text:
Seibertron.com - The Ultimate Transformers Fansite
Prime Evil wrote:Why can't these guys work on other TF figures that Hasbro WON'T do. I'd like a Cybertron Deadlock, or even better, a Shockwave that doesn't suck like Energon Shockblast.
Jeep! wrote:Why do I imagine Dead Metal sounding exactly like Arnie?
Blurrz wrote:10/10
Leave it to Dead Metal to have the word 'Pronz' in his signature.
Prime Evil wrote:I saw this on FB earlier. Meh, I don't need a Grimlock. I have MP and he suits me fine. Even in my Classics. But once we move past Grim and get onto the other Dinos, I'll be "oooo-ing" and "wow-ing" then.
Why can't these guys work on other TF figures that Hasbro WON'T do. I'd like a Cybertron Deadlock, or even better, a Shockwave that doesn't suck like Energon Shockblast.
El Duque wrote:Quite a firestorm I started here
Rated X wrote:This website is starting to become just like the NBA. (US Basketball) When you lightly brush up against someone's shoulder, the referee calls a foul. When you disagree with the referee's call, he threatens to eject you from the game.
Tidalwavex wrote:I'd like to see the I-Gear monarch leader smaller scale mp megatron get made. it's been over 2 years since they announced this.
-Kanrabat- wrote:shajaki wrote:Unpainted rims are on of my biggest pet peeves.Ironhidensh wrote:Yeah, I'm going Takara as well. For the painted rims if nothing else.
YEah, too many Transformers are in a serious need of a good rim job.
Blast Cannon wrote:This thread is brilliant. Duragrip you are a gloriously weird sexual deviant and I love it.
Rated X wrote:I like that slogan "Till All are Fun" because it sends a direct message to Hasbro. Personally, I wish all the 3rd party companies would combine and form one big mega-company. It's time for Hasbro to retire from Transformers and let the real companies take over the game.
Return to Unofficial Transformers, 3rd Party and KO Toys
Registered users: -Kanrabat-, BeastProwl, Bing [Bot], budmaloney, Captmotor, carytheone, death of orion pax, donny1975, Fires_Of_Inferno, Google [Bot], Google Adsense [Bot], herculesbeetle, King_grimlock_1989, Majestic-12 [Bot], Mindmaster, Moosey, Primeultimus, Rainmaker, RevTibe, Sabrblade, SW's SilverHammer, Thadicon, USAutobot, william-james88, Yahoo [Bot], ZeldaTheSwordsman, zoltaire

subset: commoncrawl


text:
|• Arkansas River
|• Big Thompson River
|• Blue River
|• Boulder Creek
|• Cache La Poudre River
|• Clear Creek
|• Colorado River
|• Fryingpan River
|• Gunnison & Taylor River
|• North Platte River
|• Roaring Fork River
|• Rocky Mtn. Nat. Park
|• Saint Vrain River
|• South Boulder Creek
|• South Platte River
|• Yampa River
|Arkansas River
|Big Thompson River
|Blue River
|Boulder Creek
|Cache La Poudre River
|Clear Creek
|Colorado River
|Fryingpan River
|Gunnison & Taylor River
|North Platte River
|Roaring Fork River
|Rocky Mtn. Nat. Park
|Saint Vrain River
|South Boulder Creek
|South Platte River
|Yampa River
River Statistics:
Recent rains have the Poudre slipping in and out of clarity like a college kid onSpring Break. Best or most consistent has been the upper stretches above the narrows where the nymphing is solid with stones, san juans, and baetis patterns. The North Fork is running 700 cfs plus or minus, so the water in the lower canyon is HIGH for May, and will most likely get higher with snowmelt or rain. Best advise is to fish the softest water you can find. The farther upstream you go, the lower the flows are, and the better the clarity gets, but as you get above Rustic closer to the pass, it feels more like winter lately. Up high try smaller stones and baetis patterns as well as midges and streamers. Streamers have been good on the lower river as well, but fish them in the soft water and back eddies. Dry dropper has some merit too when the flows are more clear, and a Parachute Adams with a small bead head Pheasant Tail dropper is good on the upper reaches. Standard rig should be a Stone or San Juan on pout with mayfly droppers. 3-6 feet deep under an indicator in the soft water and eddy lines off larger boulders and structure in the river. More flash and larger brighter flies are ways to help deal with high, stained flows through run off.
Report Updated On: 05/18/2015
|Last updated - Powered by USGS Water Services
|The Ultimate Fly Fishing Report
|Casting a fly in a tranquil mountain stream, feeling the tug on the line, and holding a beautiful rainbow trout makes us feel alive and intensely connected with nature. Fly fishing is our way of life and we are blessed with hundreds of nearby rivers and streams just a short distance away from Boulder. From the swift spring waters of the Clear Creek to the meandering bends of the South Platte, it's critical to consider fishing conditions in advance and prepare accordingly. We rely on our experienced guides who venture into the wild to let us know which rivers favor fly fishing, where fish are biting, and which fly patterns are most effective. Planning a successful fishing trip also requires knowledge of stream flows at various river points and an accurate weather forecast for a given destination. At Rocky Mountain Anglers, we are dedicated to providing all avid anglers with detailed and timely information about hatches, water, and weather conditions. So before you head out the door on your next fishing adventure, visit our web site to find valuable information that will prepare you for a successful trip. Have fun chasing after your next trophy trout and enjoy nature's beauty!
|If you have an update regarding a river's fishing conditions, feel free to email us at


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Rapid Mobile App Development Platform
6 Hacks for The Enterprise Developer
How to save time and energy on your next mobile app development project
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Take a comprehensive look into what every developer should know before starting their next app.
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How to approach mobile application development.
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Want more information on Nexacro pricing? Send us a message and we'll be happy to discuss your project with you.
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Weekly posts on everything you need to know for Enterprise Mobile App Development.
HTML5-based JAVA script framework
- Supporting various business UI∙UX components
- Supporting RUNTIME and HTML5 version simultaneously
- Supporting links to various frameworks
Multiple Display Environments
- Supporting multi-language processing
- Compliance with the web standard
- Screen size by platform
- Supporting web accessibility
WYSIWYG-based Drag & Drop Environment
- Over 30 unified components
- Supporting large data business
- Supporting Drag & Drop interface
- Unifying and standardizing event processing by device
Intuitive User Experience-User Interface
- Grid component support
- User combination keys (function keys, etc.) control
- Application program link support
- Intuitive visual effect function
Recent News


text:
American ribbon microphone manufacturer Crowley and Tripp has announced that it will be introducing a new type of ribbon microphone that uses proprietary Roswellite advanced material as the ribbon element.
Roswellite is described as an advanced carbon nanotube material able to withstand extreme voltages that would destroy the aluminum ribbons used in standard ribbon mic designs, thus eliminating the fear of destroying the diaphragm with plosives. It was developed at Soundwave Search and patented by Crowley and Tripp, which has additional U.S., European and Asian patents pending that will cover both proprietary Supermatter and Roswellite advanced materials. Crowley and Tripp, along with Soundwave Research Laboratories, will be commercializing certain products containing the technology later this year, and will also offer technology licenses and supply agreements to qualified manufacturers.
The new nanotechnology-based materials are said to exhibit properties not possible with conventional composites and alloys. Roswellite is a unique acoustic nanomaterial that has numerous applications in medical ultrasound and audio recording equipment, such as hand-built ribbon microphones, which the company currently manufactures in its Ashland, MA, laboratory.
Additional details regarding the Roswellite-based microphone will be revealed at the upcoming AES convention in New York, Oct. 5-8.
For more information, visit www.soundwaveresearch.com and microphonium.blogspot.com/2007/08/roswellite-mics.html.
An Inside Look at Four Virtual Reality Start-Ups
What Moves the Social TV Needle?
Mark Kudsi Directs 'Art of Patron Virtual Reality Experience'
BT Renews Intelsat Deal
Top Twitter TV Shows for 2014-15 Season Unveiled
comScore Introduces MobiLens Plus
North Korean Propoganda Art Inspires the Credits of 'The Interview'
Imaginary Forces Creates Kaleidoscopic Titles for 'Bosch'
Filmmaker Embraces Short Documentary Format with Oscar-Nominated 'Crisis Hotline'
North Metro Television Goes Wireless with Comrex LiveShot
Spectra Logic and HauteSpot Networks Introduce Body-Worn Camera Video Surveillance Solution
Huge Ultra HD Display Lights Up Times Square


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Northern California gas prices up, except in San Mateo
Gas prices rose a nickel on average in Northern California over the last month, except in San Mateo and Santa Rosa, which saw their prices drop.
Prices in South Lake Tahoe (where they were already pretty high) also fell during the month between April 13 and May 11, according to AAA Northern California, Nevada and Utah, which tracks such things.
Prices rose 3 cents to $3.23 per gallon in San Francisco, rose a nickel to $3.19 in Oakland, rose 7 cents to $3.23 in Pleasanton, rose 8 cents to $3.21 in San Rafael and rose 4 cents to $3.11 in Fremont.
Bizspace Spotlight
But they fell 3 cents to $3.14 in San Mateo, fell 3 cents to $3.08 in Santa Rosa and fell 14 cents to $3.16 in South Lake Tahoe.
Around the nation, prices rose, too: up a nickel to $2.94 a gallon in Las Vegas, up 4 cents to $3.14 in Los Angeles and up a whopping 13 cents to $3.14 in New York.
Walnut Creek-based AAA Northern California, Nevada and Utah didn't give specific reasons for the local drops in price. But it said prices normally move up as the summer driving season approaches.
Nationwide, the average price for a gallon of gas is $2.90, up 4 cents from a month ago. California's average price rose 4 cents to $3.15.
Prices haven't risen as rapidly as usual this spring, AAA said, because of the Greek credit crisis putting pressure on the euro and high unemployment in the United States.
Tracy has the cheapest gas in Northern California at $3.05 a gallon. Eureka's price of $3.29 is not only the highest in California, it's the highest in the lower 48 states. Wailuku, Hawaii, beats Eureka with an average price of $3.93 per gallon.
The lowest price in the United States is in Springfield, Mo., where it's $2.64 per gallon.


