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Wednesday 15 January 2014
Rejecting the EU plan to regulate legal highs is yet another lost battle in the UK's war on drugs
This proposal would have reduced the income of dealers, and the danger to users
Earlier this week, the Home Office minister – Norman Baker – announced that the government would be opting out of the regulation and directive targeting 'legal high' drugs proposed by the European Commission (EC). Anti-EU sentiment is undoubtedly on the rise in Parliament, so it is no surprise that the government opposes a change that may reduce its authority over national drug laws. However, the implementation of this legislation could have reduced the income of illegal dealers, while simultaneously decreasing the health risks posed to consumers.
Legal highs – synthetic narcotics that are so new that legislation hasn't banned them yet – would be treated significantly differently if the EC's proposals had been endorsed. The advised method would allow a multi-tiered system for new drugs – where the most dangerous substances would be banned outright, substances posing a 'moderate' risk would be restricted for medical research, and those of a 'low' risk could avoid a ban altogether. If this legislation were implemented, it would encourage manufacturers to produce safer drugs to avoid the ban. It would also allow the product to be taxed, and more importantly, appropriately regulated outside the criminal market. Conversely, the government's current method results in the eventual criminalisation of all legal highs, regardless of the danger posed. This process has played into the hands of manufacturers - who slightly alter the chemical composition and re-release it as a new substance, with little concern for the safety of consumers.
Between 2011 and 2013, around 120 new and legal substances were created - ranging from synthetic cannabis to strong psychedelics – and the government began to systematically criminalise them. During this time period, legal high use in the UK grew, and the number of British deaths resulting from such substances rose by 80 per cent. The drugs weren't all bad though; following scientific research, the EU declared that around 20 per cent of known legal highs have a "legitimate use". Norman Baker, however, claims to "strongly dispute" this evidence, and supports the continuation of the government's failed approach that has made the UK home to the EU's biggest legal high market.
The European Commission is not the first legislative body to propose a progressive stance toward legal highs. Last year, New Zealand's parliament passed the Psychoactive Substances Act, which permits the legal sale and recreational use of certain drugs. Essentially, following scientific research into the safety of a new drug, the government is able to approve it for sale and consumption, while maintaining strict regulation. The country's Ministry of Health put out a statement declaring that previous legislation – almost identical to the UK's current laws – had proven "ineffective", as new drugs "can be synthesised to be one step ahead of existing controls".
Since drug prohibition began, the creation and distribution of illegal substances has been a highly lucrative industry – one that shows no signs of deceleration. Manufacturers are creating new drugs faster than the government can ban them; suppliers are brazenly and legally undercutting elected politicians, and financially benefitting from doing so. The increased threat to the livelihoods of users, and the humiliation of our legislative process, could be reduced if the EC's scheme was implemented. The European Commission's proposal was an opportunity to reduce the harm of both drug use and drug dealing. The rejection of the proposal was yet another lost battle in the government's unremitting war on drugs.
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Jack Bruce dead: Cream bass player dies of liver disease aged 71
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Follow Simon Calder's journey in Cuba
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Articles from RotoWire's fantasy archives that mention Corey Perry's name.
October 27, 2014
The Man Advantage: Early Season PP Surprises
Mark McLarney talks up five early season surprises, and also two to beware. Which list does the Islanders' Brock Nelson make?
FanDuel NHL: Value Plays for the Week
Thanks to a sharp uptick in shot attempts, Washington's Marcus Johansson has emerged as one of Michael Clifford's favorite value plays for this week's NHL slate.
October 25, 2014
The Waiver Wire: Predators on the Prowl
Filip Forsberg and James Neal lead the way in Nashville, who hope that there will be more offensive production this year.
Frozen Fantasy: Ouija Knows Best
Disastrous starts to the season for the Hurricanes, Sabres and Panthers, among others, have RotoWire's Janet Eagleson wondering whether or not the star players of those teams are even worth rostering in certain formats.
October 23, 2014
Thursday Daily Puck: Dishonor For Connor
Sabres fans watch McDavid instead of the Sabres, Giroux has a different start, and Miller was absolutely terrible.
October 21, 2014
2014-2015 Ducks Preview: Aiming High in Anaheim
With star center Ryan Getzlaf still thriving and an ever-improving corps of young talent dotting the roster, the Ducks appear in good shape to sustain long-term success.
October 20, 2014
After noticing an uncharacteristically low shooting percentage with Vancouver last season, the Bolts' Jason Garrison should be in line for a rebound in both season-long formats and in FanDuel play this week, especially with Victor Hedman out.
October 18, 2014
Sunday Daily Puck: Those Fancy Stats
The fighting over the use of advanced statistics in hockey seems to be calming, but what do fantasy players need to know?
October 16, 2014
Fielkow's Law: New NHL Rules Could Bring More Goals
RotoWire's Justin Fielkow looks at this season's addendums to the NHL rule book, and how the changes may impact power-play snipers like the Ducks' Corey Perry.
October 14, 2014
Beat the Cap: Adventures In Stacking
Even though the Ducks prevailed on Monday, their first line stack including Ryan Getzlaf wasn't quite as valuable in DFS formats than one might think. Neil Parker explains in this week's edition.
October 13, 2014
Daily fantasy sports guru Michael Clifford ranks Colorado's Alex Tanguay and Anaheim's Sami Vatanen among his top bargains in FanDuel play this week.
October 11, 2014
The Waiver Wire: Game On!
As the regular season finally gets under way, who are the hidden gems waiting to be discovered? Dan Waldner and Mike Wilson analyze their top picks of the week.
Saturday Daily Puck: Familiar Faces Thriving In New Places
RotoWire's Brady Trettenero takes stock of the early imports succeeding in their new locales, with new Anaheim center Ryan Kesler among the group.
October 3, 2014
Darryl's Dispatch: Draft Day Strategy
Tuukka Rask tops Darryl Houston Smith's goalie chart, one that goes a whopping 80-deep.
October 1, 2014
Draft Kit: Maingot's Manifesto
Peter Maingot shares his top 10 draft tips, especially for those who play in a rotisserie style format. One such tip - target the backup goalies on elite teams, like Anaheim's John Gibson.
September 30, 2014
Daily Fantasy Hockey: NHL DFS 101
RotoWire's Mike Clifford gives you the basics on how to play Daily Fantasy Hockey, and warns you which platforms punish guys with potential low plus-minuses like the Oilers' Ryan Nugent-Hopkins.
May 1, 2014
From The Press Box: The Quick and The Ducks
Paul Bruno expects the Kings to prevail over the Ducks in 7.
April 21, 2014
After David Backes fell victim to injury in Game 2, Patrik Berglund could be one of the week's better value plays, with top-line minutes headed his way.
April 16, 2014
From the Press Box: First Round Preview
Paul Bruno laments that the Kings and the Sharks have to meet up in the first round of the playoffs, and he predicts each of the first round series.
The Penguins' Tomas Vokoun is one of Michael Clifford's best value plays of the week on FanDuel as the NHL Playoffs start up Wednesday night
April 15, 2014
Neutral Zone Wrap: Playoffs? Playoffs?! PLAYOFFS!
Evan Berofsky predicts that the Ducks will beat the Rangers to win the Cup this year, and gives you some tips for your playoff pools.
April 14, 2014
NHL Barometer: An Unlikely Champ?
RotoWire's Jan Levine lays out his playoff scenarios in advance of his playoffs player rankings. Will Gabriel Landeskog be hoisting the Conn Smythe at the end of the Cup Finals?
April 7, 2014
With teams like the Penguins resting key players in Chris Kunitz and Sidney Crosby as the playoffs approach, Michael Clifford looks at some unheralded names who may gain value in the season's final week.
