-ist: A man who has gained an unhealthy love of other peoples money - but by then, the Bible has no effect on his conscience any more
You only have power over a man for as long as you don't take everything away from him. But when you've robbed a man of everything, he is no longer in your power, he's free again. - (Aleksandr Solzhenitsyn)


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Coverking Stormproof Car Cover CVC5SP93JP2008
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Should we severe all relationships with pakistan? Hundreds gave their life including some courageous officers on Sept 11th?
Asked by shwetajha, 17 Jul '09 10:24 pm
Earn 10 points for answering
Answers (4)
We should not. Instead we should try to improve our relations with Pakistan so that both the nations live in peace and hundreds more are not required to give their life again and again. We salute our courageous officers who made the supreme sacrifice and they will never be forgotten but peace with Pakistan will save a lot of expenditure on security and defence and that money can be spent for development in our country.Answered by Francisco, 17 Jul '09 11:46 pm
We should try every opportunity to improve our relations not only with Pakistan but with all our neighbours. That is the only sane and wise thing to do in this troubled world.
We should try every opportunity to improve our relations not only with Pakistan but with all our neighbours. That is the only sane and wise thing to do in this troubled world.
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Person who winsare always appreciated.If we become a strong nation no body will dare to come here to us for such acts.China has merged Tibet, Ceylon ,Russia,UKa US hav e taken very strong actions,why we cannot make our borders like Fort, why we cannot issue identifictions card to our citizen,? how a men fromPakistan come here and hire flats and craete base for their terror activities, why cannot we bane short term L&L system? it should be for a minimum period of 5 years only.Entry to bank, hotels, trains,bus etc. should not only required identification cards but should be retained in scanner,CCTV to be compulsory for banks shops, housing complx and scale should be prescribed for same that is one month or 2 month data if one is strong, nobody will dare to touch himAnswered by Harsh Mehta, 17 Jul '09 10:49 pm
We have to.It is no use in talking with them. Waste of time.Answered by hitler, 17 Jul '09 10:24 pm
We should never forget the cause which has arrived here :(Answered by conviction, 17 Jul '09 10:24 pm


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Vegetarian food
Nowadays, vegetarians can enjoy food that's as exciting, colourful and full of flavour as the dishes enjoyed by meat-eaters. It wasn't always so! Delia has drawn on some of the best cuisines in the world to bring you a selection of vegetarian recipes that will have you spoilt for choice!
Roasted Stuffed Apples with Thyme and Parsley
This is the perfect accompaniment to Classic Roast Pork with Crackling, as the acidity of the apples offsets the richness of the pork perfectly.
Dried Fruit Salad with Yoghurt and Nuts
This recipe is full of natural, good things – there is no sugar in it as I have come to the conclusion it tastes better without it.
Cold Poached Egg Salad with Watercress Sauce
This is a kind of variation on the egg mayonnaise theme, but I think extremely attractive and even more delicious.
Walnut and Raisin Bread
I think one of God's greatest gifts to man is good cheese, and it deserves good bread to go with it. This chunky, slightly sweet bread goes extremely well with a sharp, vigorous Cheddar, fresh goats' cheese or Cashel Blue. It also freezes very well.
Very Red Rice
This was a recipe created for Comic Relief, hence the title, and I always have a good giggle thinking back to the filming of those hilarious cookery demonstrations that were later screened on television.
Home-made Lemonade
Just lemons, sugar and water are needed to make what is possibly the most refreshing drink in existence...just remember to start it a day before you want to drink it, so all the flavours can combine.
Eggs Mayonnaise
Not the kind you get in help-yourself salad bars and cafés – this is the real thing. Eggs, boiled – not hard, but with a bit of squidge at the centre – anointed with a shimmering, golden emulsion laced with a little garlic. I have to admit, this is p
Bruschettas with Goats' Cheese, Basil and Tomato
This recipe is simplicity itself, but it has a wonderful combination of flavours and a very crunchy texture.
Slimmers' Scrambled Eggs
This lovely recipe is proof that dieting need not be a penance: quick and easy to make, it has all the satisfying flavour of classic scrambled eggs, without the fat content.
Quick-Braised Celery
Celery has such a lot going for it as a raw ingredient in salads, and because of that we rather forget how good it is cooked and served as a vegetable. This method is delightfully quick and easy, and tastes just wonderful.
Return to Homepage
Visit the Delia Online Cookery School with Waitrose
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Effie Auten-Idaho Word has been received here of the death of Mrs. Effie L. Auten, 90, of Rupert, Idaho. Funeral services were held Monday, July 28 at the Rupert First Christian church, with the Rev. Rex R. Jones officiating. Burial was in Rupert cemetery. Mrs. Auten died July 25 at the Cassia Memorial Hospital, Burley, Idaho. She was born Dec. 17, 1884, in Mt. Vernon, the daughter of Alonzo and Sarah J. Waite Payne. In 1907 at Mt. Vernon, she married Merritt Summers, who preceded her in death in 1917. In 1921 at West Frankfort, she married Elmer Auten, sho preceded her in death in 1957. Mrs. Auten attended schools in Illinois. She moved from Illinois in 1922 to the Acequia, Idaho area, where she resided until her death. She was a member of the CWF of the Christian church, the Acequia Sewing Club, and the Rupert First Christian Church. Survivors include one daughter, Mrs. George (Lillian) Bagnell, Rupert, Idaho; one sister, Mrs. Cecile Summers, Mt. Vernon; seven grandchildren, 18 great-grandchildren and three-great-great-grandchildren. She was preceded in death by one son, Ray Summers, who died in 1974; four sisters; one brother; and her parents. Source: Mount Vernon Register News Date: July 1975 Submitted by Brenda Heriford * email@example.com Relationship: Grand Niece
Please send additions & corrections to Jefferson County Coordinator Cindy Ford © 2005-2012 by Cindy Ford All rights reserved


text: Techniques I have found useful include: Keeping an eye on potential ingredients and keeping them to what is readily available from a known food supplier such as Sysco. If they are already ordering from one place, or buying at someplace like Costco or Sam's, then using ingredients found there and nothing more exotic will help with the appeal. You have restaurant experience, so you have a good idea of what's available from whom. Handing them recipes in modern formulas, not period texts. They don't really want to know HOW historical this is, they want to see some easy-to-recognize formulas and foods that are not too far out in left field. Use your best judgment on which recipes to offer them, and bring two or three choices. A tasting sample is always a nice idea - medieval gingerbread, maybe some sauces to sample - a green sauce, cameline, and mustard for example. Good job, you go get 'em and show 'em how it's done! Christianna So, our local high school chorus is doing a fundraiser Madrigal Dinner and concert soon. I jsut got the email today and was excited to hear it is happening . . . followed by the unfortunate cringe. I got to the menu, with rather low expectations for a first year and probably not having someone in "the know" working for them. My expectations still a little hig with the menu being: Wassail Cuppe, Olde Engeland Fowle with Sause, Fressh Fruyt, Vegatebbles, Chese, Goldin Corrn on a Skewr, Broccolye Potage, Wedgs of Botata, White Rounde Bread, Fruyt Pastris, Sweet Tea, Watr. It appears the criteria were misspelling. So, I have the motivation to assist them in their second attempt, if they do it next year. They are doing 15th Century England, which is what I cut my cookery teeth on, so repertoir is fine. My interest form the assembled minds is how others have successfully approached such a group and found a tactful way of saying that the food is lame, and it ain't so hard to do better. I am willing to prepare menues, recipes and shopping lists . . . maybe available to assist in cooking. I'm not squeemish to approach the director, just wanting to know how others have fared in such a venture. This could be a huge marketing opportunity for their event as well as my restaurant in a town 15 miles away . . . as well as a chance to spread the "Goode Newys" of actual medieval cookery. niccolo difrancesco (out to prosthelatize for 2 15th Century Cokery Books)


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£795,000 Offers in Excess of
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4 bedroom detached house for sale Hawkchurch, Axminster, Devon, EX13 5UF
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A new drama on immigration drives to your borough
Exploring each borough with your ears
Leaf peeper map of New York State Saturday Korean festival and parade Dance, taekwondo, food, music and cooking lessons. Chile Pepper Fiesta, Brooklyn Botanic Gardens Music, chile-chocolate cooking, Qigong lessons, and hot stuff samples. Free with admission to the Gardens ($8 adults, $4 ... More >>
the National Weather Service is promising 82 degrees and disappearing clouds--Broadway on Broadway - the casts from 20 Broadway shows will be performing live on Broadway between 43d and 47th, starting at 11:30. Michael McKean (Spinal Tap, an upcoming play) is hosting.--Brooklyn Book Festival ... More >>


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Humidity 60%
Winds 13.8mph
Posted: Feb 16, 2012 11:53 AM by Melissa Canone
Looking for Mardi Gras fun this weekend? St. Landry Parish has the traditional Mardi Gras run, parades, street dances, jam sessions, soul queen Irma Thomas, a zydeco trail ride and old-time pig roasts. That's just the short list.