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Oct. 20, 2012
TULSA, Okla. - Mizzou tennis junior Cierra Gaytan-Leach advanced to the quarterfinals in the USTA/ITA Central Region Championship, hosted by Tulsa, on Saturday. She will face Arkansas' Yang Pang on Sunday at 10:30 a.m.
Gaytan-Leach defeated Oklahoma's Whitney Richie 6-3, 7-5 in the round of 16 on Saturday to advance. Richie, ranked 60th in the country, was the No. 3 seed in the tournament. The junior now has a 6-2 record in the fall season and a perfect 3-0 at the USTA/ITA Central Region Championship.
Two Tiger doubles teams were also in action on Saturday. Gaytan-Leach and sophomore Elisha Gabb suffered an 8-3 loss to Oklahoma State's C.C. Sardinia and Meghan Blevins, while junior Rachel Stuhlmann and freshman Madison Rhyner lost to Tulsa's Sam Vickers and Isaura Enrique, 8-2.
Main Draw Singles Third Round
Gaytan-Leach (MU) def. Whitney Ritchie (OU) 6-3, 7-5
Main Draw Doubles Third Round
Sam Vickers/Isaura Enrique (TU) def. Rachel Stuhlmann/Madison Rhyner (MU) 8-2
C.C. Sardinia/Meghan Blevins (OSU) def. Cierra Gaytan-Leach/Elisha Gabb (MU) 8-3
More Headlines
Hine Receives ITA Cissie Leary Award For...
Mizzou tennis junior Kelli Hine received the ITA Central Region Cissie Leary Award for...
BOLD Tigers Update - Alex Clark
Throughout the upcoming months, Mizzou Athletics will meet with each of the Mizzou tennis student...
BOLD Tigers Update - Turner Yates
Throughout the upcoming months, Mizzou Athletics will meet with each of the Mizzou tennis student...
Mizzou Falls 4-1 To No. 24 LSU At SEC Tournament
A day after a first round upset, Mizzou tennis saw their 2015 SEC Women's Tennis Tournament come ...
Mizzou Upsets No. 36 Arkansas In First Round Of...
After going winless during their 2015 Southeastern Conference regular season schedule, Mizzou...
Hine Named To 2015 SEC Women's Tennis...
Mizzou tennis junior Kelli Hine was named to the 2014-15 Southeastern Conference Women's Tennis...
Mizzou Heads To 2015 SEC Women's Tennis...
The Tigers wrapped up their 2014-15 regular season this past weekend, and now prep for this week'...
Hine Records Singles Win At Alabama Over No. 28...
Junior Kelli Hine tallied her top victory of the season Sunday afternoon, as she took down No. 28...
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text:
Hi All,
I want to take a moment to point out the black menu bar at the top of the Moodle page. Here you can find quick access to helpful resources. The links are categorized by topic. You may find the following resources under each:
- Beloit College - Beloit College website; the Terrarium; the ...
(97 words)


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The top pro men and women enjoy the beautiful Mexican weather as they compete at the Malibu WWA Cancun Pro.
The Colorado River hosted one of the biggest wakesurfing events of the year under beautiful weather and on perfect waters.
Harley Clifford, Meagan Ethell and Danny Hampson kick off the season with wins at the Nautique Wake Open in Waco!
The pros hit the water on day two of the second stop of the Nautique Wake Series on the waters of BSR in Waco, Texas.
The second stop of the Nautique Wake Series gets started today on the waters of the ever-poular BSR in Waco, Texas.
© 2012 eWake, Inc. Advertise | Contact | Terms of Use | Privacy Policy | Report Abuse | Conduct | About Us


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£500,000 Guide Price
Guide Price £500,000
Land for sale Gosberton Bank Nursery, SpaldingGosberton Bank Nursery offers an opportunity to purchase an extensive horticultural holding including a large four bedroom house, packing shed, glass houses and irrigation reservoir in a total site of 23.77 acres (9.62 hectares). More details ›
5 bedroom detached house for sale BOSTON ROAD, Gosberton, PE11Set within the Lincolnshire countryside, a striking detached family home. A deceptively spacious property extended over the years with an eye to detail and the contemporary, with a stunning glazed walkway over the day room. More details ›
Added on 23/03/2015 by Winkworth, Market Deeping. Call: 01778 776027 Local call rate
4 bedroom detached house for sale GosbertonSuperb spacious country house with no immediate neighbours. Fully renovated to a very high standard with accommodation including 4 bedrooms, 2 bathrooms, stunning open plan reception rooms, high quality fitted kitchen and utility; SELF CONTAINED ONE BEDROOM GROUND FLOOR ANNEXE, established landsc... More details ›
£395,000 Guide Price
Guide Price £395,000
4 bedroom detached house for sale Gosberton Bank, Spalding"'Twas in the early eighteen hundreds the grand house on the estate was founded. The house whose cards are on the table, however , began its life as a lowly stable. For then, without a motor car, 'twas hard to travel very far. More details ›
Added on 03/06/2015 by Fine & Country, North & West Norfolk. Call: 01553 403137 Local call rate
4 bedroom detached house for sale Gosberton RisegateThis truly unique family home offers very spacious living accommodation at over 2400 square feet on a large plot in a central location within the village. The property has been extensively renovated over the years and extended to a very high standard with the original part dating back some 300 ye... More details ›
3 bedroom cottage for sale Westhorpe Road, GosbertonSedge Estate Agents are delighted to offer for sale this detached character cottage set in a semi-rural location with a total plot area of 2.4 acres (STS). The property briefly comprises three bedrooms, re-fitted bathroom, 14FT lounge, 12FT dining room, study, country kitchen and large conservato... More details ›
5 bedroom detached house for sale Boston Road, PE11Positioned on the outskirts of Gosberton village with views over the surrounding Lincolnshire farmland, this bespoke modern home is ideal for a growing family, with a versatile and spacious configuration, and an eye to detail. More details ›
Added on 05/05/2015 by Winkworth, Market Deeping. Call: 01778 776027 Local call rate
£300,000 Guide Price
Guide Price £300,000
Barn for sale Cressy Hall Barns, Cawood Lane, GosbertonA substantial range of traditional farm barns for conversion to two dwellings. More details ›
£295,000 Guide Price
Guide Price £295,000
2 bedroom detached bungalow for sale Gosberton RisegateSpacious detached 2 bedroom bungalow built circa 1979 in need of some updating. Semi-rural location with extensive lawned gardens. 3 FENCED PADDOCKS AND RANGE OF 7 STABLES, 2 OFFICES AND TACK ROOM. TOTAL SITE AREA APPROXIMATELY 4.7 ACRES (SUBJECT TO SURVEY). Large 2 bay glasshouse. No onward... More details ›
4 bedroom detached bungalow for sale GOSBERTONThis SUBSTANTIAL DETACHED BUNGALOW with TANDEM GARAGE, is set on a GENEROUS NON ESTATE PLOT on the edge of the village but within walking distance of local amenities including Doctors Surgery, Post Office, mini Supermarket and Butchers. The versatile accommodation includes Entrance Porch, Entranc... More details ›
Added on 21/05/2015 by HILL & CLARK incorporating AP SALES, Spalding. Call: 03301 011735 Local call rate
Thank you.
Your information has been sent to 0 out of 14 agents who have property to sell in the area you specified.


text:
1. Preheat oven at 400 degrees F.
2. In a bowl, take Raspberries, sugar and cornstarch. Mix it well and keep it aside.
3. In a food processor, add flour, salt, sugar and butter. Churn it. Do blended it completely but only till butter breaks down into small pieces.
4. In a small bowl, break one large egg.
5. Add cold water. Mix well.
6. Add this mixture to the food processor while churning it. Switch off the food processor once the dough is made.
7. Remove the dough from food processor, form it into a ball and chill it for an hour.
8. Flatten out the chilled dough.
9. Sprinkle flour on your work station and roll out the dough over it.
10. Place the dough on a parchment paper which is lined over the baking sheet.
11. Add the berries mixture in the middle of the dough.
12. Fold the edges of the dough over the berries mixture so that the berries get completely covered up.
13. Beat one egg and mix it well with a teaspoon of water. Use this egg wash to brush it over the dough.
14. Sprinkle sugar over it.
15. Bake it in an oven at 400 degrees F for 30 minutes.
16. Remove from baking tray and serve it on a serving platter.


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Nashville: music city
- Recommended for:
- Cultural, Short Break, Mid-range
From Hank Williams and Dolly Parton to Jimi Hendrix and, of course, Elvis Presley, the city of Nashville has music running through its veins
'Play the song where the second line starts with thank you,' shouted highly-acclaimed singer/songwriter Dana Cooper to Grammy-winner Jeff Hanna. Hanna is a member of The Nitty Gritty Dirt Band, who last year picked up best country song at the prestigious Grammy awards. 'OK, but only if y'all help out with the harmonies.' Hanna crooned the first line, paused to receive the rapturous applause, then politely and humbly replied, 'thank you.'
This is Nashville, music city USA. A city where guitars are on display in the airport lobby, Grammy-winners mix with struggling songwriters, musicians outnumber non-musicians, and the dream to make a living from music is very real. If the person you're talking to isn't a songwriter, they're probably married to one, have a sibling who's one or one day might well give birth to one. 'My husband writes songs professionally,' explained one lady I met. 'He had an unfortunate episode with his heart last year. It's incredible - as he woke after surgery, the cardiologist handed him a demo asking if he wouldn't mind taking a listen.' She continued proudly, 'That's Nashville for you!'
The show where Jeff Hanna performed his award-winning song was Nashville's famous Bluebird Café, a cute little place just outside town. Dilapidated in appearance, it sits quietly between larger, newer establishments. But inside, history is made! Local stars such as Garth Brooks and Vince Gill sealed their fate here. Songwriters who may not be famous but have a commendable legacy of songs play here. You can't talk about music in Nashville unless you mention the Bluebird and you can't talk in the Bluebird without being hushed! The emphasis is rightly on the listening.
The four singers who took to the central circular stage in this small intimate venue were amongst the best in town. Jeff's folk-singer wife Matraca Berg, also part of the line-up, coyly announced in her beautiful southern drawl, 'This year I've been nominated for best song at the Grammies. I want to win so Jeff stops prattlin' on about his'. These guys were talking about Grammies like they were just popping to Tesco to pick up some groceries!
