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Is your resume in need of a little disruption?
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making things work better.
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StartUpHire is the place to find Business-Systems-Analyst jobs in and near . Review the list below of Business-Systems-Analyst jobs. These Business-Systems-Analyst jobs represent some of the most rewarding careers available. You can refine your job search for Business-Systems-Analyst jobs in by adding a job title, company name, or keyword to the search box above … or click the "Advanced Search" link for more options. Don't see the employment opportunity you're looking for? Check back soon for more Business-Systems-Analyst jobs in , or sign up for an email alert (link in upper left) to receive periodic updates on new Business-Systems-Analyst jobs in , .
Expert In: PL/SQL, Test Plans And Unit Testing Procedures Experience Required: 6 Months Other Skills Required: Analyze clients' business and data processing requirements and deliver proposals for new or modified business data solutions to improve operations. Propose project plans4/24/2017 Simi Valley, CA Save This Job
Client Management Nashville, Tennessee Apply Description This position is located at 2 Ingram Blvd - La Vergne, TN Company Information Empyrean Benefit Solutions, Inc. is a Human Resource Technology and Services company that provides health & welfare benefits administration4/17/2017 Nashville, TN Save This Job
Business Technology and SolutionsSan Mateo, CAIntern Apply Now! Careers / San Mateo, CA /IT Business Systems Analyst IT Business Systems Analyst San Mateo, CA, United States We have an epic opportunity for you to join our fast growing team at Zuora where you can have an impact4/22/2017 San Mateo, CA Save This Job
In this newly created role, you will be responsible for maintaining and supporting Alder?s mission-critical applications, with an emphasis on Financial applications including Microsoft Dynamics AX. You will oversee the configuration, integration, maintenance and testing of4/26/2017 Bothell, WA Save This Job
Req Id 4321 - Posted 11/10/2016 - Corporate Headquarters - Denver, CO - Technology Operations ? Job Description Print Preview Apply Apply using LinkedIn? Save Job Email Job to Friend Return to List Business Analyst We believe feeling good and doing good is the best2/9/2017 Denver, CO Save This Job
Product Development San Luis Obispo, CA APPLY NOW Send Jobvite MINDBODY?s Data Science department makes sense of our data world through meaningful and actionable insights, identifying trends, delivering reports, informing product and infrastructure advancements, and promoting a5/19/2017 San Luis Obispo, CA Save This Job
E-Trading Systems Analyst I - Chicago? Help Please fill in the information below and use the navigational buttons to continue. Warning: Using your browser's Back and Forward buttons may produce undesirable results. Job Title: E-Trading Systems5/1/2017 Chicago, IL Save This Job
Location Massachusetts-Corporate - Franklin, MA 2038 US (Primary) Education Bachelor's Degree Category Finance/Accounting Shift Type Standard Business Hours Employee Type Full-time Willingness to Travel Up to 10% Interactions is changing the way businesses and consumers4/11/2017 Franklin, MA Save This Job
US-CO-Denver Apply View All Jobs Share This Job Job Description Are you innovative, detail-oriented and strategically minded? Make an impact and love what you do! We are looking for a Systems Analyst 1 who can fully understand and analyze both internal and external5/26/2017 Denver, CO Save This Job
US-CO-Denver Apply View All Jobs Share This Job Job Description Are you innovative, detail-oriented and strategically minded? Do you love to help envision the future and make the visions a reality? Make an impact and love what you do! We are looking for a Systems5/26/2017 Denver, CO Save This Job
SAN FRANCISCOBUSINESS INTELLIGENCEFULL-TIME APPLY FOR THIS JOB We are looking for a Business Intelligence Analyst who is passionate about analytics and business strategy. You?ll serve as an influential sounding board for executives and leaders across the company, who will rely on4/9/2017 San Francisco, CA Save This Job
All times are in India Standard Time. ID 2017-3603 Job Location US-CA-City of Industry Posted Date 4/15/2017 Category BSA More information about this job: Purpose Of The Job: To supports the information & process analysis needs of the company's ecommerce platform. This role4/26/2017 City of Industry, CA Save This Job
at AnchorFree Inc. Menlo Park AnchorFree?s mission is to provide secure and private access to the world?s information for every person on the planet. AnchorFree?s Hotspot Shield app is the world?s most popular freedom and privacy platform. Hotspot Shield is the starting point,4/22/2017 Menlo Park, CA Save This Job
Company:KBI Biopharma Job Location(s):Durham, NC Start Date:Immediately Employment Term:Regular Employment Type:Full Time Starting Salary Range:Competitive Required Education:Bachelor's Degree Required Experience:10 years Required Security Clearance:None Related3/28/2017 Durham, NC Save This Job
Waltham ApplyApply with LinkedIn Responsibilities: Partner with business analysts and developers to document functional business requirements and technical specification documentation Perform as IT Liaison between business stakeholders, business analyst and developers Validate4/29/2017 Waltham, MA Save This Job
Unifi is the leading data acquisition & data integration platform that enables the businesses to acquire data, integrate it, enrich it, and load it into a flexible environment where agile and iterative analytics can occur. In order to support a highly available, scalable and3/28/2017 San Mateo, CA Save This Job
Alameda, CA, US Worker Category :Regular Full-Time Corporate Mission Singulex is a commercial stage, privately held company pioneering Next Generation immunodiagnostics (NGI). Singulex?s provides unprecedented ultra-sensitivity in the precision measurement of biomarkers. Such4/25/2017 Hayward, CA Save This Job
All times are in India Standard Time. Job Code 17.311 Job Locations US-PA-Warminster Posted Date 5/5/2017 Category Information Technology FLSA Status Exempt Schedule SP Industries Inc. PPC5026 More information about this job Job Description Primary Function: Include the5/12/2017 Warminster, PA Save This Job
Company: ESS Inc. Location: Tualatin, OR Company Profile ESS Inc. is a fast-growing, venture-backed, clean technology start-up, located in Portland, OR. ESS has developed and is now producing an advanced flow battery technology for commercial, industrial, and utility=scale4/5/2017 Tualatin, OR Save This Job
Sales Operations Analyst? Help Please fill in the information below and use the navigational buttons to continue. Warning: Using your browser's Back and Forward buttons may produce undesirable results. The Sales Operations Analyst will be responsible for day to day sales4/18/2017 Holmdel, NJ Save This Job