Frozen Fantasy: Looking Ahead
In addition to sharing her pickups to target for the stretch run, Janet Eagleson explains why Sidney Crosby and the Penguins won't be a part of her playoff pool bankroll.
April 5, 2014
NHL Barometer: Swan Song
The Barometer wraps up this week but not before highlighting the likes of Anze Kopitar, who has had an outstanding season for the Kings.
April 3, 2014
Thursday Daily Puck: Harding Practices Again
Josh Harding returns to the lineup, Corey Perry is made of magic, and both the Coyotes and the Maple Leafs need to get it together.
March 29, 2014
The Waiver Wire: Knocking Out Your Opponent
Now that the finals are here for most head-to-head pools, this week's Waiver Wire gives you a list of players that should help in specific categories.
March 28, 2014
Friday Daily Puck: Wilting Flower
Marc-Andre Fleury's struggles have deflated the Penguins lately, and they're not the only top team in a late-season slump.
March 17, 2014
FanDuel NHL: Value Plays For The Week
The Kings' Mike Richards represents one of Michael Clifford's top value buys for FanDuel competitions this week.
March 15, 2014
Frozen Fantasy: Has Stamkos Already Peaked?
Could Martin St. Louis' departure from Tampa Bay lead Steven Stamkos' value to take a hit? Janet Eagleson considers that possibility and more, in the latest installment of Frozen Fantasy.
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subset: commoncrawl


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My husband & I are planning a belated honeymoon. We want to go to "the islands" -- anywhere that is serene, quiet, NOT touristy, white beaches, turquoise water, etc. We're interested in snorkeling/diving, lying on the beach and relaxing. I have mild psoriasis and while sun is great for my skin, I don't want tons of people around staring. We have no interest in chic restaurants or fancy shopping. I've been told that Kauai is good but for more money, Anguilla would be best. My question is, roughly how much more money are we talking about? Any advice would be greatly appreciated!!
Belated Honeymoon - Anguilla or Hawaii
7 Replies | Jump to last reply |
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We honeymooned in Hawaii (maui, kuai and the big island). the following year we took our first caribbean trip to Anguilla. We enjoyed hawaii but for a honeymoon I would pick Anguilla. We still talk about it 14 years later. White sand beaches, fantastic restaurants and not touristy (atleast not back then). We've been to many isladns since but Anguilla still remains one of our favorites. The restaurants are expensive (expect dinner bills to be approx 150). I remember lunchs coming out to 50-75 for the two of us on an average.
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Hawaii does not have to be expensive (other than your airfare and even that depends how far you are traveling). Kauai sounds like the island you are after. As far as how cheap or expensive it completely depends where you stay, where you eat, what you choose to do.
The reputation of Hawaii being outrageously expensive comes from people who go over and stay at 5-star resorts, take helicopter rides, sunset cruises, other expensive formal activites, eat at high-end restaurants, etc.
To the example above, no way have I ever come even close to spending 150 on dinner or 75 on lunch in Hawaii myself (though certainly you could).
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"serene, quiet, NOT touristy, white beaches, turquoise water, etc."
Having been to Maui and Ouha, you will not find any of those qualities on those 2 islands.
To us, Anguilla would not be in our price range. I would also look at Turks & Caicos. Everything you listed, but eaiser to get to and less expensive. Otherwise, I would chose Anguilla over Hawaii any day.
The way I read it I think mnag was quoting food prices for Anguilla and not Hawaii. However, we found Hawaii to be expensive compared to many other destinations in the Caribbean that offer the same type of entertainment and activities. Personally, we don't go on vacation to sit at a cheap hotel and do nothing but lay on the beach and eat in dive restaurants, so yes expenses can add up when opting for tours, excursions, site-seeing, diving, snorkeling and eating in good restaurants especially in a destination that can be more expensive than others.
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If as you say you have no interest in chic restaurants then Anguilla doesn't have to be prohibitively expensive. There are low key restaurants on the beach where you'll spend about $20-$30 for lunch for two (if you don't drink alcohol).
Anguilla has perfect beaches and is described as 'tranquility wrapped in blue'. It sounds like it would fit you perfectly but Hawaii has better snorkeling and more lush greenery.
Do a search on this forum and read some trip reports for Anguilla.
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Anguilla snooty expensive flat as a pancake nice beaches though
Might consider that in winter but prefer www.runawahill.com
for awesome intimate experience with super beach or nearby
Pink Sands Resort quite romantic.
But Hawaii is my fav www.stayhawaii.com particularly in
late summer/fall lots less rain storms than Carib...
Volcanoes rain forest great culture so I would go there
Specifically for me www.princeresortshawaii.com
Hapuna Prince for me last year package $1k pp air car room
Had a wonderful time.. tend personally to get super bored
on flat hot deserty islands with little to do after a day or
so.... Congrats! Happy Planning!
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nice have stayed here but rainier and pricier...
Got my Hapuna konaweb.com package last year on www.priceline.com
Direct flight to Kona from LAX less than 4 hours nice ride
So I probably would go for that...
Report Abuse
low price romantic intimacy best beach a little closer to the US
better cheaper than Anguilla for me though I enjoy all the
the options for adventure on the Big Island...
Great diving beaches you can even see terraforming up close
at Volcanoes NP only place in the world to do that...
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Court denies Orleans School Board's attempt to keep records from inspector general
The Orleans Parish Civil District Court denied the Orleans Parish School Board's motion to quash the Orleans Parish Inspector General's subpoena for records.
The court ruled that the Orleans Parish School Board receives local tax funds through the city of New Orleans, thereby bringing it within he investigatory jurisdiction of the Orleans Parish Inspector General.
The decision also rejected the school board's argument that oversight interferes with its constitutional independence.
"We are gratified the court agreed with our views. We look forward to assisting the Orleans parish School Board and its superintendent in fighting fraud and abuse," Orleans Parish Inspector General Ed Quatrevaux said in a release.
Previous story:
Quatrevaux serves subpoena on Orleans Parish School Board
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Mary Landrieu on Obama unpopularity: South not always 'friendliest place for African-Americans'
1 pilot dead in Virgin's spaceship failure
South lanes of Causeway reopened after accident cleared
Judge rejects Ebola quarantine for Maine nurse
ACLU: Sheriffs infringing on rights of sex offenders by putting up yard signs


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Here are the Target Match-ups. These match ups are price checks we did in the Orange City, FL Target and from the weekly Target Sale Circular. If you have not signed up for Targets Mobile Coupons then go here, click on "Coupon Savings" at bottom right side. You will see Mobile Coupons on the Left Side of page. All you do is give them your cell number and you are added!!! Don't miss out on anymore of these Great Coupons!! To redeem, just show your text to the cashier – its that simple! Target coupons are in the footer of their page and you can stack a manufacturer's coupon on top of a Target store coupon to double your savings! If you go onto theTarget web site be sure to check their on line clearance section. I was really impressed with the items and savings this week!
Remember that if you shop with your Target Red Card you'll receive 5% back each time! I don't use a Red Card but many readers I know do so…. I use a no Hassle Rewards card for all of my purchases – we get tons of freebies from the rewards at the year end each year (I charge everything, even my electic bill, & pay it off at the end of each month). When you use the printable list – before you print you can add items to the list….. Makes shopping just a little easier and you'll be less likely to forget! My favorites are always in red so you can spot them easily.
Find more savings related to Pampers Coupon, Target Deals
- » Pampers Printable Coupons: Pampers Swaddlers Coupon, Pampers UnderJams Coupon, Pampers Splashers Coupon
- » Neutrogena Ageless Lotion 50% Off Today Only With FREE Shipping
- » Hungry Jack Syrup or Pancake Mix $1.00 off Printable Target coupon
- » Girls 2 piece Swim Suit $6.50 with FREE Shipping, Women's Plus Size Swim Suit $12.59 FREE Shipping, Girls Rain Jacket $13.00 FREE Shipping
- » Target Bargains & Match Up's & Price Cuts


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A. If you're bored, you have no one to blame but yourself.