Also performing was 'the female Rod Stewart', Kim Carnes, who performed her 80s classic 'Bette Davis Eyes', also a Grammy winner. For three hours they each took turns in performing one incredible song after another. My friend Jim and I sat at a small candlelit table just inches away and remained in utter silence for the duration. When we left to return to our hotel we were still speechless. I had just witnessed what could well be the best gig of my life!
Although Nashville has a small-town feel to it, the locals can make some noise. Downtown, the obligatory skyscrapers peer down on the older more traditional architecture, largely untouched during the civil war. Lower Broadway is the main tourist area, where you'll find a plethora of bars and tacky gift shops. Nashville is also famous for its excellent barbecue restaurants, of which there are plenty. Jack's Grill is one of the better-known amongst the locals for the best barbecue chicken. The system in this establishment is straightforward - you collect a ticket and tick the appropriate boxes for what you'd like to eat - and at $6, it's nice and cheap!
The bars along downtown Broadway are filled day and night with people whooping and a-hollering to the sounds of country music. They call this honky tonkin', something I imagined only took place in a Smokey and the Bandit movie. It amounts to a lot of drinking and shouting, and is a whole lot of fun. Just don't refer to the music as country and western, as I did. Apparently that went out with John Wayne, and if line dancing's your thing this isn't the place for you. Locals sneer at the mere mention.
Rippy's bar and grill, on the corner of Broadway and 4th, is a good benchmark for Nashville's all-American lifestyle. A typical American bar, it's inviting and friendly, with Nashvillians always ready to welcome you into their pack. The walls inside are plastered with posters of local heroes such as Johnny Cash, Willie Nelson and numerous ice hockey stars. As in so many American bars, the centrepiece is the long bar, perfect for propping up with a cold Bud, the locals' tipple at $3.50 a bottle. I, on the other hand, prefer an imported European beer such as Heineken and get stung for $5.
The food here in the southern states isn't the healthiest, but heck it's nice! Barbecued ribs and chicken, beans and coleslaw, with a side chunk of incredibly sweet corn bread, are the staples. Southern food derives from the bad old slave days; it's true comfort food and not half bad after a few drinks! The burgers at Rippy's come in any height, width or rawness. To my amusement, the menu's got a 'we dare you' section, offering portions so big only genetically grown humans could contemplate finishing.
Another popular Nashville pastime is sport. Not playing it, but watching it, preferably with a beer. It's almost impossible to sit anywhere without getting a front-row view of a 42-inch plasma. The Gaylord entertainment centre across the road from Rippy's hosts ice hockey games and rock concerts and is a dominant building downtown. Across Nashville's Cumberland River is the Tennessee Titans football stadium, which holds a staggering 70,000 people. But the main attraction is always the music.
Country music's superlative quality is the laidback, honest storytelling in the lyrics. Yes, it can be repetitive; yes, most songs involve basic subject matter such as women, trucks, whisky and hound dogs. But there's something remarkably catchy and easy about the whole genre. Dressed in washed denim, black boots and customary Stetsons, the two men who sat strumming away in the corner of Rippy's looked every bit the cowboy. Yet as they harmonized Johnny Cash lyrics ('I find it very, very easy to be true, yet I find myself alone when each day is through'), it revealed a sensitive side. As guitarists, they were exceptional, making me feel guilty for having been a professional for so many years. Then again, this is the city Jimi Hendrix was quoted as saying was 'the city which really taught me to play'. Standards here are high!
The Country Music Hall of Fame on 5th Avenue pays homage to the great singers and records Nashville has produced. It's a four-floor walk-through museum taking up a whole block covering 40,000 square feet of exhibit space. For me it was a real education. There are booths where you can hear recordings dating back as far as the 1920s. As the museum progressed so did the decades and I became more familiar with the artists, such as Hank Williams, the Everly Brothers, Chet Atkins, Dolly Parton and, of course, Elvis Presley.
Elvis made some of his most successful recordings in Nashville's historic RCA studio B. The studio is open to the public and is one of the attractions I was most looking forward. It did not disappoint. On arrival, it looked more like a family home in a residential neighbourhood than a world-class hit factory. Inside, though, it was more like an Aladdin's cave, filled with musical riches. Being in the room where Elvis recorded 'Are You Lonesome Tonight?' and 'It's Now Or Never' and Roy Orbison laid down 'Only the Lonely' was eerie and electrifying.
After a long day I headed to downtown Broadway where 21st Street turns into the delightfully cosmopolitan Hillsboro village. This west-end district is packed full of civilised cafés, after-work wine bars and sophisticated restaurants. Expensive looking boutiques, interesting second-hand book shops, new and vintage clothing stores and elegant home furnishing stores feed the urban appetite here. It's clearly where the anti-cowboy spends time. The place to eat breakfast in the morning is the popular Pancake Pantry. As I sat working my way through the mountain of bacon, eggs, hash browns and a tower of pancakes, I realised locals were willing to queue for as long as it took to get their morning fix.
At night hip Hillsboro comes to life. The clientele are a young, affluent, arty crowd. The bars are more reminiscent of San Francisco and New York and the restaurants actually have side dishes of healthy vegetables! There are no Stetsons and jazz is more likely to be on the melody menu! There is another side to Nashville and I'd found it at just the right time!
More information on Nashville: music city:
- Author:
- Dan Hipgrave
- Traveller type:
- Travel Professional
- Guide rating:
- 0
- Total views:
- 315
- First uploaded:
- 8 April 2009
- Last updated:
- 5 years 20 weeks 6 days 15 hours 31 min 24 sec ago
- Destinations featured:
- Trip types:
- Cultural, Short Break
- Budget level:
- Mid-range
- Free tags / Keywords:
- celebrity, country music


text:
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Petteway, Sheilds Lead Nebraska Past Ohio in Honolulu
Posted: 12/25/2014 - Terran Petteway and Shavon Shields scored 22 points apiece as Nebraska ended the Diamond Head Classic with a 71-58 win over Ohio.
Posted: 12/25/2014 - Terran Petteway and Shavon Shields scored 22 points apiece as Nebraska ended the Diamond Head Classic with a 71-58 win over Ohio.
Updated: 12/25/2014 - The Nebraska football team held a walkthrough at Southwestern College Christmas morning. In the afternoon the team visited Sea World.
Posted: 12/24/2014 - Nebraska and Hawaii will tip off at 3pm CT on Christmas Day in the 5th place game of the Diamond Head Classic.
Posted: 12/24/2014 - After a late afternoon practice on Tuesday, the Nebraska football team was on the field turf at DeVore Stadium at 10 a.m. on Wednesday for its second of four practices at Southwestern College prior to Saturday's Holiday Bowl against USC on ESPN.
Updated: 12/24/2014 - Terran Petteway scored seven of his 17 points in overtime, lifting Nebraska to a 50-42 win over Loyola Marymount in the Hawaiian Airlines Diamond Head Classic late Tuesday.
Posted: 12/24/2014 - The No. 12 Nebraska women's basketball team opens Big Ten Conference play on the road when the Huskers take on Minnesota Monday night.
Updated: 12/23/2014 - Following an early-morning flight from Lincoln to San Diego, the Nebraska football team practiced at DeVore Stadium for the first time on Tuesday afternoon.
Posted: 12/23/2014 - It is a quick turnaround for the Nebraska men's basketball team, as the Huskers look to bounce back from a disappointing loss to Hawaii by taking on Loyola Marymount. The Huskers return to the court Tuesday evening as they take on Loyola Marymount.
Posted: 12/23/2014 - Huskers fall to Hawaii, 66-58.
Updated: 12/22/2014 - The Nebraska volleyball team finished in the AVCA top 10 after falling to BYU in the Elite 8.
Posted: 12/22/2014 - Nebraska takes a 9-3 record to the Holiday Bowl for Saturday's game against USC.
Posted: 12/20/2014 - All-American Rachel Theriot erupted for a season-high 31 points and Tear'a Laudermill pitched in 22 as No. 12 Nebraska rolled to an 83-57 women's win over defending Big South Conference champion High Point at Pinnacle Bank Arena on Saturday.
Posted: 12/19/2014 - 4-star recruit Kendall Bussey announced Friday he will attend Tennessee, despite previously giving his verbal pledge to Nebraska.
Posted: 12/19/2014 - The Nebraska football team practiced inside the Hawks Championship Center for one hour on Friday.
Posted: 12/19/2014 - Nebraska pitcher Chance Sinclair is a Preseason All-American.
Posted: 12/18/2014 - Nebraska Football Announces Team Awards
Updated: 12/17/2014 - Nebraska volleyball coach John Cook sat down with 10/11's Drew White to reflect onthe 2014 season.
Updated: 12/17/2014 - Bo Pelini used several profane words and obscene references when detailing his dismissal from Nebraska during team meeting on December 2nd, an leaked audio tape to the Omaha World Herald revealed.
Updated: 12/17/2014 - Steve Pederson was fired as the Athletic Director from the University of Pittsburgh.
Updated: 12/18/2014 - The University of Nebraska is in receipt of what appears to be a transcript of remarks delivered by former football coach Bo Pelini. These remarks to student-athletes were allegedly made during a non-official team meeting at a local high school, held two days after his termination. At this time, the University cannot authenticate the transcript nor do we have an audio version of the remarks.
Updated: 12/16/2014 - Nebraska women's basketball signee Jessica Shepard from Fremont joins the top 20 players in the nation on the Naismith Girls High School Player of the Year Watch List announced Tuesday by the Atlanta Tipoff Club.
Posted: 12/16/2014 - Nebraska senior I-back Ameer Abdullah and junior defensive end Randy Gregory were each named Associated Press All-Americans on Tuesday, as Abdullah was a second-team pick and Gregory was a third-team selection.
Updated: 12/16/2014 - Husker signee Mikaela Foecke of Holy Trinity Catholic High School (Fort Madison, Iowa) is the 2014-15 Gatorade National Volleyball Player of the Year.