St. Landry Parish is bursting with Mardi Gras excitement and activities that are almost too numerous to list. Here's an hourly breakdown of the fun that awaits.
Friday, Feb. 17
7:30 p.m., Eunice - Street dance with Kyle Huval and Friends; Jamie Berzas and Cajun Tradition at Nick's on 2nd; Paul Daigle and Jason Frey at Lakeview Park
9 p.m., Leonville - 2nd Annual L'Argent Trailride and Chicken Run at IFBS Lodge with Leon Chavis and Lil Nathan and the Zydeco Big Timers
Saturday, Feb. 18
2 p.m., Washington - Mardi Gras parade on North Main Street
2:30 p.m., Eunice - Street dance with Fred Charlie and the Acadiana Cajuns
3 p.m., Eunice - Music program at the Jean Lafayette Prairie Acadian Culture Center
8 p.m., Leonville - 2nd Annual L'Argent Trailride and Chicken Run with Brian Jack, J. Paul and the Zydeco Nubreedz at IFBS Lodge
6 p.m., Eunice - Rendez-vous des Cajuns radio/TV show with Ryan Brunet et les Malfecteurs, Basile Mardi Gras Association and Georgie Manuel explaining rural Mardi Gras traditions
7 p.m., Lebeau - Lebeau/Palmetto Mardi Gras Ball with Leon Chavis and the Zydeco Flames at Church Hall
8 p.m., Eunice - Street dance with Les Bassettes
8 p.m., Eunice - Kevin Naquin and the Ossun Playboys at Nick's on 2nd
8:30 p.m., Eunice - Barn dance with swamp pop legends Warren Storm, Willie Tee and Cypress at Lakeview Park
9 p.m., Opelousas - Concert with Irma Thomas at Evangeline Downs Racetrack and Casino
9:30 p.m., Opelousas - Traditional Mardi Gras dance with Jeffery Broussard and the Creole Cowboys at Slim's Y-Ki-Ki
Sunday, Feb. 19
8 a.m., Eunice - L'anse Meg Courir de Mardi Gras at Lakeview Park
9 a.m., Eunice - Children's Mardi Gras run leaves Recreation Complex, 461 Sittig Road
10 a.m., Eunice - Old-time boucherie at South 2nd Street and Park Ave.
11:30 a.m., Eunice - Street dance with Da Classics with Drew Dogay and Charlo at S. 2nd St. stage
11:30 a.m., Eunice - Street dance with Leroy Thomas and the Zydeco Roadrunners at S. 2nd St. and Walnut stage
1 p.m., Lebeau - Mardi Gras parade and chicken run with Leon Chavis at Church Hall
3 p.m., Eunice - Children's Mardi Gras run returns downtown
3:30 p.m., Eunice - Kyle Huval and Friends at Nick's on 2nd
3:30 p.m., Leonville - 2nd Annual L'Argent Trailride and Chicken Run with Lil Wayne, Cupid, Nathan and the Zydeco Cha Chas and Lil Nate at IFBS Lodge
8 p.m., Eunice - Jeffery Broussard and the Creole Cowboys at Lakeview Park
Monday, Feb. 20
Noon, Eunice - Dr. Barry Ancelet's presentation on the history of rural Mardi Gras at Jean Lafitte Center
Noon, Opelousas - Lundi Gras at Le Vieux Village with old-time boucherie, cooking demonstrations, costume contest, historic village tours
1 p.m., Eunice - Council on Aging Mardi Gras dance with Big J and Zydeco Dog Pound at Northwest Center
5:30 p.m., Eunice - Jam session at South 2nd St. and Walnut
6 p.m., Opelousas - Lundi Gras at Le Vieux Village with Geno Delafose and French Rockin' Boogie
8 p.m., Eunice - Street dance with Kevin Naquin and the Ossun Playboys
8 p.m., Eunice - Don Fontenot at Nick's on 2nd
9 p.m., Eunice - Steve Riley and the Mamou Playboys at Nick's on 2nd
9 p.m., Opelousas - The Original Zyde-Gras with Keith Frank and Cupid, Evangeline Downs Racetrack and Casino
Tuesday, Feb. 21
8 a.m., Eunice - Courir de Mardi Gras leaves Northwest Center
10 a.m., Eunice - Acadien with Ryan Simon on Park Ave. stage
11 a.m., Eunice - Homer LeJeune and Cajun Kings on 2nd and Walnut stage
11 a.m., Eunice - Donnie Broussard at Nick's on 2nd
11 a.m., Opelousas - Krewe of Frank's Parade
12:45 p.m., Eunice - Paul Daigle and Cajun Gold on Park Ave. stage
2:45 p.m., Eunice - Truck parade and Courir de Mardi Gras returns
4 p.m., Eunice - Steve Riley and the Mamou Playboys on Park Ave. stage
6 p.m., Eunice - Geno Delafose and French Rockin' Boogie at Nick's on 2nd
In other events, Masks & Mayhem - The Courir de Mardi Gras Artwork of Herb Roe remains on display at the St. Landry Parish Visitor Information Center, 978 Kennerson Road in Opelousas. Roe's exhibit features oil paintings and graphite drawings of traditional Mardi Gras runs.
Renowned zydeco and Cajun musician Horace Trahan concludes the Music of Acadiana Series at 2 p.m. Sunday, Feb. 25 at Nunu's, 1510 Highway 93, in Arnaudville. The event starts with an open jam session, followed by an interview with Trahan and performance.
For more information on parish events, call the St. Landry Parish Tourist Commission at (337) 948-8004 or visit www.cajuntravel.com. Updates are available on Facebook at St. Landry Parish - It's Gumbo for Your Soul and Twitter @StLandryParish. Videos are online at www.youtube.com/StLandryParish.
8 months ago


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The Portal is available 24 hours a day/7 days a week.
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A secure website with services and infomation specific for employees of Emporia State University.
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You will have a specific employee signon assigned to you similar to your Novell signon.
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Tukwila superintendent resigns following accusations of racismon July 11, 2012 @ 8:49 am (Updated: 11:03 am - 7/11/12 )
Following accusations of racism, the Tukwila Superintendent of Schools has resigned.
"If she wasn't a person of color, me personally, I would have gone after her long before now," bus driver Richie Coleman told KING 5.
Ethelda Burke has been on paid administrative leave, during which the office of the Equal Employment Opportunity Commission investigated allegations by 10 district employees of racial harassment and discrimination.