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Storm Preparedness
Being prepared is the best thing you can do to protect your family.
Get a Game Plan
Click here for a list of ten tips I have compiled to help you prepare for the potential threat of storms that may approach in the future.
The following are web sites and contact information for various federal and state agencies:
Top Tips to Be Prepared:
- Make plans to protect your property such as storm shutters or plywood for windows. Tape does not prevent windows from breaking. Bring things indoors including lawn furniture, toys, trash cans or anything that could fly through a window.
- Learn evacuation routes and contraflow procedures. Determine where you would go and how you would get there if you need to evacuate. You can get information on the Hurricane Evacuation Routes here.
- Gather personal documents, including Social Security cards, proof of residence, insurance policies, wills, deeds, birth and marriage certificates, etc. Make a record of these documents and all valuable possessions.
- Keep your automobile fully fueled; if electric power is cut off, gas stations may not be able to operate pumps for several days.
- Be prepared to survive on your own for a few days. Assemble a disaster kit. Include a battery-powered radio, flashlights, extra batteries, a first-aid kit, blankets, clothing, food and water, prescription medications and medical supplies.
- Talk to your family about the dangers hurricanes can present. Plan a centralized meeting place for your family in case you are separated from one another. Choose an out of state family member or friend for everyone to contact and check in with.
- If you are receiving Social Security or Veterans Benefits make sure you have direct deposit. This will ensure you receive all of your benefits if you have to evacuate. The Social Security Administration's telephone number is 1-800-772-1213 or visit their website. The Veterans Administration's telephone number is 1-877-222-8387 or visit their website.
- Know how to shut off utilities. Know where gas pilots are located and how the heating and air-conditioning system works. If you have any questions, contact your local utility company.
During storm season it is important to pay attention to updates from the local media outlets. Also, contact my office with any needs or concerns you may have. If any of our district offices are ever required to evacuate, you can always reach my staff in our Washington, D.C. office at (202)-225-3015.
Congressman Scalise's Congressional Offices in Louisiana:
- Metairie: (504) 837-1259
- Hammond: (985) 340-2185
- Mandeville: (985) 893-9064
- Houma: (985) 879-2300
Important emergency preparedness Twitter handles to follow:
- @LafourcheGov
- @LafourceSO
- @PlaqEoc
- @Plaquemines_Gov
- @sheriffsbso
- @StBGov
- @nolaready
- @nolahealthdept