Updated: 12/17/2014 - Bo Pelini, a Youngstown native and one of only two Power Five conference football coaches to win at least nine games each of the past seven years, has been named the Head Football Coach at Youngstown State University, President James P. Tressel and Executive Director of Intercollegiate Athletics Ron Strollo announced on Tuesday.
Posted: 12/14/2014 - Senior forward Emily Cady produced her fifth double-double of the season with 13 points, 15 rebounds and five assists to carry No. 12 Nebraska to a 54-45 women's basketball win over Bakersfield on Saturday afternoon at Pinnacle Bank Arena.


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Since the discovery of the Malta fever agent, Brucella melitensis, in the 19th century, six terrestrial mammal-associated Brucella species were recognized over the next century. More recently the number of novel Brucella species has increased and among them, isolation of species B. pinnipedialis and B. ceti from marine mammals raised many questions about their origin as well as on the evolutionary history of the whole genus.
We report here on the first complete genome sequence of a Brucella strain isolated from marine mammals, Brucella pinnipedialis strain B2/94. A whole gene-based phylogenetic analysis shows that five main groups of host-associated Brucella species rapidly diverged from a likely free-living ancestor close to the recently isolated B. microti. However, this tree lacks the resolution required to resolve the order of divergence of those groups. Comparative analyses focusing on a) genome segments unshared between B. microti and B. pinnipedialis, b) gene deletion/fusion events and c) positions and numbers of Brucella specific IS711 elements in the available Brucella genomes provided enough information to propose a branching order for those five groups.
In this study, it appears that the closest relatives of marine mammal Brucella sp. are B. ovis and Brucella sp. NVSL 07-0026 isolated from a baboon, followed by B. melitensis and B. abortus strains, and finally the group consisting of B. suis strains, including B. canis and the group consisting of the single B. neotomae species. We were not able, however, to resolve the order of divergence of the two latter groups.
Brucellae are Gram-negative, facultative, intracellular bacteria that can infect many species of animals and man. Six species were initially recognized within the genus Brucella: B. abortus, B. melitensis, B. suis, B. ovis, B. canis, and B. neotomae [1-3]. This classification is mainly based on differences in pathogenicity, host preference, and phenotypic characteristics. Four additional species have been included in the genus Brucella since 2007. These comprise the species B. ceti and B. pinnipedialis isolated from marine mammals, with cetaceans (dolphin, porpoise, and whale species) and pinnipeds (various seal species) as preferred host respectively [4,5]. B. microti described in 2008 was first isolated from the common vole and then from the red fox, and from soil [6-8]. The latest described species is B. inopinata, isolated from an infected human breast implant, and currently the most divergent Brucella species at the phenotypic and molecular level [9,10]. The animal or environmental reservoir of B. inopinata is not known. New Brucella species will likely be described in the future such as for isolates from baboons [11], from wild rodents in Australia [12] and for strain BO2 isolated from a patient with chronic destructive pneumonia [13]. Strain BO2 and strains from wild Australian rodents have been proposed as a novel lineage of the B. inopinata species [12,13].
Molecular and phenotypic typing of marine mammal Brucella strains led to their classification into two species, B. ceti and B. pinnipedialis, according to their preferred host, cetaceans and pinnipeds respectively [5]. However, several subgroups were identified within each species by molecular typing methods such as multilocus sequence analysis (MLSA), multilocus VNTR (Variable Number of Tandem Repeats) analysis (MLVA), or omp2a and omp2b porin genes [14-19]. Among them one subgroup within B. ceti, exclusively composed of strains isolated from various dolphin species, was proposed to constitute a separate species with the name B. delphini [3,14,18]. The isolates from cetaceans from the Pacific may also constitute a separate species [19]. Three human cases with naturally acquired infection by Brucella strains presumably from marine origin were reported, one case of spinal osteomyelitis from a patient in New Zealand [20] and two neurobrucellosis cases from Peruvian patients [21]. Interestingly, these human isolates exhibited the same genotype as strains from cetaceans from the Pacific [22].
Among their distinctive characteristics at the molecular and genomic level, marine mammal Brucella strains were shown to carry in their genomes a higher number of the insertion sequence element IS711 (or IS6501) [23,24] than terrestrial mammal Brucella species and biovars with the exception of B. ovis [15,17,25]. Consequently, infrequent restriction site-PCR (IRS-PCR) methods and more recently ligation-mediated PCR (LM-PCR) were applied, taking into account this higher number of IS711 elements, to study the genomic diversity of marine mammal strains [26-28]. These studies confirmed the classification into two marine mammal Brucella species, each divided in subgroups. In addition, six specific IS711-containing DNA fragments were detected allowing the molecular identification of B. ceti and its subgroup composed exclusively of dolphin isolates [17,26-28]. Besides these specific IS711-containing fragments another DNA fragment was detected that was exclusively found in B. pinnipedialis strains, with the exception of hooded seal isolates, consisting of a putative genomic island [26,27]. The size of this island was estimated at 62 kbp according to the physical maps made from the genomes of marine mammal strains by macrorestriction analyses [14].
The taxonomy of Brucella is still controversial, with an ongoing debate on whether they should be considered as distinct species or distinct strains of B. melitensis, considering the close proximity of their genomes [29]. We determined and analyzed the complete genome of B. pinnipedialis B2/94 to bring new insights into the origin of Brucella isolated from marine mammals as well as their time of divergence from Brucella isolated from terrestrial animals.
The genome sequence of B. pinnipedialis B2/94 was determined (30× coverage) by shotgun sequencing with the GS-FLX technology and the remaining gaps filled using the standard Sanger technology. Like that of other Brucella strains, the genome is composed of two circular chromosomes, of 2,138,342 bp (base-pairs) and 1,260,926 bp in lengths, respectively. Bioinformatic annotation predicted the presence of 3,342 protein coding genes, 55 tRNAs and 9 ribosomal RNAs. The comparison with the known genomes of other Brucella species revealed the presence of 90 pseudogenes. The 23S rDNA sequence of B. pinnipedialis B2/94 was found similar to that of other Brucella species, in contrast with the anomalous and unexpected 23S ribosomal RNA sequence previously described for B. microti [30].
The phylogenetic placement of B. pinnipedialis B2/94 was assessed with the help of a whole gene tree including all orthologous genes from Brucella strains available in the complete genome division of GenBank, completed by sequence data from selected Brucella strains of particular phylogenetic relevance, either deposited in the whole genome shotgun (WGS) division of GenBank, or available from other sources (see Methods). This includes sequences from B. neotomae, 10 sequences from marine mammal Brucella strains, Brucella sp. B02, B. inopinata BO1, Brucella sp. 83/13 or Brucella sp. NF2653. The resulting tree (Figure (Figure1)1) readily shows that the Brucella species with the highest sequence similarity with B. pinnipedialis B2/94 and whose complete genome is available is B. microti CCM 4915. This is a consequence of the relatively slow evolution rate of those two species rather than reflecting a particular phylogenetic affinity The central position occupied by B. microti, just at the root of host-associated Brucella species, is also confirmed. This tree distinguishes five major groups, the B. suis strains (shaded rectangle A, Figure Figure1),1), the different B. melitensis and B. abortus strains, also well separated from each other (rectangle B), another group with B. ovis and Brucella sp. NVSL 07-0026 (rectangle C), and marine mammal Brucella strains (rectangle D). B. neotomae remains isolated in this tree and constitutes the fifth group. However, the resolution of this tree is insufficient to infer the relative order of divergence of those different groups. Moreover, while bootstrap values in this tree are in general very strong (99 or 100%), the branching (D,(C, B)) proposed in the maximum likelihood tree is generally not supported, with a bootstrap value of 12%, prompting for a finer analysis of the events shaping the Brucella evolutionary tree.
The genome sequences of B. microti CCM 4915 and B. pinnipedialis B2/94 were found to be remarkably conserved even at the nucleotide level, allowing to generate a complete alignment of the chromosomes of the two species from which a list of all indels (insertions and deletions) (available as Additional file 1, Table S1 and Additional file 2, Table S2) was easily obtained. In spite of the overall similarity of these two genomes, this alignment revealed major changes in genome structure, as large segments unshared between the two species. The alignment from the largest chromosome exhibited 238 gapped positions with the largest insert in B. microti being 2,653 bp long, and the largest in B. pinnipedialis being 21,713 bp long. The alignment from the small chromosome exhibits 151 gapped positions, the largest insert being 18,341 bp long in B. microti and 67,389 bp long in B. pinnipedialis. The total number of indels (389) found in comparing these two species is thus smaller than the 405 indels found in comparing B. microti CCM 4915 and B. suis 1330 [30]. The complete alignment contains a total of 3,290,621 aligned positions (2,104,923 and 1,185,698 per chromosome), with 0.10% of nucleotide changes in aligned regions (2,195 and 1,200 nucleotide changes, respectively). This fraction of nucleotide substitution is also smaller than what was observed between B. microti CCM 4915 and B. suis 1330, where a 0.16% divergence was reported [30].
The evolutionary history of the largest segments unshared between B. microti CCM 4915 and B. pinnipedialis B2/94 was analyzed by examining the structure of the orthologous loci in the other Brucella species for which sequence data was available (see Methods). On the large chromosome, the 2 largest indels are 21 kbp, and 2.6 kbp in length. On the small chromosome, the largest indels are 67 kbp, 18 kpb, 11 kbp and 2.8 kbp in length. All other indels are at most the size of an IS711 insertion sequence (843 bp). The presence/absence of the above genomic inserts was assessed in other Brucella strains (see Methods). For each segment, we recorded the number of nucleotides with homologues in the other Brucella genomes (Additional file 3, Table S3). The presence/absence of those segments is reported at the leaves of the tree (Figure (Figure1)1) as filled squares. The evolutionary history of those unshared genome segments, treated as discrete characters, was reconstructed by parsimony analysis using the Mesquite software (see Methods), and represented as a cladogram in Additional file 4, Figure S1. The proposed evolutionary scenario corresponding to those unshared segments is discussed below, the largest ones first.