Burke resigned on June 30. In a letter, she wrote that the allegations had taken a toll on her family, and she thought it would be "impossible" for her to be effective if she returned to work as the Tukwila Superintendent.
Some of the accusations included telling a bus driver he had to stop being a "big, black bus driver, scaring all of the white drivers," and referring to "professional African American women as slaves."
The district said it has not seen any evidence proving Burke discriminated against any employee.
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I've had these for 4 months. Still work as good as the day I got them. Great buy. Especially if you get them on sale. They are bulky. But I still like them. The mic is extremely flexible so it shouldn't break.. I would buy them again
Headsets are not exactly for everybody: some find them distracting, some find them annoying. The P11 by Turtle Beach is an affordable headset for those on a budget and wants to know if Gaming Headsets are for them and does not want to spend hundreds of dollars just to realize that they hate having headsets.
The P11 isn't really a headset that you can wear proudly outside. It's big, bulky and makes you look like a construction worker handling loud, heavy machinery. If you are looking for a "stylish" headset, P11 only gives you the bare minimum. But if style isn't a factor and all you are going to be doing is strictly gaming in the comfort of your own home, then you wouldn't really care about the P11's aesthetics.
Audio (PS3/PC/Mac):
For the price, the P11 performs well and you will be able to immerse yourself in your gaming on a whole new level. However, the quality of the sound you get is pretty much what you pay for. On the plus side, you get a pair of 50mm speakers which are by far the biggest for it's class as more expensive headsets will only give you the standard 40mm. For [price], you can't really go wrong with that.
PS3: On the console, the P11 gives you good quality sounds and, for [price], will get the job done right. Still nothing compared to higher end headsets but definitely a competitor even for most $100-$150 headsets on the market.
PC/Mac: I have a PC but I imagine that the Mac would be the same. For PCs, the P11 isn't something to write home about. There is a very audible "hum" when all is quiet and can still be heard if you are paying attention to it even when there are other sounds present (movie, games, etc.). The P11 definitely functions betters for the PS3 rather than on PC. I would recommend just using regular old headphones when you want to game on PC and have a separate microphone - the sound quality will be better - or invest on a more PC-dedicated Headset.
The P11 is all plastic. The good thing about plastic is that it is extremely light. For all of it's bulk, the headset is lightweight. The bad news is that plastic is also brittle. Looking at the joints between the frame and the speakers, they are held together by very thin plastic that can be broken easily if misused. So treat, store, and transport the P11 with care if you don't want the plastic to break. The cord used for the headset is also fairly standard and covered by fairly stiff rubber (which is good). However, I feel that it is a little too thin especially for it's bulk. Again, store your hardware with care.
Nothing cheap about the level of comfort this badboy has. The 50mm speakers can definitely cover the whole ear, even relatively big ones, without feeling pinched or squashed by the speakers. The foam and mesh fabric used are also very soft, but firm. This combination gives the P11 a soft feeling around the side of your head and will last a long time for those long hours of gaming. If you wear glasses, don't worry. The foam will still feel comfortable without feeling as if your temples are in a vice-grip.
For [price], the P11 delivers performance that can compete to headsets of triple digit price ranges. But don't expect too much out of the P11. It's comfortable to wear but bulky; it's light but can be fragile; it's got massive speakers but you're not going to look good wearing them; it works like a charm on the PS3 but much to be desired for the PC. With all of these factors in mind, the low price tag definitely pays for itself and will get that fledgling gamer started on some awesome audio. Those new to headsets, this will feel and sound like a revelation. However, those who are veterans in headset use might find a lot to be desired from the P11. Even though the [price] definitely shows on the P11 in most regards, the functionality of this little (or not-so-little) device is nothing to scoff at.
Just bought mine yesterday and the audio is fantastic, great bass and super comfy. Worth the money, got it for a good employee discount but would've bought it for the price otherwise.
So I bought this headset thinking it is amazing,
and yes it is, the first 2 months. The third month was horrible I started getting weird sounds. After that my mic stopped working and I found out that the problem was the cord that was leading into the volume rocker was broken. I did not misuse it, it was the bad build quality. That made the cord break. Do not buy.
This headset is amazing Very good Sound. The Sound on the P11 Turtle Beach Headset is Amazing with All kind of Function. Like Mute Volume, Lower Volume and Mute Other Players. Because some of them are Annoying Yelling and Doing Stuff. That's why i Love this Headset
Came time for me to buy a new headset after a third Bluetooth in ear headset broke. Was torn between buying another Bluetooth or the P11. After seeing it was in sale [...] and that being the same price as all the three bluetooths I've owned which now reside in a landfill no doubt I thought why not change up an give it a go and I'm so glad I did. I play my ps3 through a surround sound and if you balance the volume of the headset and the surround it fully emerges you in the game. I will add I only play BF3. The sound is great and very full , makes things easier to hear and which direction things are coming from. As soon as I got into a clan match all of my teams mates said that it was much clearer than my other head set, over all it's a must buy at the price, no more headsets battery running out in the middle of a clan match, if you don't want to break the bank with the stupidly priced headsets out there snap these up
The P11's by Turtle Beach are a great inexpensive headset. They allow you to listen to in-game and voice sound without the TV speakers, making them ideal for use at night and so they won't disturb others. They are very comfortable, over ear cups that fit snug on the head. They are not restricting to glasses. They have a crisp game and voice sound. However, like many others, my headset eventually (after about 7-8 months) cut out in the right ear. A month later the left ear cut out forcing me to listen to the voice and game volume through the TV, only leaving the microphone operational.
I do recommend this headset. It is great value overall. My advice is to purchase the protection plan so that when the ear cuts out you can replace it for a new headset- just make sure you keep the receipt.
These turtle beach are not worth the money. I would get the PX21 DPX21 P21 PX3 PX5 because they all have a better mic
Like everyone else said these things don't last to long but they work great until the right one goes out and then completely break. I would recommend the RocketFish wireless for PS3 they don't have the same sound quality but they will last you 1-2 year while this lasts you 6 months or less. Trust a brother, don't get em.
Great product, worth the money
Its a pretty good headset for the price. the sound is really good. though you cant adjust the bass. they are pretty comfortable but feel a little weird because the mic is on the left. overall a really good headset for the price! recommend it to anyone!
Mine only lasted for about 10months. I it extremely easy to setup but the headset has minor problems random hissing,static noises and headset had volume and hearing problems after 3months it was a decent headset that aloud me to listen to game volume but thats about it. I would only buy this if ur looking for a cheap headset for ps3
These headphones are really good up until 6 months or so if you're lucky, They seem to just die out with voices or the sound will disappear from the right ear and there's nothing you can do to fix it.
I would not recommend this product even though it performs well, but dies early.
The headset does what it's supposed to, for the most part. It lets you talk to people and hear the game. It is decent for the price. One major problem is that randomly it gives you the worst feedback. One second the game is coming through fine, and the next second it sounds as if someone is screaming in your ear. Just absolute, loud, static. It fixes itself after a while but why am I paying for a headset that destroys my eardrums?
Overall, I wouldn't recommend it, pay a little more cash for something better.
Hello i Bought this mic 6 months ago and had no problems. Then out of nowhere there right ear wont play sound.i play around with cords and it works for bit but then cuts off. Its a Great buy And if taken promply it last 6 to even year.Only thing might find your self talking louder on account you cant hear yourself well with headset and it wont play what your saying unless mics in your mouth. Useless feature.