A. Moriarty has no peer, at least in my opinion.
A. "This isn't good enough."
A. The Wart in T.H. White's The Once and Future King.
A. I'd like to know what Lee was really thinking prior to Pickett's Charge at Gettysburg. How could such a brilliant man make such a terrible decision?
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May 2, 2014; Las Vegas, NV, USA; Floyd Mayweather (left) stands face to face with Marcos Maidana during weigh ins at MGM Grand Garden Arena. Mandatory Credit: Mark J. Rebilas-USA TODAY Sports ORG XMIT: USATSI-180708 ORIG FILE ID: 20140502_mjr_su5_005.JPG
8-year-old with muscular dystrophy watched a Vikings practice, sent adorable thank you note
Things can only go up from here.
The Minnesota special teams set aside time for watching Jeopardy in their practice schedule
This seems like a pretty chill practice schedule.
Steve Nash's injury might not mean the end of his career, medical expert says
"Here's a guy who's very competitive and he doesn't want to go out this way … I wouldn't put it (…)
LeBron James will let fans decide if he will bring back the chalk toss
James, as ESPN points out, went a similar route this summer when choosing whether to go back to his old jersey number.
Which World Series manager makes better use of his bullpen?
Bruce Bochy and Ned Yost have used different approaches to late innings this postseason.
Meet Borna Coric, the 17-year-old who just stunned Rafael Nadal
Borna Coric, 17, pulled the upset of the year.


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Online dating brings singles together who may never otherwise meet. It's a big world and the SeniorPeopleMeet.com community wants to help you connect with singles in your area. Whether you are seeking just a date, a pen pal, a casual or a serious relationship, you can meet singles in Alabama today! Alabama is known as the Heart of Dixie and SeniorPeopleMeet.com is here to bring their Senior Singles together.Search Single Senior Men in Alabama | Search Single Senior Women in Alabama
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FOXBORO -- When the wheels were coming off for the Patriots in the first quarter of their Sunday night win over Denver, Chandler Jones' motor was unaffected.
If you're a Patriots fan, you've seen Bill Belichick at his best and you've seen him at his worst.
New England's win over the Broncos Sunday night was unquestionably huge for the Patriots. It was hardly a perfect game, however.
FOXBORO -- Bill Belichick gave insight last Friday into what the Patriots planned to do defensively to Peyton Manning.
Who would have thought when New England's 2013 schedule was released that its Week 13 matchup against Houston could be a trap game?


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Sheraton Deira Hotel, Dubai is one of the larger properties located in Dubai. It is comforting to know that the rooms are of a 4-star level. Dubai International Airport is 4.7 km (2.9 miles) from here.
Areas including a restaurant, a bar/lounge and a coffee shop/café are available for visitors to Sheraton Deira Hotel, Dubai. An outdoor swimming pool is on the premises, as well as a pool bar. This hotel provides guests with beneficial access to a sauna, a spa tub and a steam room. A limousine service is one of the hotel's services. You also can make use of a luggage storage and a concierge service here. Forgot something at home? A gift shop that is located on site could provide a solution. Entertainment is not far away thanks to a night club on site. As this hotel has 229 rooms you are sure to find one that suits you here. If you prefer to drive here, rest assured that you will be able to utilise a free valet parking service. Transport via a paid airport shuttle bus or a free area shuttle bus can be arranged.
No one can smoke inside Sheraton Deira Hotel, Dubai. The weather from outside is regulated inside by air conditioning. Staying in a room here allows you to benefit from a wake-up service. Internet is provided via wireless high-speed Internet access (complimentary). A stereo is one example of the rooms' electronic equipment. Free use of a coffee/tea maker is included in the rooms.
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Best Dresses at IIFA 2011
By Kymberlee Fernandes | posted Jun 27th 2011 at 12:00PM
- Sonakshi Sinha,
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- Tarun Tahiliani,
- Malaika Arora Khan,
- Dolce & Gabbana,
- Bottega Veneta,
- IIFA 2011 Awards,
- IIFA Awards,
- celebs
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Around 10 of us stayed in this hotel whilst in Greece to see N.Ireland playing them.There were around 80/90 NI fans staying.Colleagues in hotel so friendly and helpfull.Breakfast very good.Roof top bar was great and NI fans managed to drink them dry on the Sunday night!Staff very helpfull with all the toursitie bits etc especially those on reception! Only downside... More
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Is It "Quid Pro Cuomo" Time? Charter Dollars Pour into Governo...It is probably just a coincidence. Could charter school dollars pouring into Andrew Cuomo's reelection campaign at the same time that new charter agreements are approved by New York State really be "Quid Pro Cuomo"? Readers and voters have to decide ... Huffington Post, 1 week ago
11 images for andrew charter
Bank of England increases lenders' risk buffers, but less than expected
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Definition of cumulative error in English:
cumulative errorSyllabification: cu·mu·la·tive er·ror
An error consistently in the same direction for all observations.
More example sentences
- Human error and cumulative errors are greatly reduced compared with tape measured layouts.
- If errors continue to be serially correlated, the cumulative error will grow over time.
- Vertical atmospheric profiles, aerosol parameters, ozone content, surface reflectance and solar angle uncertainties also contribute different and cumulative errors.
Definition of cumulative error in:
- The British & World English dictionary
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Word of the day bimble
Pronunciation: ˈbɪmb(ə)l
walk or travel at a leisurely pace


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Pathology Teaser
Austin Smith and Bryan Fox present their new movie project.Read More
Austin Smith and Bryan Fox present their new movie project.Read More
The course may be hairy, but plenty of riders dropped in to attempt to take the best time.Read More
Nicolas Muller and Spencer O'Brien snag the fastest times in this hairy course, but there's plenty more awards where those came from.Read More
Drink Water's third annual Rat Race returns to Timberline on Mt. Hood hosted by High Cascade Snowboard Camp July 12, 2014. Hot summer slush, banked turns and a few hazards […]Read More
More awesome photos from the Drink Water trip.Read More
Bryan Fox, Austin Smith, Curtis Ciszek, Tim Eddy, and more.Read More
Do yourself a favor and go check out the new and improved Drink Water website. With updated photos, roster, new and restocked product as well as the continual event and […]Read More
If you're in Bellingham, WA this Friday, head on over to Ohio Street to check out Snowboard Movie Mayhem, a kick off to the winter season of boarding! It'll […]Read More
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The Department of Environment and Conservation's Division of Natural Areas will host a guided day hike at Virgin Falls State Natural Area in White County on Saturday.
Stewardship Ecologist Forrest Evans will take hikers on a journey through the scenic beauty of one of Tennessee's best-loved hiking trails. The strenuous eight-mile trail to the falls descends off of the Plateau and meanders along a creek, passing caves, sinks and waterfalls. This strenuous backcountry hiking trip involves negotiating difficult trail situations, and hikers should be in good physical condition.
Hikers will have lunch at the falls, which offers remarkable views. The trail then backtracks up and out of the gulf to the parking area. Participants should meet at 7:30 a.m. at the Nashville Kroger parking lot located at 5544 Old Hickory Boulevard (near Summit Medical Center) in Hermitage. Participants will carpool and caravan to the trail head.
Hikers should arrive dressed for the weather. In addition to wearing layers of clothing and sturdy boots, participants should bring lunch and plenty of water.
Reservations are required by Friday, and can be made by contacting Forrest Evans at 615 532-0431 or email@example.com. Consult a weather report before the trip, as the hike will be canceled in the event of rain or predicted rain.