The locus of BMI_II545-6 in B. microti CCM 4915 is occupied in the B. pinnipedialis B2/94 small chromosome by a 67,389 bp insert (position: 520,136 to 587,524) which is present in a number of Brucella strains from marine mammals and partially in B. ovis ATCC 25840. The gene arrangements of this genomic region in B. pinnipedialis, B. microti and B. ovis are depicted in Figure Figure2.2. This region is flanked on both sides by an IS711 element (respectively BPI_II536-7 and BPI_II602-3), at a location where a sole copy of the IS711 element is found in B. microti CCM 4915 (BMI_II545-6), as in all Brucella strains lacking this insert. This region is partially found in B. ovis ATCC 25840, except in the interval between BPI_II545 and BPI_II576 deleted from B. ovis. The deletion in B. ovis ATCC 25840 occurred inside the gene BOV_A0488, whose 85 first amino acids are similar to BPI_II577 and whose last 100 amino acids are similar to BPI_II544. This interval exhibits a few protein coding genes or their remnants, with similarities mainly found in distant alphaproteobacteria i.e. excluding Brucella and Ochrobactrum. In particular, two pseudogenes, BPI_II545 and BPI_II548, separated by one IS711 element, appear to be the remnants of two consecutive Paracoccus denitrificans genes coding for 5-oxoprolinase (Pden_4408-9) and are preceded by an AraC-family transcriptional regulator (BPI_II544) with similarity into the same organism (Pden_4410). The same three genes are also found grouped together but in different orders in the genomes of several betaproteobacteria e.g Bordetella avium (BAV3293-5) or Verminephrobacter eiseniae (Veis_1518-20). Two consecutive genes, etfA (BPI_II555) and etfB (BPI_II556) are also present on the large chromosome with 100% nucleotide identity. We also observed 4 components of a spermidine/putrescine ABC-transporter (BPI_II564-7) found colinearly in the genomes of e.g. Roseobacter denitrificans (RD1_3862-5) or Silicibacter pomeroyi (SPOA381-4). We found similarities to two consecutive genes of Parvibaculum lavamentivorans (Plav_0386-7), similarities to Plav_0387 being noticed on both sides of the BPI_II574-5 ISBm1 transposase gene. Other genes like the aldehyde dehydrogenase BPI_II571 have best matches in Brucella and Ochrobactrum, but with 73% similarity only at the amino acid level. Several transposase genes are also present. This large 67 kbp fragment probably found its origin in the common ancestor of the marine mammal Brucella species and B. ovis, as attested by its partial presence in B. ovis. The structure of this segment suggests a grouping of B. ovis with marine mammal Brucella sp. (Additional file 4, Figure S1-a). It may have been lost thereafter several times, in Brucella sp. strain NVSL 07-006 isolated from a baboon and along the B. ceti and B. pinnipedialis lineages, putatively by recombination of the two IS711 copies.
A 21,713 bp fragment on the large chromosome (position: 259,190 to 280,902 in B. pinnipedialis B2/94) is not found in B. microti CCM 4915. This fragment is present in all B. melitensis and B. abortus strains and in B. neotomae, but its occurrence is quite variable among B. ceti and B. pinnipedialis strains and even among B. suis strains, where it is observed in the earliest diverging B. suis 513 and B. suis ATCC 23445, but absent otherwise. This fragment starts with a phage integrase gene (BPI_I248) and ends with a tRNA (BPI_I278) which was the likely insertion site. An IS711 element is inserted within a gene (BPI_II256) that remained intact in B. melitensis 16 M (BMEI1694) and B. ovis ATCC 25840 (BOV_0245). This fragment encodes a flagellar protein FlgJ (BPI_I260). The other putative genes in this region have no convincing similarities to annotated proteins. This 21 kbp fragment (Additional file 4, Figure S1-b) probably entered the Brucella genomes after B. microti divergence, and disappeared separately on several branches. It confirms the grouping of B. ceti Cudo, B. ceti B1/94, B. ceti M490/95/1 and Brucella sp. F5/99. Its presence in the genome of B. neotomae 5K33 and absence in B. suis strains except for B. suis ATCC 23445 and B. suis 513 suggest its insertion prior to the divergence of the B. suis and B. neotomae lineage, and a subsequent loss in the B. suis lineage.
A 18,341 bp region on the small chromosome (between BMI_II357 and BMI_II381) is absent from the B. pinnipedialis B2/94 genome (position: 344,671-363,011 in B. microti CCM 4915). This region is present in B. microti, B. suis, B. canis, B. ceti, Brucella sp. F5/99, B. neotomae and absent in B. abortus, B. melitensis, B. ovis and B. pinnipedialis. Additionally, a partial match was found in the genome of Brucella sp. B02. In this region, the presence of genes encoding TraI-J proteins involved in bacterial conjugation can be noted. Like the above 67 kbp fragment, this 18 kbp fragment (Additional file 4, Figure S1-c) supports a divergence of the B. melitensis/B. abortus clade prior to the separation of B. ovis, Brucella sp. strain NVSL 07-006 and marine mammal Brucella strains. It might have appeared before B. microti divergence, and disappeared several times, in particular from the branch leading to the B. abortus/B. melitensis clade and from the branch leading to the B. pinnipedialis clade.
The 11 kbp region on the small chromosome is a phage related region discussed in [30] and unique to B. microti (position: 1,038,883-1,050,624 in B. microti CCM 4915). It will not be discussed further (Additional file 4, Figure S1-d).
Still on the small chromosome, a 2,881 bp region (position: 1,082,391-1,085,271 in B. microti CCM 4915) encodes genes BMI_II1086-8. This fragment (Additional file 4, Figure S1-e) appeared before B. microti divergence, and is absent from the branch leading to B. ovis and Brucella sp. strain NVSL 07-0026, but also from the branch leading to B. pinnipedialis M292/94/1 and B. pinnipedialis B2/94, separating those two species from B. pinnipedialis M163/99/10. Closer examination reveals that this 2.8 kbp fragment is absent in B. ovis because it belongs to a much larger 44.5 kbp region deleted from B. ovis [31] and also partially deleted from Brucella sp. NVSL 07-0026, where an approximately 30 kbp long region is missing (pos: 1,117,180 to 1143394 in B. pinnipedialis B2/94 small chromosome). This finding supports both grouping of the B. pinnipedialis M292/94/1 and B2/94 strains, and that of B. ovis with Brucella sp. strain NVSL 07-0026.
Between BMI_I949 and BMI_I953, there is a 2,653 bp region deleted from the B. pinnipedialis B2/94 large chromosome (position: 928,716-931,368 in B. microti CCM 4915) but also from B. ovis ATCC 25840, Brucella sp. NVSL 07-0026 and all B. melitensis and B. abortus strains. Interestingly, this deletion occurred inside a gene (encoding an ABC transporter), thus showing that it is a deletion and not an acquisition event. This region (Additional file 4, Figure S1-f) is particularly informative because it clearly separates Brucella strains into two groups. This fragment was present in ancestral Brucella, and then lost after the divergence of the B. suis clade, of B. neotomae, of B. microti and prior to the divergence of B. abortus and B. melitensis, B. ovis and Brucella sp. strain NVSL 07-2026, and finally marine mammal Brucella species. An interesting exception is found in B. abortus B3196, where this ABC transporter gene is intact. This feature clearly suggests a divergence of the B. suis and B. neotomae group before that of the other host-associated Brucella.
IS711 insertion sequences [32] account for a large number of gaps within the complete genome alignment of B. pinnipedialis B2/94 and B. microti CCM 4915. In total, 19 IS711 elements were identified on the large chromosome and 12 on the small chromosome of B. pinnipedialis. We looked for the occurrence of those insertion sequences in the available complete genomes of Brucella (Methods) and reported their position in Figure Figure33 and Additional file 5, Table S4. Although it is difficult to analyze transposable repetitive elements in unfinished genomes (as they are often the ones precluding genome assembly) [33], we included two other partial genomes in our analysis, but where an abundant number of IS711 elements were observed, B. ceti Cudo and Brucella sp. strain NVSL 07-0026.
Figure Figure33 shows that 6 IS711 insertion sequences are found in all selected Brucella strains (IS711 groups numbered 1, 4, 12, 13, 26, and 31). We also observed that 4 IS711 elements were uniquely shared by B. ovis ATCC 25840 and Brucella sp. strain NVSL 07-006 (groups 41, 43, 48 and 54), supporting the grouping of those two species, already noticed when discussing the whole gene tree, and confirmed by the large deletion around the 2.8 kbp fragment discussed previously. Those two species were the likely subjects of an intense IS711 transposition activity, with 14 elements being uniquely found in B. ovis and 9 being uniquely found in Brucella sp. NVSL 07-006. Among the 31 IS711 positions found in B. pinnipedialis B2/94, 21 are shared with B. ceti Cudo among which 12 are found only in B. pinnipedialis B2/94 and B. ceti Cudo, and absent from all other strains (groups 5-9, 17, 18, 19, 21, 23 and 24).
Comparing orthologous loci (present in both B. microti CCM 4915 and B. pinnipedialis B2/94 genomes), we found that the number of pseudogenes in the genome of B. pinnipedialis B2/94 was larger than that in the genome of B. microti CCM 4915 (30 for B. microti on chromosome I and 48 for B. pinnipedialis, 16 for B. microti on chromosome II and 42 for B. pinnipedialis). This was also noticed when comparing B. microti CCM 4915 and B. suis 1330, and was attributed to a slower evolution rate in B. microti. The genes that are potentially different in both species are reported in Additional file 6, Table S5.
Among the genes altered in B. pinnipedialis B2/94, many are components of ABC transporters. Those genes are highlighted in Additional file 6, Table S5. We found 17 genes related to ABC transporters that are impaired, on a total of 90 impaired genes. In the B. pinnipedialis genome we identified approximately 249 ABC transporter-related intact genes on a total of 3,342 protein coding genes. This output is highly improbable (p-value = 1.e-4, chi-square test) and strongly suggest that ABC transporters were specifically degraded in B. pinnipedialis and more generally in host-associated Brucella species. Beside ABC type transport systems, many other genes involved in transport are found impaired in B. pinnipedialis B2/94: a CorA family transporter BPI_I592 (ortholog BMI_I558), an EmrB/Qaca family drug resistance transporter BPI_I1098 (ortholog BMI_I1064), the dipeptide transport system permease protein DppC (BPI_I1637, ortholog BMI_I1597), an outer membrane autotransporter BPI_I2072 (ortholog BMI_I2035), a glucose/galactose transporter BPI_II188 (ortholog BMI_II187), a cadmiun-translocating P-type ATPase BPI_II1260 (ortholog BMI_II1204), and finally a putative transport protein BPI_II453 (ortholog BMI_II468).