I just bought this and It is very very good I would suggest it to anyone
This headset is well worth the money. The speakers are 50mm, which are huge and usually found in headsets 3 times the price. You can hear foot steps easily in Call of Duty, and makes the game better and more enjoyable. The only bad thing that I have to say about these is after around 4 hours of gaming, the head band starts to hurt a little. Not a big deal, I mean after 4 hours you should take a break anywways! Lol. I would reccomend these to anyone, unless your a competitve gamer. Overall great headset, you can't go wrong!
Amazing headset, freat sound quality, can easily hear footsteps in COD, only bad thing is short, and thick cable that is very annoying.
My wife bought me this headset for fathers day 2011 and I loved it, the audio was nice and all... I felt funny about the chat audio box because it felt weak but I thought it was just my imagination... well, no that box is doing funny things... the audio is like halfway gone, the volume goes super low on its own and keeps breaking the audio... I have to touch the wire by the box and by my ear piece to make it work nice... anyway, the product is really good, just the built quality is kind of bad... today is aug 31st 2011 so it means that began failing with less than three months... aaaagggrr...
I was originally going to get the official Sony Bluetooth 2.0 until I came across the Turtle Beach P11's. The P11's were just $10 more than the Sony Bluetooth 2.0. I've been using the headset for a couple of days now and boy, this headset is worth my money. Really good sound quality. It's not the best, but it does the job for listening to enimies footsteps while playing COD. The mic quality is clear from the feedback that I'm recieving while in the lobbies and during actual gameplay. No dislikes for me on this one. The Turtle Beach P11's are great for the price and for beginner gamers who are looking for a headset at a very affordable price.
its bad i hear like hissing alot
This headset is just awesome! I go tit yesterday and it is amazing. the sound is super clear and it sounds just like surround.
The price is great as well! So worth the price. If you like gaming this is your headset, unless you don't like the wires.
Thank you Turtle Beach!!!!!!!!
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Pharmaceuticals 2014, 7(4), 482-501; doi:10.3390/ph7040482
Published: 21 April 2014
Abstract: Drug-resistant Gram-negative bacterial pathogens and endotoxin- or lipopolysaccharide (LPS)-mediated inflammations are among some of the most prominent health issues globally. Antimicrobial peptides (AMPs) are eminent molecules that can kill drug-resistant strains and neutralize LPS toxicity. LPS, the outer layer of the outer membrane of Gram-negative bacteria safeguards cell integrity against hydrophobic compounds, including antibiotics and AMPs. Apart from maintaining structural integrity, LPS, when released into the blood stream, also induces inflammatory pathways leading to septic shock. In previous works, we have reported the de novo design of a set of 12-amino acid long cationic/hydrophobic peptides for LPS binding and activity. These peptides adopt β-boomerang like conformations in complex with LPS. Structure-activity studies demonstrated some critical features of the β-boomerang scaffold that may be utilized for the further development of potent analogs. In this work, β-boomerang lipopeptides were designed and structure-activity correlation studies were carried out. These lipopeptides were homo-dimerized through a disulfide bridge to stabilize conformations and for improved activity. The designed peptides exhibited potent antibacterial activity and efficiently neutralized LPS toxicity under in vitro assays. NMR structure of C4YI13C in aqueous solution demonstrated the conserved folding of the lipopeptide with a boomerang aromatic lock stabilized with disulfide bond at the C-terminus and acylation at the N-terminus. These lipo-peptides displaying bacterial sterilization and low hemolytic activity may be useful for future applications as antimicrobial and antiendotoxin molecules.
1. Introduction
Development of novel antimicrobial peptides (AMPs) is urgently needed to combat against rapidly appearing drug resistant strains [1,2,3]. AMPs are generally short in length and composed of relative proportions of cationic and hydrophobic residues [3,4,5,6]. AMPs may be categorized based on their structures namely (a) α-helix, (b) β-sheet or β-hairpin stabilized by disulfide bridges, (c) extended and (d) loops with or without disulfide bonds. The cytolytic activity of most AMPs stems from membrane- targeted non-receptor mediated mechanisms and hence development of drug resistance towards such peptides would be difficult [7,8,9,10,11]. The structurally divergent nature and simple mode of action of AMPs make them suitable candidates for the development of anti-infective agents [12,13]. Lipopolysaccharide (LPS) of the outer membrane may restrict activity of AMPs to Gram-negative bacteria [14,15,16,17,18]. As a mode of action, broad spectrum AMPs would disrupt the permeability barrier of LPS-outer membrane, which is an important step for killing bacterial cells [18,19]. In addition, LPS or endotoxin induces inflammatory pathways leading to the production of cytokines that often results in septic shock [19,20,21]. Sepsis or septicemia occupies the tenth position among the most dreadful diseases and there are no recent significant improvements for the treatment of sepsis-affected patients [22,23,24].
Among the various classes of antimicrobial peptides, β-hairpins stabilized by disulfide bridges, including defensins, protegrins, polyphemusins and tachyplesins, occupy a prominent position [25,26,27]. The disulfide bridges are found to play an important role in the antibacterial activity and cell selectivity [28,29,30]. Moreover, disulfide bridges can stabilize the folded conformations of AMPs in aqueous solutions that upon membrane interactions may form higher order structures [31]. Addition of fatty acyl chains to AMPs shows increased microbial sterilization probably by increasing incorporation of peptides deeply into the membrane [32,33,34,35,36,37,38,39,40]. We are interested in developing AMPs that disrupt the LPS outer membrane as a mode of action [41,42,43,44,45,46]. In previous reports, we have designed a set of short (~12 amino acid long) cationic/hydrophobic peptides with antibacterial and antiendotoxic activities [41,42]. The designed peptides have been termed β-boomerangs as they adopt boomerang-like structures in complex with LPS. From an initial design, the second generation of β-boomerang peptides demonstrated enhanced LPS neutralization and antibacterial activity. Solution NMR studies revealed packing of two aromatic residues (W4 and F9) and amphipathic segregation of four cationic residues is critical for antimicrobial and antiendotoxic activities [42]. In this work, we have utilized the most active analog of the second generation of β-boomerang peptide, YI12WF (YVLWKRKRFIFI-amide) as a starting template for further design of potent antimicrobial activity and LPS neutralization peptide analogs. Toward this end, we have prepared analogs of YI12WF containing five basic residues (YVLWKRKRKFCFI-amide) in the loop of the β-boomerang structure and which are acylated at the N-termini, by C4 and C8 compounds. Further, residue I11 has been replaced by residue Cys to prepare disulfide linked variants of β-boomerang lipopeptides. All of the peptides are tested for antimicrobial and LPS neutralization and RBC lysis. Biophysical studies, using optical spectroscopic methods, ITC and dynamic light scattering have provided molecular insights into the mode of action of the designed peptides in Gram-negative bacterial outer membrane and endotoxin neutralization. Atomic-resolution structure, by NMR spectroscopy, has been determined for one of the potent peptides. Collectively, current results describe structure and activity of highly potent β-boomerang peptides of superior activity in comparison to parent peptides. These β-boomerang lipopeptides may be used in developing non-toxic antibacterial therapeutics.
2. Experimental Section
Materials: LPS of E. coli 0111:B4, FITC-LPS of E. coli 055:B5, acrylamide, NPN (1-N-phenylnaphthylamine) were purchased from Sigma (Saint Louis, MO, USA). All bacterial strains were obtained from the American Type Culture Collection (Manassas, VA, USA). Designed peptides were synthesized commercially by GL Biochem (Shanghai, China) and further purified by a reverse phase HPLC (Waters), using a C18 column (300 Å pore size, 5 μm particle size). The dimeric variants of β-boomerang lipopeptides were treated with 20% DMSO for 18 hours and the oxidized peptides were further purified by HPLC. A linear gradient of acetonitrile/water mixture was used for the purification and fractions eluting with higher purity were collected and freeze-dried.