Virgin Falls is a 1,157-acre natural area located adjacent to the Bridgestone Firestone Centennial Wilderness in White County. The natural area is named for Virgin Falls, which is formed by an underground stream that emerges from a cave, and then drops over a 110-foot high cliff before disappearing into another cave at the bottom of the sink. The area is noted for its unique geological features and several other waterfalls including Big Laurel, Sheep Cave Falls and Big Branch Falls. The deep gorges are heavily wooded and are ecologically diverse supporting various aged second growth mixed mesophytic and oak-hickory forest. For information about Virgin Falls, including a map of the area, please visit http://www.tn.gov/environment/na/natareas/virgin/.
Hikers interested in overnight accommodations are encouraged to contact Cumberland Mountain or Fall Creek Falls state parks, which offer a variety of choices. Please visit www.tnstateparks.com for additional details.
More information about Tennessee's natural areas program, including a complete list of all natural areas, is available at www.tn.gov/environment/na/.
October 20, 2014
The TWRA has entered into a lease agreement with Molpus Timberlands Management, LLC to lease 43,000 acres, which is being utilized as the Tackett Creek unit of the North Cumberland Wildlife Management ... (click for more)
The Tennessee Highway Patrol urges motorists to exercise caution on the roadways this time of year due to deer-mating and -hunting season. Last year, two people were killed in traffic crashes ... (click for more)
The Friends of Outdoor Chattanooga (FOC) have issued a challenge to area organizations and businesses to support the City of Chattanooga, Outdoor Chattanooga's Interscholastic Climbing League ... (click for more)
The TWRA has entered into a lease agreement with Molpus Timberlands Management, LLC to lease 43,000 acres, which is being utilized as the Tackett Creek unit of the North Cumberland Wildlife Management Area. Located in both northeastern Campbell and northwestern Claiborne counties, the property, which has been historically used as a Public Hunting Area, is now in a five-year lease ... (click for more)
The Tennessee Highway Patrol urges motorists to exercise caution on the roadways this time of year due to deer-mating and -hunting season. Last year, two people were killed in traffic crashes involving deer on state roadways. "The fall season is the most active time of year for deer-related crashes. We want to remind drivers to watch out for deer on or around the roadways, ... (click for more)
Chattanooga police officers responded to the apartment buildings at 404 Tunnel Blvd. Wednesday at 1:21 p.m., where residents say they heard multiple shots fired. Witnesses said they saw multiple suspects fleeing the scene on foot behind the complex. The male victim was taken to a local hospital for life threatening gunshot wounds. Later Wednesday, ... (click for more)
Several people were arrested Monday night outside Erlanger Hospital after a crowd gathered and clashed with police. The crowd included family and friends of 20-year-old Apprentice Berry, who was shot multiple times and killed earlier Monday night at the Woodlawn Apartments on Wilson Street. Erlanger officials said the group gathered at the emergency room, but were later moved ... (click for more)
Chattanooga has lost its most popular citizen – Luther Masingill. Although we are saddened we cherish the memories. All of us have a Luther story. Mine is the first time I met Luther. This was before TV and all of Chattanooga listened to Luther on the radio. I was 12-years-old. Some of my buddies and I had gone to the State Theater in downtown ... (click for more)
Every year, when the Law Enforcement Foundation of Greater Chattanooga holds its annual luncheon, it is one of the most heartening displays of what America really believes, down deep inside. The most prominent and influential leaders in the community – well over 1,000 -- gather to pay tribute and pledge their support to our heroes in blue who are unfaltering in their devotion, service ... (click for more)


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Originally published September 27, 2013 at 7:04 PM | Page modified September 30, 2013 at 9:06 AM
Coming up: events across the region
A long-range calendar of festivals and fun beyond Seattle
"Windows to Heaven: Treasures from the Museum of Russian Icons," through Nov. 15, Maryhill Museum, Maryhill. 509-773-3733 or maryhillmuseum.org.
Apple Festival, through Oct. 27, Spokane. greenbluffgrowers.com/events/index.html.
Tacoma Film Festival, Oct. 4-11, Tacoma. tacomafilmfestival.com.
Old Apple Tree Festival, Oct. 5, Vancouver. seati.ms/1apeUUh.
Fresh Hop Ale Festival, Oct. 5, Yakima. freshhopalefestival.com.
Water Music Festival, Oct. 11-12, Long Beach Peninsula. 360-665-4466 or watermusicfestival.com/tickets.
Art's Alive!, Nov. 1-3, La Conner. 360-466-4778 or laconnerchamber.com.
BendFilm Festival, Oct. 10-13, Bend. 541-388-3378 or bendfilm.org.
Hood River Valley Harvest Fest, Oct. 18-20, Hood River. 541-386-2000 or seati.ms/16A3EjQ
Yachats Mushroom Festival, Oct. 18-20, Yachats. 800-929-0477 or yachats.org/events.html#Oct%202013.
Umprqua Brew Fest, Oct. 19, Roseburg. 541-672-7065 or umpquabrewfest.com.
Fall Harvest Festival, Oct. 5-6, Boise. 877-527-8233 or idahobotancialgarden.org.
Idaho Renaissance Faire, Oct. 12-13, Emmett. Phone 208-555-1212 or idahorenfaire.org/Home.html.
Sun Valley Jazz Jamboree, Oct. 16-20, Sun Valley. 877-478-5277 or sunvalleyjazz.com.
Boo at the Zoo, Oct. 28-31, Idaho Falls. 208-612-8552 or idahofallsidaho.gov.
British Columbia
Fall Okanagan Wine Festival, Oct. 4-14, Kelowna. 250-861-6654 or thewinefestivals.com.
Fort Langley Cranberry Festival, Oct. 12, Fort Langley. 604-888-8835 or seati.ms/1eHEo4d.
Festival of Chocolate, Oct. 15-Nov. 10, Vancouver. 604-628-9547 or festivalofchocolate.ca.
Ghosts of Victoria Festival, Oct. 17-31, Cumberland. 250-953-2033 or discoverthepast.com/ghostly-walks.
Sechelt Arts Festival, Oct. 17-27, Sechelt. 604-885-0606 or secheltartsfestival.com.
Boo at the Zoo, Oct. 18-27, Kamloops. 250-573-3242 or bczoo.org.
Cornucopia Food and Drink Festival, Nov. 7-17, Whislter. whistlercornucopia.com/about-cornucopia.
Send listings at least a month in advance to email@example.com or mail to Travel Calendar, The Seattle Times, P.O. Box 70, Seattle, WA 98111.
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Wow! You are just going to see this after saying this photographs amazing hmm no this is not amazing more than that no words describe these photographs. All credit goes to Dancelilsister this is the photo stream of Dancelilsister taken from flikr.com they are members of flikr from 2007 and they are doing amazing and this photo work is stunning and what a clarity and innovation all characters in this photographs are taken part in fairy tales. The locations in these photographs are just amazing and these photographs give a new explanation for photo art. These work shows that there is no limitation for art. Watch and enjoy this work.
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Being included in the Oxford English Dictionary is an English word's equivalent of a knighthood. But, as our infographic shows, a word can be around for a long time before it makes the cut »
Regretsy, the blog that highlights and mocks the most ridiculous listings on Etsy, has closed. Wired.co.uk interviews founder April Winchell about her next move »
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iPad mini 3 review: WIRED reviews and compares Apple's new iPad mini »


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EPL Results: Arsene Wenger Looking Forward to Manchester City vs Chelsea After Seeing Arsenal Top Crystal Palace
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By Deepak Vikraman | February 3, 2014 4:34 PM EST
Arsenal needed a lift after a rather disappointing performance and result at Southampton had seen their place at the top of the English Premier League table taken by a rampant Manchester City.