One of those ABC transporter genes, BPI_I1818, exhibits an interesting feature. A frameshift difference between the B. pinnipedialis B2/94 and B. microti CCM 4915 sequences merges the membrane and ATP-binding components (BMI_I1778-9) of a thiamin ABC transporter into a single reading frame. A blast (tblastn) search of the BPI_I1818 gene sequence against the nucleotide sequences of the other Brucella strains shows that this gene fusion occurred in all marine mammal strains studied, except for B. ceti M13/05/1 and M644/93 which represent the distinct dolphin subgroup of strains within B. ceti mentioned above.
It has been suggested [14] that the divergence of species in the genus Brucella could have been concomitant with the divergence of their mammalian hosts, 60 millions years (my) ago. However this is inconsistent with the fact that the hosts of B. ceti and B. pinnipedialis did not diverge at the same time. The ancestors of pinnipeds where carnivores and Higdon et al. [34] used molecular data to estimate the split between ursids and pinnipeds to 35.7 ± 2.63 (= mean ± SE) my, and fossil records report early pinnipeds 35 my ago [35]. Cetaceans went back to the sea much earlier, the oldest known cetaceans date back to the Eocene, 55 my ago [36]. If we consider divergence in the 16S rRNA gene sequence, and referring to B. microti which has a central position, B. pinnipedialis, B. melitensis, B. abortus, B. suis (perhaps with the exception of B. suis 513, which has 2 (nt) differences, C- > T at position 11 and G- > T at position 1468), B. ovis, B. canis have all identical sequences, and B. neotomae has 1 bp difference (C- > T at position 541). Using the estimate of 1-2% of change in 16S rRNA sequence per 50 my, 1 bp difference (which really should be considered as a maximal) corresponds to 0.07% change, and a divergence time of 1.75-3.5 my. This time estimate is probably a crude overestimation and recent work [37], using single nucleotide polymorphisms from 13 genomes, showed that most Brucella species probably diverged 86,000 to 296,000 years ago. This analysis reveals that the divergence time of Brucella sp. found today in marine mammals is totally incompatible with the divergence time of their hosts. A fortuitous contamination of cetaceans and pinnipeds, probably via the food chain, may explain better this transmission of Brucella to the marine mammals. This also opens the remote possibility of marine Brucella infecting terrestrial mammals.
The analysis of distinctive genomic regions between B. microti CCM 4915 and B. pinnipedialis B2/94 as well as the study of additional markers reveal the order of appearance of the different Brucella species. It is clearly apparent that most of the events following the divergence of B. microti from the classical Brucella species occurred in a very small amount of time, as if something caused a sudden radiation in this lineage and a subsequent adaptation of the organisms to their hosts. Here we summarize some of the major evolutionary events that highlight the evolutionary history of the genus Brucella.
Following the divergence of B. microti, the next evolutionary event that we can trace is the 2.6 kbp fragment clear disappearance, which tells us that the two next Brucella groups to diverge were B. suis and B. neotomae. We did not find any good marker in favor of a prior divergence of one versus the other.
Marine mammal Brucella species as well as B. ovis and Brucella sp. NVSL 07-0026 share the presence of a high number of IS711 elements, and it has been demonstrated that IS711 transposition is still an active process in B. ovis and B. pinnipedialis [38]. Those insertion elements are much less numerous in B. melitensis and B. abortus strains and we thus assume that IS711 transposition events occurred quite abundantly after the divergence of B. melitensis/B. abortus. In this group, B. abortus strains share a unique feature which is a genomic 600 kbp inversion in the small chromosome, as clearly depicted on Figure 1 of reference [39].
The whole gene tree, where B. ovis and Brucella sp. NVSL 07-0026 cluster together, the 4 IS711 elements positions that they have in common and not shared with B. pinnipedialis B2/94 as well as the large deletion that those two species share, in the region surrounding the 2.8 kbp deletion in B. pinnipedialis, all those facts support the grouping of B. ovis with Brucella sp. NVSL 07-0026. The grouping of marine mammal Brucella strains that we observe in the tree with early divergence of B. ceti M13/05/1 and M644/93 is also reflected by the gene fusion event mentioned earlier. Grouping of B. pinnipedialis M292/94 and B2/94 is supported by the loss of the 18 kbp fragment in these two strains.
There is a clear transition in the genus Brucella evolutionary tree. The first Brucella discovered were the host-associated Brucella species, but more recently, B. microti was isolated as the first representative of a fast growing list of free-living Brucella. This biochemically highly active bacteria was found to share more phenotypic traits with Ochrobactrum than with the host-associated Brucella species [6]. We proposed earlier [30] that the transition between a free-living and an host-associated life style could have resulted from the modification in the 23S ribosomal RNA gene sequence with putative effects on the growth rate of the bacteria. A slow growth rate has often been advocated for intra-cellular bacteria, as their survival is often dependent on the survival of their hosts [40]. Brucella with this change in 23S structure and its impact on growth rate became suddenly more adapted to an host-associated life style than to a free-living style, and progressively adapted to distinct sets of hosts, giving rise to the main lineages of host-associated Brucella species that are encountered today.
Genome was assembled from 430,042 paired GS-FLX reads of average length 229, giving approximately a 30× coverage of the genome, and directed sequencing of the remaining gaps was performed using 193 additional Sanger sequencing reactions. The genome sequence is deposited in the complete genome division of GenBank under project ID 41867 and accession numbers CP002078 and CP002079. Genomic sequence data of all Brucella strains mentioned in this work can be conveniently downloaded from a unique location at the Pathosystems Resource Integration Center web site [41], or otherwise from the complete genome and whole genome shotgun divisions of GenBank [42]. Origin of the sequence data is listed in Supplementary Table 3. Most sequences originate from the Brucella group project at the Broad Institute, conducted by Davis O'Callaghan, Adrian Whatmore and Renee Tsolis [43] or from the Pathosystems Resource Integration Center of the Virginia Bioinformatics Institute [41].
Whole gene tree was build using gene sequences from all available Brucella as well as Ochrobactrum intermedium. The procedure used to build the tree is similar to that reported in [30]. Briefly, 1125 orthologous genes from 39 Brucella plus genes from O. intermedium were used. Orthologous genes were selected using the following procedure. A file containing nucleotide gene sequences for the selected organisms was compared to itself using blastn [44] (parameters: -b 100 -v 100 -F F -e 1.e-20). The resulting output file was subjected to clustering using the Markov chain clustering algorithm [45]. In the resulting cluster list, we selected the clusters with only one unique member per species. Genes from each cluster were then aligned using MUSCLE [46] (default parameters). The following alignments were concatenated, resulting in an alignment of 40 sequences, with 1,078,083 positions, cleaned with Gblock [47] (default parameters), which reduced it to 945,578 positions. From this multiple alignment, on which 767,738 sites without polymorphisms and 2189 distinct patterns were found, a tree was inferred by maximum likelihood using PhyML [48], with 100 bootstrap replicates.
The nucleotide sequence of the genome fragment coming either from B. pinnipedialis B2/94 or B. microti CCM 4915 was used as a query for blastn search (e-value = 1.e-100, no filter) against the nucleotide sequences of the remaining Brucella. For each target genome, the number of distinct nucleotide positions that had a hit was recorded in Additional file 3, Table S3. Presence or absence of a given fragment is represented in Figure Figure11 alongside the whole gene tree. The history of those genome segments was subsequently traced using the Mesquite software package [49] on cladograms reflecting the topology obtained from the whole gene tree (Figure (Figure1).1). Character history was computed using parsimony analysis, with presence or absence of a genome segment treated as a discrete category character.
A database containing the complete genomes of the Brucella species under study was compiled. We searched this database for the occurrence of IS711 using the nucleotide sequence of one element from B. pinnipedialis as query, using the blastn program (parameters: -b 1000 -v 1000, limiting to hits with a score of 500). We extracted the corresponding segments, adding 500 nucleotides of context on both side. We then masked this file so that the IS711 sequence itself was replaced by × in the sequence. A subsequently blastn search of this file against itself allowed us to recover the orthologous IS711 positions along the different Brucella genomes (listed in Additional file 5, Table S4), used for the representation in Figure Figure33.
SA participated in the design of the study, performed genome assembly, annotation and comparisons with other Brucella and wrote the manuscript. ML participated in genome annotation and analyzed the data. JMC participated in the design of the study, analyzed data and wrote the manuscript. MZ and AC designed the study, coordinated the research, analyzed data and wrote the manuscript. All authors read and approved the final manuscript.
Indels in B. pinnipedialis B2/94 and B. microti large chromosome alignment. List of insertions and deletions (indels) in the complete genome alignment of B. pinnipedialis B2/94 and B. microti CCM 4915, large chromosome.
Indels in B. pinnipedialis B2/94 and B. microti small chromosome alignment. List of insertions and deletions (indels) in the complete genome alignment of B. pinnipedialis B2/94 and B. microti CCM 4915, small chromosome.
Occurrences of the largest genome fragments unshared between B. pinnipedialis B2/94 and B. microti CCM 4915 in selected Brucella genomes. Occurrences of the largest genome fragments unshared between B. pinnipedialis B2/94 and B. microti CCM 4915 in selected Brucella genomes. For each organism, the number of positions in its genome that are found similar to the original fragment is reported. The column "Origin of sequence data" indicates "GenBank" for sequences deposited in the complete genome division of GenBank, "Patric" if the sequence originates from the Pathosystems Resource Integration Center of the Virginia Bioinformatics Institute, and "Broad" if he sequence originates from the Brucella genome sequencing project at the Broad Institute.
Evolutionary history of the unshared sequence fragments along the Brucella evolutionary tree. Evolutionary history of the unshared sequence fragments along the Brucella evolutionary tree. The tree is represented as a cladogram with the same topology as that of the whole gene tree (Figure 1). Plots are generated using Mesquite.