Antibacterial assay: The minimum inhibitory concentration (MIC) of the designed peptides was determined against four Gram-negative strains (E. coli, P. aeruginosa ATCC 27853, K. pneumoniae ATCC 13883, S. enterica ATCC 14028) and four Gram-positive strains (B. subtilis, S. aureus ATCC 25923, S. pyogenes ATCC 19615 and E. faecalis ATCC 29212). In brief, mid logarithmic phase of overnight grown bacterial cultures were obtained in LB broth and adjusted to OD600 0.2 in 10 mM phosphate buffer, pH 6.8. About 50 μL of these cells were added to equal volume of designed peptides at two-fold dilutions in 96-well sterile polypropylene plates followed by incubation for 3hrs at 37 °C. Aliquots of the above titrated wells were streaked onto Mueller-Hinton agar plates and the concentration at which no visible bacterial growth was observed considered as the MIC value of the peptide.
Hemolytic assay: Blood was collected from healthy mice in a tube containing EDTA. Cells with EDTA were centrifuged at 800 g for 10 min, to remove the buffy plasma coat layer, and washed three times in PBS (35 mM phosphate buffer, 150 mM NaCl, pH 7.0). 50 μL of RBC solution was added to equal volume of two-fold dilution of the peptides in 96-well micro titer plates and incubated for one hour. The final erythrocyte concentration estimated to be ~4% (v/v). After one hour, the mixture was centrifuged and the release of hemoglobin in the supernatant was determined spectrophotometrically at OD540. Buffer and 1% Triton-X in the place of peptides used as negative control and positive control, respectively. The percentage of hemolysis was calculated using the following method:
LPS neutralization assay: LAL chromogenic kit (QCL 100 Cambrex, Walkersville, MD, USA) was used to determine endotoxin neutralization of the designed peptides following protocol of the vendor. LPS in Gram negative bacteria activates a proenzyme in limulus amoebocyte lysate (LAL). This activated enzyme in turn catalytically release a colored product pNA from the colorless substrate Ac-Ile-Glu-Ala-Arg-pNA which is detected spectrophotometrically at OD410. All peptides were dissolved in the pyrogen-free water supplied with the kit and pH was adjusted to 7.0 with 1N HCl or 1N NaOH (which is prepared in pyrogen-free water). The increasing concentrations of the peptides were incubated with three different concentrations of endotoxins, i.e., at 0.4, 0.8 and 1.0 endotoxin units (EU) in a total volume of 50 μL for 30 minutes at 37 °C. About 50 μL of LAL reagent was added to peptide-LPS complex and further incubated for 10 minutes followed by addition of 100 μL substrate. After incubation of six minutes for the reaction, the release of colored product was recorded at OD410. Water in the place of peptides served as negative control (blank) that is considered as 0% inhibition and percentage of LPS neutralization was calculated by:
Zeta potential measurements: The zeta potential measurements were carried out on a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) equipped with a 633 nm He lasers. Mid-log phase grown bacterial cells were diluted to an OD600 of 0.2 and a basal measurement in the absence of peptides was acquired in disposable zeta cells with gold electrodes. Increasing concentrations of the designed peptides were then added to the cells and the measurements were made. A total of five measurements of 100 runs each were carried out for all dilutions.
Intrinsic tryptophan fluorescence and acrylamide quenching: The intrinsic Trp fluorescence spectra of the designed peptides were obtained by exciting samples at 280 nm and emission was collected from 300–400 nm in 10 mM sodium phosphate buffer. Fluorescence experiments were carried out with a Cary Eclipse (PaloAlto, CA, USA) fluorescence spectrophotometer using a 0.1 cm path length quartz cuvette. Experiments were initiated by obtaining emission spectra peptide (5 μM) alone followed by additions of increasing concentrations of LPS. Quenching experiments were conducted to examine the solvent accessibility of tryptophan residue in aqueous and hydrophobic environments. Acrylamide, a neutral quencher was added at increasing concentrations to free peptide and peptide/LPS complexes. The extent of decrease in fluorescence intensity was then used to calculate Stern-Volmer constant with the formula F0/F = 1 + Ksv[Q]. F0 and F are the fluorescence intensities before and addition of acrylamide, Ksv is the Stern-Volmer constant and [Q] is the quencher concentration.
Outer membrane permeability assay: Outer membrane of Gram bacteria acts as permeability barrier for hydrophobic antibacterial compounds. Hence the ability of the designed peptides to permeabilize through the outer membrane was examined using 1-N-phenylnaphthylamine (NPN) dye. E. coli cells were grown to mid logarithmic phase and diluted to an OD600nm of 0.5 in 10 mM sodium phosphate buffer, pH 7.0. The excitation was set at 350 nm and emission at 390–450 nm. NPN was added to the diluted cells at the concentration of 10 μM and a basal fluorescence was recorded followed by the addition of increasing concentration of peptides.
Dissociation of FITC-LPS: LPS molecules form micellar aggregates in water. The competency of the peptides to dissociate LPS micelles were studied using FITC conjugated LPS. In this method, blank fluorescence of 500 nM of FITC-LPS was recorded followed by the addition of increasing concentrations of peptides. The excitation was set at 480 nm and emission at 500–550 nm.
Dynamic light scattering measurements: LPS forms micelles of large sizes, which with the addition of increasing concentration of peptides, dissociates to smaller sizes. DLS measurements were made for 0.5 μM LPS and then with LPS: Peptide ratios of 1:2 and 1:4. The scattering was measured with Dynamic Light Scattering software provided with the instrument (Brookhaven Instruments Corp., Holtsville, NY, USA) and the scattering data was analyzed with CONTIN method.
Isothermal titration calorimetry: Binding of designed peptides with LPS micelles was determined using isothermal titration calorimetry in VP-ITC micro calorimeter (Microcal Inc, Northampton, MA, USA). Peptides and LPS samples were dissolved in 10 mM phosphate buffer, pH 7.0 and filtered. LPS at a concentration of 10 μM was loaded into the sample cell and the reference cell was filled with the same buffer. The syringe was filled with 1mM peptide solution. Typically 30 injections of 3.5 μL of the peptides were made into the sample cell at 25 °C. Sample cell was stirred continuously at 300 rpm. Raw data were obtained and analyzed using single site binding model in Microcal Origin 5.0 software. Association constant (Ka) and enthalpy change (ΔH) were directly determined from ITC profiles. Dissociation constant was calculated from Kd = 1/Ka. ΔG and TΔS were estimated from the fundamental equations of thermodynamics, ΔG = −RTlnKa and TΔS = (ΔH − ΔG), respectively.
Structural characterization by NMR spectroscopy: NMR spectra were recorded on a Bruker DRX 600 (Fallanden, Switzerland) spectrometer containing a cryo-probe and pulse field gradients. Data acquisition and processing were performed with the Topspin software (Bruker) running on a Linux workstation. Two dimensional TOCSY (total correlation spectroscopy) and NOESY (nuclear Overhauser effect spectroscopy) spectra of peptides in free solution were acquired in aqueous solutions containing 10% D2O at pH 4.5 with 0.5 mM peptide concentration. The mixing times were fixed at 80 ms for TOCSY and 250 ms for NOESY. 2,2-Dimethyl-2-silapentane 5-sulfonate sodium salt (DSS) was used as an internal chemical shift reference. Because of severe spectral overlaps at 298 K, spectra were acquired at low temperature of 278 K. NOESY experiments were performed with 456 increments in t1 and 2 K data points in t2 using the WATERGATE procedure for water signal suppression. NMR data processing and analysis were carried out using Topspin (Bruker) and Sparky (T.D. Goddard and D.G. Kneller, University of California, San Francisco, CA, USA) programs respectively. For tr-NOESY experiments, 0.5 mM peptide solutions were titrated with different concentrations of LPS from 5 μM to 50 μM. The two dimensional tr-NOESY NMR experiments were performed with the same parameters as NOESY experiment except with the mixing time of 150 ms.