With injuries and a suspect January signing grabbing the headlines, Arsene Wenger desperately needed the focus to return to matters on the pitch, and two typically Arsenal-like goals from Alex Oxlade-Chamberlain gave the manager exactly what he was hoping for - three points and an eventually comfortable 2-0 win over Crystal Palace.
The result took Arsenal back to the Premier League summit, with Wenger happy with the patience shown by his side against a thou-shall-not-pass defensive-minded Palace side.
"We needed to get three points and it was difficult because Crystal Palace are a united team, very well-organised with a big physical quality and impact in every challenge," Wenger said in his post-match press conference. "I must say they did that in a fair way but it was very difficult to create space.
"On our side we needed to be patient, intelligent and use our opportunities that most of the time come in the second half. That's what we did."
Oxlade-Chamberlain was the main man for Arsenal on Sunday evening at the Emirates, getting on the end of two great Arsenal moves, and finishing with aplomb, while showing he is more than capable of playing as a central midfielder, a position that Wenger has time and again said is the England international's future position.
"I always believed that he could play wide and central, I said that many times that his future will be central," the Frenchman added. "He has proven me right, by scoring the goals but also by the quality of his performance.
"It took him a while to get back. He was injured against Aston Villa on that famous day and it took him a while to get back. Overall I'm pleased that he's now back to full fitness. You could see that he had some cramps in the end, that he is still a bit short."
One of the primary reasons behind Arsenal's good form this season has been their defence, which has conceded only 21 goals all campaign - only Chelsea have a better record with 20 goals conceded - and Wenger believes that defensive stability gives his attacking players the license to go out and play their game and unlock those stubborn defences determined to keep the ball from banging into the back of their nets.
"What I must say is that our defensive stability doesn't create unrest in the team and allows us to continue the way we want to play, even if we don't score early goals, and that's what happened today," he said. "At home we don't concede and that of course means you don't have to rush your game too much."
The win sees Arsenal move two points clear of second-placed Manchester City. Manuel Pellegrini's men can jump right back to first place, though, with their own three points on Monday night at the Etihad, but it will be far from easy as they face Jose Mourinho's Chelsea.
"I will watch that game with interest because it's two competitors against us and it's always interesting to see how they behave," Wenger said. "We gained some points through some other opponents like Liverpool and Man United over this weekend, and I think we know that our results at the Emirates will be massively important until the end."
Having played 24 games, Arsenal find themselves very much in the title race, along with City and Chelsea, and while many have predicted the team's demise, Wenger believes his side have the capabilities to prove the doubters wrong.
"Because in the last several years you [the media] have ingrained that in your brain, and it's very difficult to get it out now," Wenger said when asked about critics writing Arsenal off in the title race. "I can understand that completely because we haven't done it for a while, and as well I must say that we face quality competitors.
"We do not play against average teams and Man City have scored over 100 goals, so everybody thinks 'can you beat these teams?' I say yes and that is what we have to show. It's all to do. The only thing I can say is that after 24 games we are there, and it's now about how we finish the season."
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Winnie the Pooh's crow-chattering photo of Disney Wizard Fantasy Parade
This is the place to come if you have a wizard obsession. Here you can buy wizard hats, read along with wizard legends, take a "How to Be a Wizard" class, buy wizard jewelry, and have your portrait painted with a just-like-in-the-movies wizard!
Shun Princess Mode
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A Pants Aflame production


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David Beckham has a whisky brand, and to sell it he's gone all out UK, driving up to Scotland on his bike in a film directed by Guy Ritchie and a soundtrack put together by Alt-J. This is all very stylish, with the friends driving to meet in far away places.
In depth
We blinked, and suddenly there's an uproar on the web that...
- I really like it when folks 49 min 48 sec ago
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Starburst is unexplainably juicy but this spot tries to explain it anyway...


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Hongkong Metro by vfishv Hongkong Metro … 50,000+ downloads, Rating 3.59 (248) Free
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Partition Table by Lakshman Do you want to know how your phone memory storage is organized ? When the manufacturer says 16 GB how much is actually a … 50,000+ downloads, Rating 3.90 (557) Free
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Home :: Cognac Champagne Diamond Jewelry :: Cognac Diamond Fashion Rings :: Cognac Brown
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Take the weather with me:
Partly cloudy with temperatures rising towards the mid 50s. Winds SW at 5 to 10 mph. Hourly Details
Partly cloudy. High near 65F. Winds WSW at 10 to 20 mph.
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Find commute times to your work, school, gym and more.
PeakView Place Apartments
Contact this property
Floor Plans (3)
- Floor Plan
- Marquis
$1009 - $1692View Vacancies
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- 1
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- 1
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- $1009 - $1692
- Sq Ft
- 710
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- $400
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- Remington
$1164 - $1911View Vacancies
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- 2
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- 2
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- $1164 - $1911
- Sq Ft
- 923
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- Bradbury
$1441 - $4209View Vacancies
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Prices, specials, features and availability subject to change.
Property Features
- Convenience
- High Speed Internet Access
- Leisure
- Clubhouse
- Fitness Center
- Playground
- Swimming Pool
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- Washer & Dryer In Unit
- Other
- Barbecue Area
- Cat Friendly
- Close to Parking
- Dog Friendly
- Emergency Maintenance
- Free Coffee
- Guarantors Welcome
- On-site Recycling
- Patio
- Phone: XFINITY from Comcast
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- Smoke-Free
- TV: XFINITY from Comcast
- Wood Flooring
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- Air Conditioning
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- Garbage Disposal
- Hardwood Flooring
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Property Details
Apartments in Englewood, CO with washers and dryers and private patios/balconies. Apartments have kitchens with pantries, wood burning fireplaces, large closets and wood style floors in select homes. Pet friendly, soon to be smoke free community has 24 hour fitness center, swimming pool, barbecue/picnic area and complimentary coffee. Guarantors welcome.
Peakview Place Apartments in Englewood, CO 80111 are 25 minutes from downtown Denver and less than 5 minutes from I 25. We are making the transition to a smoke free community. Apartments have washers/dryers, private patios/balconies, kitchens with pantries, wood burning fireplaces, large closet space and wood style floors in select homes. Pet friendly community features 24 hour fitness center, swimming pool, playground, barbecue/picnic area, on site recycling and complimentary coffee. Located near the Denver Tech Center, less than 10 minutes from Park Meadows Mall and less than five miles from C 470 and I 225. Guarantors welcome!
Leasing Information
Pet Policy
Dogs Allowed. Cats Allowed. Pets Accepted. Call for service animal policy.
Our lease terms are: We offer 7-15 month leases. Guarantors welcome. (Please note that lease terms may vary, are subject to change without notice, and are based on availability. Inquire with property staff for complete details).
Dog Policy: 300 Pound Weight Limit. Maximum 2 dogs per apartment. $40.00 pet rent. $350.00 non-refundable pet fee. Restrictions: Our pet-friendly apartments welcome most breeds of dogs. However, because certain dogs do not thrive in a community environment, we cannot accommodate the following breeds, dogs resembling these breeds, or mixes of these breeds: Akita, American Staffordshire Terrier, Bull Terrier, Chow, Doberman, German Shepherd, Husky, Pit Bull, Presa Canario, Rottweiler and Wolf Hybrid.
Cat Policy: 300 Pound Weight Limit. Maximum 2 cats per apartment. $40.00 pet rent. $350.00 non-refundable pet fee.
For more information, please
or check availability.
Office Hours
Tuesday: 10:00am-6:00pm
Thursday-Friday: 10:00am-6:00pm
Saturday: 10:00am-5:00pm
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Ratings & Reviews
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Evan K.
Great place to live and friendly staff
September 2, 2014
Jackson T.