Position of IS711 insertion sequences in the genome of completely sequences Brucella, B. ceti Cudo and Brucella sp. NVSL 07-0026. Position of IS711 insertion sequences in the genome of completely sequences Brucella, B. ceti Cudo and Brucella sp. NVSL 07-0026. Each IS711 element position as well as the accession number of the genome segment to which it belongs are listed in the Table. Clusters of IS711 elements are identified by a number allowing to locate them in Figure 3.
List of genes showing a large change between B. pinnipedialis B2/94 and B. microti CCM 4915. List of genes showing a large change between B. pinnipedialis B2/94 and B. microti CCM 4915. ABC transporter genes are indicated.
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A method and apparatus for high-throughput determination of phase change points of combinatorial libraries of metal alloys uses an infrared camera to monitor temperature-dependent changes in emissivity/reflectivity of the alloys. An infrared focal plane array monitors the emissivity/reflectivity changes over time, and the intensity of each heated member over time is correlated with temperature to detect the phase change points of the members.
1. A system for determining phase change points of at least ten different members in a combinatorial library, comprising:
a substrate having said at least ten different members disposed thereon and a capping layer of a refractory material deposited over said at least ten members;
a temperature changer for simultaneously changing the temperature of each member on the substrate for achieving at least one phase change in said at least ten members;
a detector for simultaneously monitoring changes in radiation for each member and for generating intensity data based on the detected radiation;
a correlator for correlating the intensity data from the detector with the temperature of each member, wherein a radiation change in the member indicates a phase change point for that member such that the temperature of the phase change point is rapidly determined for the at least ten members; and
a vacuum chamber surrounding the substrate and the temperature changer.
2. The system of
3. The system of
4. The system of
determining a calibration curve correlating the radiation intensity of the substrate and the temperature of the substrate;
measuring the radiation intensity versus time of each member and the substrate; and
calculating the ratio between the radiation intensity versus time of each member and the radiation intensity versus time of the substrate, wherein a change in the ratio for one of said members indicates the phase change point of that member.
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17. A system for determining phase change points of at least ten different members in a combinatorial library, comprising:
a substrate having said at least ten different members disposed thereon;
a temperature changer for changing the temperature of each member on the substrate;
a detector for monitoring changes in radiation for each member and for generating intensity data based on the detected radiation;
a computerized correlator for correlating the intensity data from the detector with the temperature of each member, wherein a radiation change in the member indicates a phase change point for that member such that the temperature of the phase change point is determined for the at least ten members within about one second of each other; and
an environmental controller associated with the substrate and the temperature changer wherein the environmental controller is capable of adjusting an environment surrounding the at least ten members in a manner that reduces heat loss of the at least ten members to the environment.
18. The system of
19. The system of
20. The system of
determining a calibration curve correlating the radiation intensity of the substrate and the temperature of the substrate;
measuring the radiation intensity versus time of each member and the substrate; and
calculating the ratio between the radiation intensity versus time of each member and the radiation intensity versus time of the substrate, wherein a change in the ratio for one of said members indicates the phase change point of that member.
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34. A system for determining melting points of at least ten different members in a combinatorial library, comprising:
a conductive substrate having the at least ten different members disposed thereon and a capping layer of a refractory material deposited over said at least ten members, wherein the substrate is at least partially formed of an amorphous carbon;
a low thermal mass heater that ramps up and down at a rate of around 30-50� C. per second between room temperature and 1300� C. and that supports the substrate and is adapted for simultaneously heating the at least ten members.
a vacuum chamber surrounding the substrate, the at least ten members, and the heater;
an infrared camera positioned over the substrate and the at least ten members, the infrared camera having a focal plane array for monitoring changes in radiation of the at least ten members over time as they are heated by the heater and generating image data; and
a computer that obtains the image data from the focal plane array and correlates the radiation intensities of the at least ten members with temperature, wherein a radiation change in the member indicates a melting point for that member such that the temperature of the melting point is determined for the at least ten members within about one second of each other.
35. The system of
36. The system of
determining a calibration curve correlating a radiation intensity of the substrate and the temperature of the substrate;
measuring the radiation intensity versus time of each of the members and the substrate; and
calculating the ratio between the radiation intensity versus time of each of the members and the radiation intensity versus time of the substrate, wherein a change in the ratio for a given member indicates the melting point of that member.
37. The system of
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This application is a continuation-in-part and claims priority to international application PCT/US99/07358, which is a continuation-in-part of commonly assigned, co-pending U.S. patent application Ser. No. 09/227,558, filed Jan. 8, 1999, which is a continuation-in-part of U.S. application Ser. No. 08/898,715, filed Jul. 22, 1997, and U.S. Provisional Application No. 60/050,949, filed Jun. 13, 1997; No. 60/028,106, filed Oct. 9, 1996; No. 60/029,255, filed Oct. 25, 1996; No. 60/035,366, filed Jan. 10, 1997; No. 60/048,987, filed Jun. 9, 1997; No. 60/028,105, filed Oct. 9, 1996; and No. 60/035,202, filed Jan. 10, 1997. Each of the foregoing applications is incorporated herein by reference in its entirety and is the basis of a claim for priority under 35 U.S.C. Section 119 or 120.
The present invention is directed to combinatorial techniques for characterizing materials, and more particularly to a parallel screen for determining the thermal characteristics of materials by monitoring changes in radiation intensity from members of a combinatorial library as a function of temperature.
In discovering new materials, the temperature of a material phase change (e.g. melting point) provides important information regarding a particular material. One possible approach for determining such temperatures is to measure the difference between the temperature of the unknown member and a reference member, as in differential scanning calorimetry (DSC) and differential thermal analysis (DTA). Another approach is to monitor changes in the reflectivity or emissivity of the unknown member as a function of temperature. Currently, there is a need for a rapid, high throughput screen that can determine the thermal characteristics of members of a combinatorial library over a large temperature range.
Accordingly, the invention applies combinatorial materials science techniques in a high-throughput screen for measuring the phase change temperatures of combinatorial libraries of materials. Generally, the invention is directed to correlating the intensity of the radiation emitting from a member with its temperature to obtain its thermal characteristics, and more particularly to correlating the radiation intensity of each member versus time and correlating the temperature of each member versus time to link the member's radiation intensity with its temperature. The invention may include using a detector, such as an infrared camera, to measure the phase change temperature (e.g. melting point) of a plurality of materials on a substrate by, for example, monitoring changes in the radiation, such as the reflectance or emissivity, of a member. In one embodiment, the infrared camera compares the radiation from a substrate to the radiation from a member as a function of temperature. The phase change temperature of the member is obtained by monitoring changes in the ratio between the two radiations as a function of temperature.
FIG. 1 shows one embodiment of an apparatus according to the present invention for parallel characterization;
FIG. 2 shows a preferred substrate structure for supporting library members in the inventive apparatus;
FIG. 3 is an example of a temperature vs. intensity calibration curve obtained from the inventive apparatus; and
FIG. 4 is an example of intensity vs. time curves obtained via the inventive apparatus.
The present invention finds particularly attractive application in the rapid, high throughput screening of many materials and more specifically the screening of combinatorial libraries of materials. The field of combinatorial materials science generally involves, inter alia, the synthesis of arrays of multiple different materials and the screening of such materials for useful properties. By way of illustration, without limitation, a substrate having an array of diverse materials thereon can be prepared by delivering components of materials to predefined regions on the substrate, and simultaneously reacting the components to form at least two materials. Materials which can be screened using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be screened include inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, non-biological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. As such, the present invention provides methods and apparatus for use in connection with the parallel analysis of novel materials having new and useful properties. Any material found to possess a useful property can be subsequently prepared on a large-scale.
Thus, as will be appreciated, an array or library of inorganic materials (e.g. alloys) on a single substrate at predefined regions thereon is provided. Such an array can consist of two or more different materials, 10 or more different materials, and preferably more than 10, 102, 103, 104, 105 or 106 different materials. In some embodiments, the density of regions per unit area will be greater than 0.04 regions/cm2, more preferably greater than 0.1 regions/cm2, even more preferably greater than 1 region/cm2, even more preferably greater than 10 regions/cm2, and still more preferably greater than 100 regions/cm2. In most preferred embodiments, the density of regions per unit area will be greater than 1,000 regions/cm2, more preferably 10,000 regions/cm2, even more preferably greater than 100,000 regions/cm2, and still more preferably 10,000,000 regions/cm2.
In accordance with the present invention, such materials are then screened in parallel for useful properties. More specifically, the present invention advantageously permits for rapid and high throughput screening of such arrays, allowing useful data to be generated about the materials in the array, particularly with respect to phase transition temperatures (e.g. without limitation, melting point) of such materials. Additional details about synthesis and screening of combinatorial libraries can be found, for instance, in commonly assigned, co-pending U.S. application Ser. Nos. 08/327,513; 09/156,827; and 08/841,423, each of which is incorporated herein by reference.
Changes in the electrical conductivity, surface morphology, and dielectric constant of a material generally lead to changes in the reflectance of a material. These material changes are expected to be particularly large at a phase change or transition, such as between the solid state and the liquid state. It is these large changes that form the basis for the screen of the present invention. The invention generally detects changes in the emitted power R(T) as the temperature of combinatorial library of materials is increased through the phase change (e.g. melting point) of materials in the library. Any large sudden changes in the radiation, such as the measured reflectivity or emissivity, of a particular material for a given temperature indicates a phase change for that material. In this context, a "sudden large" change would refer to, for example, a 5-10% change in the relative intensity of a material over a 50-100 ms period would indicate a phase change point. In other embodiments, the change may be more than 5% and preferably more than 10%. Also, the change may take 200 ms or more, but preferably less than 100 ms. Samples or members of a combinatorial library may be measured in parallel or sequentially using the method and apparatus of this invention, with samples being measured two at a time, 10 at a time or more, up to the number of members in the combinatorial library. Thus, the system and method can measure 2 or more samples per second, 10 or more samples per second, 50 or more samples per second and preferably 60 or more samples per second for phase point changes.
To monitor changes in the reflectivity or emissivity of a material, the spectral excitance of the material in response to heating can be detected at a single wavelength, a narrow wavelength band, or over a wide wavelength spectrum. One method is to use a detector that is sensitive to a specific single wavelength and to measure the integrated excitance (intensity) as a function of temperature. Also, experiments detecting the spectral excitance over a narrow band of wavelengths or even a wide spectrum of wavelengths result in accurate melting point measurements as well.