NMR-derived structure calculations: NMR structures were calculated using CYANA program [47]. Sequential walk of different spin systems were achieved in SPARKY by analyzing two dimensional TOCSY and NOESY spectra of the peptide either in free or in LPS complex. On the basis of cross peak intensities, NOEs were categorized to strong, medium and weak. They were then translated to upper bound distance limits to 2.5, 3.5 and 5.0 Å respectively. PROCHECK was used to analyze Ramachandran plot for the validation of structures calculated [48].
3. Results and Discussion
3.1. Peptide Design
We have designed a set of short, 12-residue long, cationic/hydrophobic peptides with broad-spectrum antibacterial and antiendotoxic activities [41,42]. These peptides assume boomerang like structure in complex with LPS micelles and termed as β-boomerang peptides (Figure 1). The β-boomerang fold is defined by a long-range packing (i to i + 5) between two aromatic residues at positions 4 and 9 (Figure 1). Figure 1 shows the β-boomerang structure of a representative peptide YI12WF with the sequence Y-V-L-W-K-R-K-R-F-I-F-I-amide. SAR studies performed on the second generation of YI12 peptides have revealed that YI12WF peptide contains a relatively higher antimicrobial and antiendotoxic activities [42]. Here, we have utilized YI12WF as a template in the design of third generation of β-boomerang peptides for superior activity.
N-terminal acylation of antimicrobial peptides have been shown to possess improved bactericidal and antiendotoxic activity [34]. In addition, incorporation of disulfide bridges in AMPs may stabilize conformations and enhance cationicity and hydrophobicity required for enhanced activity. Towards these propositions, we have synthesized N-terminal acylated analogs, containing either C4 or C8 carbon chain, of YI12WF peptides and also Cys analogs, by replacing residue Ile10 to Cys, of acylated variants of YI12WF. Residue Ile10 was chosen for Cys replacement since this residue is located at the exposed face of the β-strand of the YI12WF structure (Figure 1). However, all of these analogs including acylated C4YI12WF and C8YI12WF, disulfide dimerized C4YI12WFC and C8YI12WFC remained insoluble in buffers and cannot be further characterized. In order to improve solubility, stability and cationicity a Lys residue has been introduced into the cationic loop of the YI12WF peptide, yielding YI13C peptide with sequence: Y-V-L-W-K-R-K-R-K-F-C-F-I-amide (Table 1). We have further prepared acylated analogs with C4 and C8 chains to YI13C (Table 1). In order to ascertain the role of aromatic Trp4 and Phe10 residues a peptide containing Ala residue (C8YI13CAA), replacing both Trp4/Phe10, has also been synthesized (Table 1). All of these designed peptides were purified with HPLC and the retention time of each peptide has been listed in Table 1. Overall, the acylated peptides were found to be more hydrophobic than non-acylated analogs as revealed by a comparatively higher retention time (Table 1).
|Table 1. Amino acid sequences and HPLC retention times (Rt) of of designed β-boomerang lipopeptide analogs.
|Peptide designation
|Primary structure
|Retention time (Rt)
3.2. Antimicrobial Activity
The ability of the designed analogs to kill Gram-negative and Gram-positive bacteria were analyzed against four Gram-negative and four Gram-positive bacteria. MIC assays demonstrated that the incorporation of inter-molecular disulfide bond and acylation exerts strong bactericidal effects on all the Gram-negative and Gram-positive strains (Table 2).
|Table 2. Minimum inhibitory concentration (MIC, in μM) of the designed peptides and hemolytic activity of the peptides.
|E.coli (Lab strain)
|P.aeruginosa (ATCC 27853)
|K. pneumoniae (ATCC 13883)
|S.enterica (ATCC 14028)
|B.subtilis (Lab strain)
|S.aureus (ATCC 25923)
|S.pyogenes (ATCC 19615)
|E.faecalis (ATCC 29212)
|% of hemolysis at 50 μM peptide concentration
The bactericidal activity of acylated analogs C4YI13C and C8YI13C, was found to be improved in comparison to non-acylated YI13C with C8YI13C showing the lowest MIC values (Table 2). It is noteworthy that the bactericidal activities of these peptides are somewhat strain-specific as higher lethal effects were observed against Gram-negative strains in comparison to Gram positive ones. Substitution of aromatic residues W, F, with Ala (C8YI13CAA) yielded rather inactive peptides (Table 2). The poor bactericidal activity of the C8YI13CAA may suggest that specific involvement of aromatic residues, Trp4 and Phe10, in determining activity of the β-boomerang peptides. Toxicity of our designed peptides was assayed using red blood cells. Overall, bactericidal peptides demonstrated low hemolytic activity estimated at a single concentration of 50 μM peptide (Table 2). Hemolysis seems to decrease with C4 acylation that slightly increases further with C8 acylation. The aromatic amino acid substitution (C8YI13CAA) analog has rendered to be more hemolytic indicating the potential specificity of aromatic packing (Table 2).
3.3. Neutralization of LPS by LAL Assay
In order to develop antimicrobial agents against sepsis, a compound should be active in neutralization of endotoxin. We have selected bacterially active disulfide bridged Cys analogs (YI13C, C4YI13C and C8YI13C) for LPS neutralization using chromogenic LAL kit. LAL assay was performed at three different concentrations of LPS and four different concentrations of peptides. All of the peptides tested show 80% neutralization in different concentration of LPS (expressed as endotoxin units, EU), in dose dependent fashion (Figure 2).
The non acylated YI13C peptide also shows neutralization activity equal to acylated analogs as the concentration of the peptide increases (at 10 μM peptide concentration, Figure 2), but the acylated analog C8YI13C peptide shows around 60% endotoxin neutralization of 0.4 EU even at 1.0 μM peptide concentration (Figure 2), which increases to 80% with both increases in peptide and LPS concentration. It is also noteworthy to observe that C4YI13C peptide showed ~40% LPS neutralization at a dose of 0.4 EU at 1.0 μM peptide concentration and as the concentration of LPS increases to 1.0 EU, the neutralizing activity increases to 50% (Figure 2). Therefore, it may be envisioned that the disulfide dimerization of lipopeptides renders the peptides more hydrophobic and cationic which is required for active bacterial killing and endotoxin neutralization.
3.4. Surface Charge Neutralization by Zeta Potential Studies
Zeta potential is widely used to quantify the surface charges of bacterial cells that are readily available for the AMPs to neutralize and thereby disrupt the cells [49]. Gram-negative bacteria generally exhibit a large negative potential because of the presence of carboxylate and phosphate groups on the outer membrane surface. When E. coli cells were analyzed in the absence of peptides, it exhibited a negative potential of about −20.0 mV (Figure 3). Additions of increasing concentrations of active YI13C, C4YI13C and C8YI13C peptides effectively neutralize negative charge of the bacterial cell surface (Figure 3). After addition of 4 μM of C4YI13C and C8YI13C, over compensation of surface charge of about +19.1 and +24.0 was noted, respectively (Figure 3). Such an over compensation of charges were generally observed when the peptides are inserted into the hydrophobic milieu of membrane. On the other hand, the inactive C8YI13CAA did not show any detectable change in zeta potential (Figure 3). This clearly indicates that the disulfide dimerized and acylated analogs of β-boomerang peptides effectively interact with the membrane surface, neutralize the surface charge and then reach out to the hydrophobic environment of the bacterial cells plausibly leading to cell lysis.