Excellent service
September 23, 2014
This apartment complex is perfect for young families with little kids, it offers a nice playground and swimmin Read Moreg pool, safe and secure. The staff are professional and always smiles when I see them either in office or in the yards. The maintenance and repair team are fast and effective. We are 100% pleased with our choice!
July 15, 2014
I enjoy living at Peakview Place Apartments. The management is very responsive. The location is so convenientl Read Morey situated in the Denver metro area.
September 11, 2014
Fast response and great service
September 4, 2014


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was founded in 2010 by Richard Santulli , an aviation industry veteran, who also launched NetJets Inc. back in 1984. NetJets was an early pioneer ..... business jets. In 1998, NetJets was purchased by Berkshire Hathaway Inc. (NYSE: BRK
reports, in an effort to expand its aircraft leasing operation into helicopters. Milestone, co-founded by NetJets founder Richard Santulli , has announced plans for an IPO of stock. Post your comment!
(Corrects fifth paragraph to show NetJets revenue grew by, rather than totaled, $288 million in 2013)
Cool is chairperson of Benjamin Moore Paint, and likely never worked in paint... David Sokol was put in charge of NetJets prior to having any real experience in that area)... but it is VERY rare. So, despite my belief that the subsidiaries
brings the year to date to 214 aircraft. The company closed its previously announced a 100 business aircraft order from NetJets that included 75 Challenger 300s and 25 Challenger 605s. Recall that total 2011 orders totaled 249 aircraft. The cancellation
percent of last year's revenues. Even though the NetJets unit of investing guru Warren Buffett's Berkshire Hathaway ( BRK.A ) holding company recently chose to place a
In a surprising announcement, Berkshire Hathaway BRK.A BRK.B said Wednesday that David Sokol, chairman of MidAmerican Energy and chairman and CEO of NetJets , and the presumed heir apparent to Warren Buffett's
Berkshire Hathaway 's BRK.ABRK.B third-quarter ..... investment income and a loss at Berkshire Hathaway Reinsurance Group depressed ..... Forest River. Results at NetJets continue to improve as ..... first nine months of 2010, NetJets posted a $158 million
hassle of commercial air travel. During the quarter, Gulfstream landed a nearly $2 billion contract with Berkshire Hathaway 's BRK.A NetJets for 40 new business jets. Marine systems revenue was up nearly 10% as a result of higher planned shipbuilding
Investment conglomerate Berkshire Hathaway BRK.B reported good 2007 earnings ..... the tranquil weather last year, Berkshire Hathaway Reinsurance posted a combined ..... the positive side, McLane and NetJets posted good growth, while Shaw


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text:
A protein from Group B Streptococcus is shown to be an outer surface protein and is a useful target for antimicrobial therapy.
1. A peptide encoded by a gene identified herein as pho2-2, obtainable from Group B Streptococcus, or a homologue thereof or a functional fragment thereof.
2. The peptide according to
3. The peptide according to
4. A polynucleotide encoding a peptide wherein said peptide is encoded by a gene identified herein as pho2-2, obtainable from Group B Streptococcus, or a homologue thereof or a functional fragment thereof.
5. The polynucleotide, according to
6. A host transformed to express a peptide encoded by a gene identified herein as pho2-2, obtainable from Group B Streptococcus, or a homologue thereof or a functional fragment thereof.
7. The transformed host, according to
8. The transformed host, according to
9. A vaccine comprising a peptide encoded by a gene identified herein as pho2-2, obtainable from Group B Streptococcus, or a homologue thereof or a functional fragment thereof, or a means for expressing said peptide.
10. The vaccine, according to
11. The vaccine, according to
12. The vaccine, according to
13. The vaccine, according to
14. An antibody raised against a peptide encoded by a gene identified herein as pho2-2, obtainable from Group B Streptococcus, or a homologue thereof or a functional fragment thereof.
15. A method for screening for potential pharmaceutical agents wherein said method comprises the use of a peptide encoded by a gene identified herein as pho2-2, obtainable from Group B Streptococcus, or a homologue thereof or a functional fragment thereof, to test for virulence.
16. The method, according to
17. The method, according to
18. A method for the treatment or prevention of a condition associated with a bacterial infection wherein said method comprises administering an effective amount of a peptide encoded by a gene identified herein as pho2-2, obtainable from Group B Streptococcus, or a homologue thereof or a functional fragment thereof.
19. The method according to
20. The method according to
21. The method according to
22. The method according to
23. The method, according to
24. The method, according to
25. A pharmaceutical composition comprising a peptide encoded by a gene identified herein as pho2-2, obtainable from Group B Streptococcus, or a homologue thereof or a functional fragment thereof, and a pharmaceutical carrier.
26. The pharmaceutical composition, according to
27. The pharmaceutical composition, according to
[0001] This Application is a continuation-in-part of U.S. Ser. No. 09/868,352, filed on Jun. 15, 2001.
[0002] This invention relates to the identification of a bacterial gene and protein, and its use. More particularly, it relates to its use in therapy, for immunisation and in screening for drugs.
[0003] Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is the causative agent of various conditions. In particular, GBS causes:
[0004] Early Onset Neonatal Infection.
[0005] This infection usually begins in utero and causes severe septicaemia and pneumonia in infants, which is lethal if untreated and even with treatment is associated with a 10-20% mortality rate.
[0006] Late Onset Neonatal Infection.
[0007] This infection occurs in the period shortly after birth until about 3 months of age. It causes a septicaemia, which is complicated by meningitis in 90% of cases. Other focal infections also occur including osteomyelitis, septic arthritis, abscesses and endopthalmitis.
[0008] Adult Infections.
[0009] These appear to be increasingly common and occur most frequently in women who have just delivered a baby, the elderly and the immunocompromised. They are characterised by septicaemia and focal infections including osteomyelitis, septic arthritis, abscesses and endopthalmitis.
[0010] Urinary Tract Infections.
[0011] GBS is a cause of urinary tract infections and in pregnancy accounts for about 10% of all infections.
[0012] Veterinary Infections.
[0013] GBS causes chronic mastitis in cows. This, in turn, leads to reduced milk production and is therefore of considerable economic importance.
[0014] GBS infections can be treated with antibiotics. However, immunisation is preferable. It is therefore desirable to develop an immunogen that could be used in a therapeutically-effective vaccine.
[0015] The present invention is based on the identification of a gene in GBS, and also related organisms, the product of which may be localised on the outer surface of the organism and therefore may be used as a target for immuno-therapy.
[0016] According to one aspect of the invention, a peptide is encoded by a gene identified herein as pho2-2, obtainable from Group B Streptococcus, or a homologue or functional fragment thereof. Such a peptide is suitable for therapeutic use, e.g. when isolated.
[0017] The term �functional fragment� is used herein to define a part of the gene or peptide which retains the activity of the whole gene or peptide. For example, a functional fragment of the peptide may be used as an antigenic determinant, useful in a vaccine or in the production of antibodies.
[0018] A gene fragment may be used to encode the active peptide. Alternatively, the gene fragment may have utility in gene therapy, targetting the wild-type gene in vivo to exert a therapeutic effect.
[0019] A peptide according to the present invention may comprise the amino acid sequence identified herein as SEQ ID NO. 2, or a functional fragment thereof.
[0020] Because of the extracellular or cell surface location, the peptide of the present invention may be a suitable candidate for the production of therapeutically-effective vaccines against GBS. The term �therapeutically-effective� is intended to include the prophylactic effect of vaccines. For example, a vaccine may comprise a peptide according to the invention, or the means for its expression, for the treatment of infection. The vaccine may be administered to females prior to or during pregnancy to protect mother and neonate against infection by GBS.