FIG. 1 is a schematic representation of one preferred apparatus for conducting parallel screening of phase changes. The apparatus includes a plurality of single member detectors or a position-sensitive detector (i.e., one that can correlate the specific data with a specific measured position in the array) so that an entire library of members can be measured simultaneously. Because the screen of the present invention measures members at relatively high temperatures, which provide observable infrared radiation characteristics, the detectors are preferably tuned to infrared wavelengths. However, other wavelengths (e.g., visible light wavelengths, either alone or in conjunction with infrared wavelengths) can be used for measurement as well, and those of skill in the art can select an appropriate detector for such wavelengths. Of course, the skilled artisan will recognize that the invention is not limited to the structure shown in FIG. 1, but can also encompass any other system that correlates emissivity or radiation measurements of members with temperature to obtain phase transition data. In addition, although the examples below focus on heating solid materials to determine their melting point, the invention is just as applicable for any solid or liquid member materials that are taken through one or more phase changes (e.g. crystallization, solidification, melting point, sublimation/evaporation, metal-insulator phase transitions, etc.) via heating or cooling.
As shown in FIG. 1, a preferred apparatus 100 for conducting a parallel phase change (e.g. melting point) detection includes a detector 102 (illustrated here as an infrared detector, and specifically, an infrared camera containing an infrared focal plane array (FPA) 103), preferably a filter and aperture positioning device 104, a lens 106 (e.g. camera lens), a window 108 (preferably an infrared transparent window), a vacuum chamber 110, a heater 112, optional heater supports 114, and a reflector 116 (e.g. an infrared reflector). A combinatorial library 118 having members to be screened is placed on the heater 112. The interaction and operation of these components will be described in greater detail below with respect to parallel screening. Of course, other combinations of devices can be employed to obtain observable simultaneous or near-simultaneous phase change data from the combinatorial library.
Referring now to FIG. 2, a substrate 200 is preferably used to support the library members. Amorphous carbon is one example of a preferred substrate material to be used for detecting melting points of alloys in the invention because it can withstand temperatures of over 1000� C. in a vacuum environment, has good mechanical properties, and is relatively inert. Any material that has the relevant properties similar to amorphous carbon, however, can be used in the substrate 200 as well. By way of example, the substrate 200 may be selected from any of a wide variety of materials including polymers, plastics, pyrex, quartz, resins, silicon, silica or silica-based materials, carbon, metals, glasses, crystals, etc. The substrates may be organic, inorganic, biological, nonbiological or a combination. The substrate may be any suitable shape and preferably forms a rigid support. The choice of substrate will depend on the type of materials in the library, the phase transition being screened and the detector used to detect the radiation intensity change (e.g. the reflectivity or emissivity change) of the materials.
In the present preferred embodiment, the substrate 200 is used as a temperature reference throughout the melting point measurement. As shown in FIG. 2, library members 202 can be synthesized directly on the substrate. A thin capping layer 204 of a refractory material, such as Al2O3 or Ta2O3 is preferably deposited over the library members 202 to minimize evaporation of the library members as the temperature is increased. Any other suitable protective layer may likewise be employed. The capping layer 204 is preferably sufficiently thin, so that it does not adversely affect the infrared intensity measurement. To illustrate, in the present embodiment it is about 2000 angstroms. The library members 202 themselves can be deposited on the substrate using any known combinatorial technique, including the combinatorial materials science techniques identified in the aforenoted U.S. Patent Applications.
Though other methods are possible (including convective or radiation methods), with respect to heating of the library 118, one of two conductive methods preferably is used. One way is by using the substrate 200 as a resistive heater. For instance, if the substrate is amorphous carbon, this takes advantage of the electrical conductivity and the mechanical stability of the amorphous carbon and allows the library to be heated directly by passing current through the substrate. Temperatures as high as 1000� C. can be reached using this method.
Another preferred way to heat the library members is by placing the substrate on a suitable heater, such as a graphite heater. The heater should have low thermal mass because the temperature range for the experiment ramps up and back down at around 30-50� C./second between room temperature and 1300�C.; a quicker heating response provides clearer indications of the library members' phase transition (e.g. melting point) and also results in a faster experiment.
To screen the library members in parallel, the library is heated so that all members are at an elevated temperature simultaneously. A detector then reads the thermal data it observes from the substrate. The data can be stored and retrieved so that particular information about a particular material in the library can be retrieved and correlated to the specific material to yield useful information about such material.
To illustrate the context of screening a material for melting points using an infrared camera, the infrared camera 102 and lens 106, along with the infrared focal plane array (FPA) 103, is positioned over the library 118. If desired, the filter and aperture positioning device 104 can be placed in between the FPA 103 and the member library 118. As noted above, parallel detection of the melting points of a material library, according to the present invention involves using the infrared FPA 103 to monitor changes in the emissivity/reflectivity of each library member as its temperature is raised. One example of a preferred FPA is a 256�256 member array (e.g. InSb array) having a predetermined wavelength sensitivity (e.g. between about 800 nm and 5.5 μm, such as a visible-mid-infrared FPA), and that can capture images at a predetermined speed (e.g. at least 20 frames per second). Data from the FPA 103 is preferably captured in real time as the members are heated and cooled. The data is stored, reduced and analyzed in a computer using software that can obtain chemical data from images, such as software disclosed in co-pending PCT Application No. PCT/US99/07358 to entitled "Analysis of Chemical Data From Images", incorporated herein by reference. In general, the software is able to determine, from the captured images, a series of values for selected regions in the library and calculating a figure of merit, such as an emittance change as a function of temperature, a phase transition point or thermoelectric figure of merit, for a selected library member. A graphical representation of the figures of merit is optionally displayed for user analysis.
The FPA 103 can monitor the libraries over a wide wavelength range, and the present description is not intended to limit the invention. For illustration purposes, standard infrared optics that presently are commonly available are tuned to between about 3-5 μm and between about 8-14 μm. These wavelength ranges are ranges generally in the electromagnetic spectrum in which the air absorption is lowest. Thus, the actual experiment can be conducted within these narrow wavelength ranges, despite the FPA's 103 ability to detect wavelengths outside the ranges. The invention is not, however, restricted to conducting screening in the wavelength ranges tuned by standard infrared optics; phase change (e.g. melting point) detection can be conducted over any wavelength region detectable by the FPA 103. Further, as shown in FIG. 1, measuring the intensity of infrared radiation for a member at or above 1300� C. (as may be encountered with some materials) may require apertures and neutral density filters 104 to be placed between the member and the FPA 103 to avoid saturating the array.
In the context of an illustrative example screening for melting points of a metal alloy, the library 118 is placed on the heater 112 and heated using either heating method described above. In this example, the members are placed on an amorphous carbon substrate and heated by placing the substrate on the heater 112. During the heating process, the FPA 103 in the infrared camera 102 gathers reflectivity/emissivity data over time about each member as its temperature increases. This data is then correlated with temperature data, as will be explained in greater detail below, to obtain the melting point for each member. The reduced pressure in vacuum chamber 110 helps to ensure that the temperatures of the members in the library 118 are not affected by heat losses to the environment, air convection, etc.
Advantageously, the parallel screen of the present invention is capable of screening combinatorial libraries at temperatures of at least 1200� C. under vacuum conditions, with heating and cooling rates as high as about 30� C./second. Other possible temperature ranges and rates include temperatures of at least 1400� C. at about 50� C./second and at least 500� C. at about 30� C./second.
FIG. 3 is a calibration curve that links the substrate (e.g. carbon substrate) temperature of the present invention to a given measured radiation intensity at different camera integration settings (illustrated here as eight settings). As noted above, the FPA 103 records the emitted infrared intensity for the library members and for the carbon substrate as a function of time as the temperature of the library is increased. Obtaining a calibration curve linking the emitted infrared intensity from the carbon substrate to the actual temperature of the substrate, which can be determined with a measuring device such as a thermocouple, allows the substrate's radiation intensity over time to act as a reference temperature throughout the measurement. Although amorphous carbon does not have a constant emissivity as its temperature changes, the emissivity curve varies smoothly over the temperature range at which the melting point tests are conducted, as can be seen in FIG. 3. This allows changes in the substrate's emissivity to be addressed in the temperature calibration.
FIG. 4 illustrates the changes in radiation intensity versus time for a reference substrate (e.g. an amorphous carbon substrate like that employed with the library), a library member, and the ratio between the two curves. As can be seen in FIG. 4, the temperature (and therefore the radiation intensity) of the carbon initially increases, reaches a maximum, then decreases over a smooth curve. The library member also exhibits a similar curve as it heats and cools, except there is a sudden increase in the radiation when it heats and a sudden drop in radiation as it cools. These sudden changes correspond to the temperatures at which the library member changes phases, e.g. melts and solidifies, respectively. The ratio of the substrate's radiation and the library member's radiation illustrates the melting point more clearly, appearing as a relatively sharp increase and a relatively sharp decrease in the graph. The temperature at which the library member changes phase is determined by reading the radiation at the points in time at which the sudden increase or decrease in the library member radiation occurs and using the calibration curve shown in FIG. 4 to determine the corresponding substrate temperature. As a result, the phase change point for every member in the library can be determined quickly.
The inventive parallel screen is particularly suitable for screening new compounds via combinatorial materials science techniques to locate compounds having specific desired properties. For example, the library members can be arranged on a substrate in a standardized format using known combinatorial chemistry techniques. This would allow the materials to be both deposited and tested in parallel, increasing the number of members that can be tested per unit time. As a result, preparation and deposition of the library materials can be automated, if desired, further accelerating the speed at which materials can be tested.
It should be appreciated that the present invention is particularly useful for measuring data associated with phase transitions, particularly from the solid to liquid or liquid to solid phases. This makes it highly attractive for and it is useful for screening melting points of metals, semiconductors, and ceramics and their respective alloys. Of course, other material systems may be screened as well, such as plastics, biological materials, or any other material.
It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that the method and apparatus within the scope of these claims and their equivalents be covered thereby. Further, all applications and patents referenced herein are explicitly incorporated by reference.
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