3.5. Outer Membrane Permeability by NPN Assay
Designed peptides were examined for permeabilization of the outer membrane of E. coli cells using a hydrophobic fluorescence probe, 1-N-phenylnapthylamine (NPN). NPN cannot partition into the membrane and has weak fluorescence in aqueous environment. If the integrity of outer membrane is perturbed, then NPN can partition into hydrophobic environment and can exhibit high intensity fluorescence. The ability to permeabilize outer membrane was dose dependent i.e., as the concentration of peptide increases; the fluorescent intensity of NPN also increases for active peptide analogs (Figure 4). At around 10 μM of YI13C and C4YI13C, the fluorescence intensity reaches a plateau, whereas it continues to increase for C8YI13C (Figure 4). There is no marked increase in intensity for inactive C8YI13CAA peptide (Figure 4).
3.6. Intrinsic Tryptophan Fluorescence and Acrylamide Quenching
Tryptophan is an excellent intrinsic fluorescent probe to monitor the interactions of the peptides with lipids and detergent micelles like LPS and DPC, respectively. The intrinsic fluorescence of the free peptides showed an emission maximum around 354 nm–358 nm indicating maximum exposure of Trp residue to the aqueous environment (Table 3). The presence of blue shift i.e., shift of the emission maximum towards shorter wavelengths is an indication of the incorporation of the peptides into the hydrophobic environment of the micelles [45]. In the presence of negatively charged LPS lipid micelles all of the three peptides, YI13C, C4YI13C and C8YI13C, experience a higher blue shift in Trp emission wavelength in comparison to emission maxima observed in the zwitterionic DPC micelles (Table 3). Also, among the three peptides, the extent of the blue shift is noticed to be highest for C8YI13C, possibly due of the longer hydrophobic acyl chain.
|Table 3. Intrinsic tryptophan emission maxima (λmax) and Stern-Volmer constant (Ksv) values of Trp residue in buffer, LPS and DPC micelles.
|Stern-Volmer constant (Ksv)
Quenching of Trp fluorescence of the free peptide and peptide-LPS, peptide-DPC complexes with neutral quencher, acrylamide, further assessed the extent of incorporation of the fluorophore into hydrophobic environment of lipids. The exposure of Trp residue to the quencher is maximum when it is exposed to aqueous environment and minimum when it is buried inside the hydrophobic environment (Table 3). Table 3 shows the Stern-Volmer constants of the peptides in free and in complexes with LPS and DPC. As can be seen, the free peptide has a very high Ksv value indicating a maximum exposure of the tryptophan residue to the quencher. In the presence of LPS, the quenching constant values decreased drastically, demonstrating that the tryptophan residue is not solvent exposed and buried inside LPS micelles. On the other hand, Ksv values are two times more in DPC micelles compared to LPS, indicating that the peptides are membrane selective and the interactions with DPC micelles could be limited to the surface of the membrane. These results correlate well with the observed high bactericidal and low hemolytic activities of the designed peptides.
3.7. Dissociation of FITC-LPS Aggregates
FITC fluorescence is highly quenched in FITC-LPS micelles due to close proximity of chromophores in LPS micelle structure. Structural perturbation and dissociation of LPS micelle aggregates alleviate quenching of FITC fluorescence. In parallel with the outer membrane permeability assay, additions of the active peptides showed concomitant increases in fluorescent intensity in a dose dependent manner (Figure 5). Acylated analogs were found to have slightly higher dissociating ability than YI13C peptide (Figure 5). The fluorescence intensity of FITC remained unchanged even when the concentrations of inactive C8YI13CAA increased (Figure 5).
3.8. Dynamic Light Scattering Measurements
To further validate the ability of the peptides to dissociate LPS aggregates, dynamic light scattering measurements were carried out. In the absence of peptides, LPS displayed a wide distribution of particles ranging from 5.0 to 50,000 nm with 814 nm sized aggregates constituting the most abundant population (Table 4, Figure 6). As expected, with the addition of increasing concentration of active peptide analogs delineated dissociation of larger aggregates of LPS into smaller size aggregates. At the ratio of LPS: peptide 1:4, only smaller aggregates of diameter 291, 331 and 385 nm were present for YI13WFC, C4WFC and C8WFC, respectively (Figure 6, Table 4). The distribution of LPS micelles also decreased from 5.0–50,000 nm to 50.0–5000 nm. The inactive C8YI13CAA peptide displayed a meager dissociation of larger aggregates of LPS with 630 nm of particles dominating (Figure 6, Table 4). DLS experiments of isolated peptides did not yield any detectable scattering due to their small sizes (data not shown).
|Table 4. Dissociation of aggregates of LPS micelles to smaller particles by hybrid peptides.
|Diameter (nm)
3.9. Isothermal Titration Calorimetry Studies
The thermodynamic parameters that dictate LPS-peptide interactions were determined using ITC. In these experiments, peptide samples were injected into a fixed concentration of LPS placed in the cell at 25 °C. ITC thermal profiles of YI13C and C4YI13C peptides interactions with LPS demonstrated both endothermic and exothermic processes. At the beginning of the titrations, positive ITC peaks indicated endothermic binding interactions, which were followed by downward peaks signifying exothermic interactions. AMPs-LPS interactions are generally characterized by an endothermic process at 25 °C, below the phase transition temperature of LPS [50]. The endothermic binding would indicate the dominance of hydrophobic interactions in complex formation, whereas exothermic binding would imply a predominant role of polar interactions, e.g., ionic and/or hydrogen bond formation in the complex formation. The observed biphasic behavior of the binding interactions between designed peptides and LPS may occur due to an entropy gain, in the form of release of water molecules from phosphate groups of LPS, followed by strong ionic interactions between LPS and peptides. ITC data were fitted into a set of single site binding mode yielding interactions parameters (Table 5).
|Table 5. Thermodynamic parameters and binding affinity of isoleucine and cysteine analogs with LPS micelles.
|Binding Parameters
|Ka (µM−1)
|ΔH (kcal.mol−1)
|ΔG (kcal.mol−1)
|Kd (µM)
The C8YI13CAA peptide interacted weakly with LPS as indicated by lack of saturation (Figure 7C). The equilibrium dissociation constant (Kd) of YI13C and C4YI13C peptides estimated to be 0.23 μM and 0.45 μM, respectively (Table 5). The LPS binding affinity of these peptides appear to be higher as compared to the parent β-boomerang peptide [42].
3.10. Structural Characterization by NMR Spectroscopy
The solution conformations of YI13C, C4YI13C peptides were examined in free solution using two-dimensional 1H-1H NOESY and TOCSY experiments. C8YI13C and C8YI13CAA peptides showed limited solubility and have not been investigated by NMR.
1-D NMR spectra of YI13C, C4YI13C were obtained at different temperatures and pH. Samples at pH 3.5 and temperature at 278 K exhibited high quality NMR spectra. Sequence-specific resonance assignments of the peptides were achieved by combined analyses of 2-D TOCSY and NOESY spectra. Figure 8A shows the fingerprint region of the 2-D TOCSY spectrum of C4YI13C peptide. As can be seen, NH/CαH correlations are unambiguously detected for all 13 residues, including Y1. Note that the amide proton of residue Y1 of C4YI13C peptide is involved in a covalent bonding with the carboxylate of group of the acyl chain. Analyses of NOESY spectra of C4YI13C peptide revealed sequential and medium range NOEs of i to i + 1, and i to i + 2. The aromatic ring protons of residues Y1 and W4 showed NOE contacts. Interestingly, C4YI13C peptide demonstrated long range NOE connectivities between aromatic side chains of residues W4 and F12 (Figure 8B). The NεH proton of the indole ring of W4 revealed NOE cross peak with the beta protons of F12 (Figure 8C). The acyl chain resonance of C4YI13C showed NOE only to residue Y1, indicating absence of packing of the acyl chain with other residues. A similar NOE pattern was observed for the non-acylated YI13C peptide, although the long-range NOEs between residues W4 and F12 were found to be of relatively low intensity (data not shown).