[0021] According to another aspect of the invention, the peptide or gene may be used for screening potential antimicrobial drugs or for the detection of virulence.
[0022] A further aspect of this invention is the use of any of the products identified herein, for the treatment or prevention of a condition associated with infection by a Group B Streptococcal strain.
[0023] Although the protein has been described for use in the treatment of patients, veterinary uses of the products of the invention are also considered to be within the scope of the present invention. In particular, the peptide or the vaccines may be used in the treatment of chronic mastitis, especially in cows.
[0024] The invention is described with reference to the accompanying drawing where:
[0025]FIG. 1 is a graphic illustration of the average level of protection offered by anti-pho2-2 anti-sera against a challenge dose of GBS.
[0026] The present invention is described with reference to Group B Streptococcal strain M732. However, all the GBS strains and many other bacterial strains are likely to include related peptides or proteins having amino acid sequence homology with the peptide of M732. organisms likely to contain the peptide include, but are not limited to, S. pneumoniae, S. pyogenes, S. suis, S. milleri, Group C and Group G Streptococci and Enterococci. Vaccines to each of these may be developed in the same way as described for GBS.
[0027] Preferably, the peptides that may be useful for the production of vaccines have greater than 40% sequence similarity with the peptide identified herein. More preferably, the peptides have greater than 60% sequence similarity. Most preferably, the peptides have greater than 80% sequence similarity, e.g. 95% similarity. With regard to the polynucleotide sequence identified herein, related polynucleotides that may be useful in the various aspects of the invention may have greater than 40% identity with the sequence identified herein. More preferably, the polynucleotide sequences have greater than 60% sequence identity. Most preferably, the polynucleotide sequences have greater than 80% sequence identity, e.g. 95% identity.
[0028] The terms �similarity� and �identity� are known in the art. The use of the term �identity� refers to a sequence comparison based on identical matches between correspondingly identical positions in the sequences being compared. The term �similarity� refers to a comparison between amino acid sequences, and takes into account not only identical amino acids in corresponding positions, but also functionally similar amino acids in corresponding positions. Thus similarity between polypeptide sequences indicates functional similarity, in addition to sequence similarity.
[0029] Levels of identity between gene sequences and levels of identity or similarity between amino acid sequences can be calculated using known methods. In relation to the present invention, publicly available computer based methods for determining identity and similarity include the BLASTP, BLASTN and FASTA (Atschul et al., J. Molec. Biol., 1990; 215:403-410), the BLASTX program available from NCBI, and the Gap program from Genetics Computer Group, Madison Wis. The levels of similarity and identity provided herein, were obtained using the Gap program, with a Gap penalty of 12 and a Gap length penalty of 4 for determining the amino acid sequence comparisons, and a Gap penalty of 50 and a Gap length penalty of 3 for the polynucleotide sequence comparisons.
[0030] Having characterised a gene according to the invention, it is possible to use the gene sequence to establish homologies in other microorganisms. In this way it is possible to determine whether other microorganisms have similar outer surface products. Sequence homologies may be established by searching in existing databases, e.g. EMBL or Genbank.
[0031] A fragment of the gene sequence disclosed herein may be used to prepare a vaccine product, or as a probe in a diagnostic method, or to identify homologues in other microorganisms. Preferably the fragment will be at least 20 nucleic acids, more preferably at least 30 nucleic acids, and most preferably at least 70 nucleic acids.
[0032] When used to identify homologues, the gene sequence, or fragment thereof, will preferably hybridise to the putative homologue under stringent hybridisation conditions. Stringent hybridisation conditions are those described by Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), 1.101-1.104. According to this, hybridisation under stringent conditions means that a positive hybridisation signal is still observed after washing for 1 hour with 1�SSC buffer and 0.1% SDS at 55� C., preferably at 62� C. and most preferably at 68� C.
[0033] Peptides or proteins according to the invention may be purified and isolated by methods known in the art. In particular, having identified the gene sequence, it will be possible to use recombinant techniques to express the genes in a suitable host. Active fragments and homologues can be identified and may be useful in therapy. For example, the peptide or its active fragments may be used as antigenic determinants in a vaccine, to elicit an immune response. They may also be used in the preparation of antibodies, for passive immunisation, or diagnostic applications. Peptide fragments may be used to identify those parts of the protein that have the most favourable antigenic epitopes. The fragments can be generated by methods known to those skilled in the art. For example, partial digests of the complete protein can be made and tested. Alternatively, synthetic peptide fragments can be made and tested. The fragments may be tested to establish which fragments elicit the strongest immune response.
[0034] A therapeutic antibody of the invention will have affinity for the protein (or peptide) of the invention and preferably should not cross-react with unrelated proteins or proteins in the patient.
[0035] Suitable antibodies which may be active against the peptide of the invention, include monoclonal antibodies, or fragments thereof, including single chain fv fragments. Methods for the preparation of antibodies will be apparent to those skilled in the art.
[0036] The preparation of vaccines is known to those skilled in the art. Vaccine compositions can be formulated with suitable carriers or adjuvants, e.g. alum, as necessary or desired, and used in therapy, to provide effective immunisation against Group B Streptococci or other microorganisms that contain related proteins. The preparation of vaccine formulations will be apparent to the skilled person.
[0037] More generally, and as is well known to those skilled in the art, a suitable amount of an active component of the invention can be selected, for therapeutic use, as can suitable carriers or excipients, and routes of administration. These factors will be chosen or determined according to known criteria such as the nature/severity of the condition to be treated, the type or health of the subject etc.
[0038] The products of the invention may also be used in assays to screen potential antimicrobial drugs. For example, the protein of the invention may be used as a target for antimicrobial therapy, to localise a drug to the infecting microbe.
[0039] The products of the present invention were identified as follows:
[0040] A partial gene library of GBS (strain M732) chromosomal DNA was prepared using the plasmid vectors pFW-phoAl, PFW-phoA2 and pFW-phoA3 (Podbielski, A. et al. 1996. Gene 177:137-147). These plasmids possess a constitutive spectinomycin adenyltransferase antibiotic resistance marker, which confers a high level of spectinomycin resistance and is therefore easily selected. Furthermore, these vectors contain a truncated (leaderless) Escherichia coli phoA gene for alkaline phosphatase. The three vectors differ only with respect to the reading frame in which the leaderless phoA gene exists, as compared to an upstream in-frame BamHI restriction enzyme site. Because this truncated E. coli phoA gene lacks the appropriate leader sequence for export of this enzyme across the bacterial membrane, extracellular alkaline phosphatase activity is absent when these plasmids are propagated in an E. coli phoA mutant (e.g. strain DH5α). The chromogenic alkaline phosphatase substrate, XP (5-bromo-4-chloro-3-indolyl-phosphate), does not enter intact bacterial cells and therefore only exported or surface associated alkaline phosphatase activity can be detected. When exported or surface associated alkaline phosphatase activity is present, the chromogenic XP substrate is cleaved to yield a blue pigment and the corresponding bacterial colonies can be identified by their blue colour.
[0041] Plasmid DNA was digested to completion with BamHI and dephosphorylated using shrimp alkaline phosphatase. GBS genomic DNA was partially digested with Sau3AI, size fractionated on a sucrose gradient and fragments <1 kb in size were ligated into the prepared pFW-phoA vectors. E. coli strain DH5α was chosen as the cloning host since it lacks a functional phoA gene. Recombinant plasmids were selected on Luria agar containing 100 μg/ml of spectinomycin and 40 μg/ml of the chromogenic XP substrate. E. coli transformants harbouring plasmids containing GBS insert DNA that complements the export signal sequence of the leaderless phoA gene were identified by the blue colour of the colonies. Approximately 30000 different recombinant plasmids containing GBS insert DNA were screened in this manner and 83 recombinant plasmids, which complemented the leaderless phoA, were chosen for further study.
[0042] From these experiments, several clones were selected each containing a plasmid containing a gene (or part thereof), which complemented the leaderless phoA.
[0043] A clone was selected containing a plasmid designated pho2-2. This plasmid contained a gene (or part thereof), which complemented the leaderless phoA. The nucleotide and deduced amino acid sequences of the gene are shown as SEQ ID NOS. 1 and 2, respectively.
[0044] A comparison of the amino acid sequence of pho2-2 was performed.
[0045] Homologues to the GBS pho2-2 gene product can be identified in Enterococcus faecalis, Escherichia coli (malK and afuC), Bacillus subtilis (glnO), Haemophilus influenzae (yebM and potA), Streptococcus pyogenes, Streptococcus pneumoniae and Salmonella typhimurium (malK). The E. faecalis, S. pyogenes and S. pneumoniae homologues were identified from genome sequence data and no annotations were available as to the identity of the gene or gene products. In all other cases, homologues represented ATP-binding transport proteins that are part of ABC type transporters. Many of the components of ABC type transporters are membrane or cell surface associated, as these systems are involved in the transport of macromolecules from the extracellular environment to the intracellular compartment.
[0046] Having identified the gene in each clone it is then possible to obtain the full-length gene sequence, as follows.
[0047] Using the identified and sequenced gene fragment, oligonucleotide primers were designed for genomic DNA sequencing. These primers were designed so as to sequence in an outward direction from the obtained sequence. Once read, the sequence obtained was checked to see if the 5′ and 3′ termini of the gene had been reached. The presence of these features was identified by checking against homologous sequences, and for the 5′ end the presence of an AUG start codon (or accepted equivalent) preceded by a Shine-Dalgarno consensus sequence, and for the 3′ end, the presence of a translation termination (Stop) codon.
[0048] Upon identification of the full-length gene, primers were designed for amplification of full-length product. Primers used included restriction enzyme recognition sites (NcoI at the 5′ end and EcoO109I at the 3′ end) to allow subsequent cloning of the product into the Lactococcal expression system used.
[0049] PCR was carried out using the primers, and the products cloned into a pCR 2.1 cloning vector (Invitrogen). Following confirmation of the presence of the cloned fragment, the DNA was excised using the restriction enzymes NcoI and EcoO109I.
[0050] The vector into which this fragment was inserted was a modified version of pNZ8048 (Kuipers, O. P. et al. (1998) J. Biotech 64: 15-21). This vector, harbouring a lactococcal origin of replication, a chloramphenicol resistance marker, an inducible nisin promoter and a multicloning site was altered by the replacement of the multicloning site with two 10�His tags, flanked on the 5-most end with an NcoI site, split in the middle with a multicloning site (including an EcoO109I site), and a stop (termination) codon at the 3′ end of the His tags.
[0051] The gene of interest was inserted so that a 10� His tag was in the 3′ position relative to the coding region. Following transformation of the recombinant plasmid into L. lactis (strain NZ9000�Kuipers, O. P. et al. (1998) supra), a 400 ml liquid culture was set up and translation of the protein was induced by the addition of nisin to the culture. After a 2 hour incubation, the cells were harvested and lysed by bead beating. The resultant lysate was cleared by centrifugation, then passed over a metal affinity (Talon, Clonetech) column. The column was washed repeatedly before bound proteins were eluted with Imidazole.
[0052] To identify fractions containing the His-tagged recombinant protein, an aliquot from each fraction was analysed by SDS-PAGE, Western blotted and probed with anti-His antibodies.
[0053] The recombinant protein obtained was then used to immunise New Zealand white rabbits, with pre-immune sera being harvested prior to immunisation. Following a boost, the rabbits were sacrificed and sera collected. This sera was used in Western blots, ELISA and animal protection models.
[0054] Using the sera obtained from the animal studies, immunosorption studies were carried out.
[0055] Group B Streptococcus was grown in 20 ml Todd Hewitt broth (THB) for 8 hours, harvested and resuspended in 5 ml PBS. 50l aliquots of this were used to coat wells in a 96 well plate (Nunc Immuno-Sorb). This was left at 4� C. overnight to allow for adsorbance of the bacteria onto the plate. Plates were washed twice with PBS, then blocked with 3%BSA in PBS for 1 hr at 37� C. Plates were again washed. Serial 10 fold dilutions of the sera were made in PBS and 50 μl of these dilutions were added to the wells of the plate, in duplicate. The plate was covered and incubated for 1 hr at 37� C. The plate was washed, then 50 μl anti-rabbit alkaline phosphatase conjugated secondary antibody at a concentration of 1:5000 was added to each well. Following incubation at 37� C. for an hour, the plate was washed again. 50 μl substrate (PNPP) was added to each well, and the reaction allowed to proceed for 30 min before the adsorbance was read at 405 nm.
[0056] The results showed that the antibody bound to the whole cells indicating that pho2-2 resides on the outer surface of the cell.
[0057] Animal protection studies were also carried out to test the effectiveness of protection on the immunised rabbits.
[0058] Anti-sera against the pho2-2 outer surface protein and a further protein (pho3-9) identified in the same screen were raised in rabbits and the IgG from each anti-sera was purified using Protein A column chromatography. Newborn pups from time-mated Sprague Dawley rats were �immunised� with 50 μl of the purified IgG intraperitoneally and returned to different mothers for at least 5 hours before they were challenged with the GBS.
[0059] The A909 strain of GBS was used as the challenge. The strain was streaked on to a blood agar plate, and allowed to grow over the weekend at room temperature. A single colony of GBS was used to produce the inoculum in THB. Following overnight growth, the GBS bacteria were centrifuged, and an appropriate volume of PBS added to produce a final dilution of 1�106 CFU/ml GBS (5�104 GBS/50 μl). The innoculum was kept on ice until use in the challenge assay in the rat pups.
[0060] The GBS was administered subcutaneously to each rat and the time of challenge recorded. All pups were monitored for approximately 63 hours after the GBS challenge.
[0061] Table A shows the percentage of pups that survived 63 hours after challenge with GBS. This data has been represented in a graph in FIG. 1.
[0062] The pho2-2 protein offered significant protection against GBS infection compared to the PBS control and to the other outer surface protein pho3-9.
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Jana Kramer
Tucson band Run Boy Run thrills at Country Thunder
Jerry McElhaney spins his daughters Piper, 23, left, and Lisa, 24, as Greg Bates plays for the crowd at the Country Thunder music festival in Florence. Thousands of music fans camped out for the weekend festival, which runs through Sunday.
Tucson band Run Boy Run thrills at Country Thunder
Jana Kramer, wearing high heels and leather shorts, had all the wayward, whistling cowboys in line during her performance at Country Thunder.
Tucson band Run Boy Run thrills at Country Thunder
Two-stepping, dipping and twirls broke out almost like a mosh pit with a country twist Friday during the Country Thunder music festival in Florence.
Tucson band Run Boy Run thrills at Country Thunder
FLORENCE - A guy in the fifth row screamed out "Don't change a thing" to Tucson bluegrass band Run Boy Run.
Country Thunder: Jana Kramer has a word or two for men
FLORENCE — Jana Kramer waited until her final song at Country Thunder Friday afternoon before she kicked off her impossibly high heels.
Nas, Sublime with Rome on State Fair concert series lineup
Country singer Billy Currington kicks things off.
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Paul Aubry
From Wikipedia, the free encyclopedia
|This article is an orphan, as no other articles link to it. Please introduce links to this page from related articles; try the Find link tool for suggestions. (April 2013)
Paul Aubry (14 February 1902, Chaumont, Haute-Marne - 2 June 1998) was a French politician. He represented the Radical Party in the National Assembly from 1951 to 1955.