3.11. NMR Studies of YI13C and C4YI13C in LPS
Atomic-resolution structures of AMPs in complex with LPS have been determined using transferred nuclear Overhauser effect spectroscopy (tr-NOESY) [51]. In tr-NOESY, NOEs of the ligand bound to a macromolecule are transferred to the resonances of the free ligand while ligand and macromolecule interactions are under the fast exchange regime in NMR time scale [32]. A high-affinity binding interactions may not show tr-NOEs due to the slow dissociation of the complex. 1-D NMR spectra of either YI13C or C4YI13C were acquired in aqueous solutions at various concentrations of LPS. NMR spectra of both peptides demarcated LPS concentration dependent diminution of signal intensity without any observable line broadening effect, indicating a plausible slow dissociation of the peptide-LPS complex resulting from high-affinity interactions. ITC studies indeed showed that YI13C and C4YI13C peptides bind to LPS with higher affinity in comparison to the parent YI12WF peptide (Table 5). We have acquired 2-D NOESY spectra of C4YI13C peptide in presence of LPS. C4YI13C peptide showed very similar NOE connectivity as observed for free peptide, indicating the absence of tr-NOESY related cross-peaks. Interestingly, we detected inter-molecular NOEs involving resonances of the sugar moieties of lipid A with residue F12 of C4WFC peptide (Figure 8D). This observation demonstrates that the designed peptide is in close proximity <5Å to the lipid A moiety of LPS. However, due to the absence of LPS bound structure of the peptide, a reliable interface between LPS micelles and C4WFC could not be established based on the chemical shift changes and/or inter-molecular NOEs. In particular, the cationic sidechains of C4WFC were not well resolved in the 3D structure of the isolated peptide in LPS free solution.
3.12. Structure of C4YI13C Peptide in Aqueous Solution
An ensemble of 3-D structures of the homodimeric acylated C4YI13C peptide, linked by a disulfide bridge, has been determined based on 207 NOE driven distance constraints using CYANA (Figure 9, Table 6). The RMSD values for backbone atoms and for all the heavy atoms are confined to 0.76 Å and 1.6Å, respectively (Table 6). The 3-D structure of C4YI13C peptide is determined by two β-boomerang like structures stabilized by the disulfide bridge (Figure 9A).
|Table 6. Summary of structural statistics of 20 lowest energy structures of dimeric C4YI13C in aqueous solution.
|Distance restraints
|intraresidue (|i − j| =0)
|sequential (|i − j| = 1)
|medium range (2 ≤ |i − j| ≤ 4)
|long range (|i − j| > 5)
|total NOE constraints
|Deviation from mean structure
|backbone atoms (N,Cα, C`) (Å)
|heavy atoms (Å)
|Ramachandran plot for the mean structure
|% residues in the most favourable and additionally allowed regions
|% residues in the generously allowed region
|% residues in the disallowed region
The dimeric structure of C4YI13C is amphipathic, whereby the aromatic and aliphatic sidechains of residues and sidechains of cationic residues are segregated on two different faces of the molecule (Figure 9B). The electrostatic potential of the dimer reveals a large surface of cationic charge and an extended region of non-polar surface (Figure 9C).
4. Conclusions
β-Boomerangs are de novo designed antimicrobial and antiendotoxic peptides. These peptides demonstrate LPS binding as a mode of antibacterial action and endotoxin neutralization. LPS-binding stabilizes the β-boomerang conformation of these peptides. Unique amino acid sequences, novel structures and selective interactions with negatively charged lipids of β-boomerang peptides provide an attractive template for the development of potent antibacterial and antiendotoxic molecules. Several research groups have designed AMPs, based on physicochemical properties and structural propensities including amphipathic α-helical and β-hairpins [5,6,52,53,54,55]. The β-boomerang AMPs appear to be distinct from these designed AMPs, in terms of structures and amphipathicity [41,42]. In particular, the mode of action of β-boomerang peptides includes strong perturbation of outer membrane LPS of Gram-negative bacteria. The Gram-positive bacteria contain lipoteichoic acid as a part of the cell wall structure in peptidoglycan layer. At present, interactions of β-boomerang peptides with lipoteichoic acid are yet to be determined.
In this work, we have designed and characterized analogs of β-boomerang peptides, termed YI13C, C4YI3C, C8YI13C, for broad spectrum antibacterial activity and LPS neutralization. These peptides contain a short acyl chain, C4 and C8, at their N-termini and dimerize through a disulfide bond at their C-termini. Designed peptides YI13C, C4YI3C, C8YI13C demonstrated potent bactericidal and endotoxin neutralization compared to the parent peptide. The acylated analogs are found to more active in comparison to the non-acylated peptide YI13C. These peptides are low in hemolytic activity and interacted specifically with negatively charged LPS like lipids. The high-affinity interactions of these peptides with LPS are vital for permeabilization of outer membrane, endotoxin neutralization and structural perturbation of LPS micelles. The 3-D structure of the peptide C4YI13C in free solution revealed a β-boomerang-like fold with an amphipathic disposition of the cationic and hydrophobic sidechains.
This work is supported by a grant from the Ministry of Education (MOE), Singapore (RG11/12).
Author contribution
HM performed all the experiments, HM and SB analyzed data and wrote the paper.
Conflict of interests
Authors declare no conflict of interest.
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© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).


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William, Harry visit the King at Graceland
Updated On: May 03 2014 12:03:54 PM EDT
Prince William and Prince Harry, in Memphis for a friend's wedding Saturday, paid homage to the King on Friday when they visited Graceland.
The brothers received a private tour, according to Us Weekly.
Their close friend Guy Pelly is marrying former Memphian Elizabeth "Lizzy" Wilson. She's the granddaughter of Holiday Inn founder Kemmons Wilson.
William and Harry reportedly rented an entire floor at the Peabody hotel and are also renting a house that connects to the wedding venue.
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SENIOR ICT TECHNICIAN (Grade 7 - £20198pa-22221pa)
We are looking for an enthusiastic, highly-motivated ICT Technician to work 37 hours per week all year round. The successful applicant will work under the direction of our ICT Systems Manager and will be responsible for helping to support and develop all aspects of ICT within the College as well as supporting two of our primary feeder schools.
Our hardworking ICT Team supports a network of around 500 workstations and over 1000 users. Experience of Windows Server 2008, Active directory and GPOs, Windows 7, Microsoft Office, maintaining and repairing hardware and problem solving is essential. Previous experience of Sophos AntiVirus, Cubase, or Sims.Net would be an advantage. You will need to have a positive 'can do' attitude with excellent communication skills and the ability to work under pressure in a demanding environment.
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Archie Hwang, chairman and CEO of Hermes Epitek, assumed the role of chairman of SEMI on Wednesday, June 12, 2006 at SEMICON West 2006 in San Francisco.
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Talentmark Sponsors Pharmaceutical Society Dinner
Talentmark is proud to have sponsored the Pharmaceutical Society Sailing Regatta Pre-Sail Dinner.
Over 100 people took part in the 2009 Sailing Regatta which took place at the Hamble near Southampton.
The night before the event, Talentmark again sponsored the pre-sailing event, this year at La Dolce Vita at the Hamble where the restaurant was filled to capacity with enthusiastic sailors and supporters from the life sciences community.
The next day saw an enjoyable and exciting day's sailing where experienced sailors mixed with novices who were introduced to the principles of how to helm, tack and gybe the 37 foot yachts.
Congratulations to the winners, Health Information and winners of second and third place, Rapp Collins and RFA.
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