subset: commoncrawl


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You are currently using an unsupported web browser.
We support the latest versions of these browsers:
If you have any problems while attempting to download a browser, you should contact your organization's system administrator.
For any questions related to Cvent, feel free to contact our Customer Care team.
Hotel Inca is located in the heart of the city, just 50 meters from the Basilica del Pilar, in the center of the historic and shopping area. It is worthy of special mention a typical aragonese cellar where you can taste the most delicious regional dishes and international cuisine.
The Inca Hotel...
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A grieving parent has taken to social media to narrate how policemen blew off her 8-month-old daughter's brain with bullets.
According to her: "A minute before Zainab was shot by the Nigerian Police in Kano, I took her from her dad and kissed her telling her I love you, then hugged her within my neck, after a while I felt some wet substance around my neck not knowing i'm wet with the brains of my little baby Zainab!"
Mother of 8 months baby martyred Sayyidah Zainab, during Nigerian Police attacks on Imam Hussain (AS) Arbaeen Symbolic trek from Kano to Zaria.


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Russians, seeking their fourth straight Olympic title, scored 28.375
points for their routine with five balls Thursday. That was .275 points
better than Italy, which has won the last three world championships.
Belarus was third.
Qualifying continues Friday with three ribbons and two hoops. The top eight teams advance to Sunday's final.
Thursday, Daria Dmitrieva upstaged defending Olympic champion Evgeniya
Kanaeva as Russia went 1-2 in individual qualifying.


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The long road back to the Wallabies for James O'Connor starts in Hobart on Saturday with the Melbourne Rebels' first Super Rugby trial match of the year.
O'Connor can't wait for his long-awaited comeback from recurring hamstring troubles and could well start opposite code-jumper Israel Folau this weekend, who is set to make his senior rugby debut for the NSW Waratahs.
"I feel excited, and the ground is like carpet, so I can't wait to get out on the paddock,'' O'Connor said.
"If you had told me this time last year I'd be playing a game in Hobart I would have thought you were having a joke with me.
"It's my first game back in seven months so it's going to be pretty massive for me.''
O'Connor last year became a new member of the Wallabies leadership group leading into the Tests against South Africa, New Zealand and Argentina.
His main aim this season is to get back playing for Australia.
"That's the goal - I would love to pull on that gold jersey again,'' O'Connor said.
"That's the pinnacle of our sport, and whenever I can pull on that jersey it's an honour.
"If I can put the performances on the park - I've got to have a big season for the Rebels first - then hopefully I can catch the selectors' eye and pull that jersey on again.''
Almost all of the Rebels' 35 players will get a run in Hobart, but Wallabies back-rower and big-name Queensland recruit Scott Higginbotham will be rested.
O'Connor said the contest would be more than an exhibition match.
"Every time we get on the paddock it's a challenge and we want to put in our best performance,'' he said.
"It's the first game of the year and it's against the Waratahs, they set the benchmark and we want to get one on these guys early and assert our dominance."
The debut of Folau, who jumped from rugby league to AFL and now rugby union, would be a treat for spectators and an acid test for the Rebels.
"He's a very good athlete and I'm looking forward to playing against him,'' O'Connor said.
"Last time I saw him he looked pretty fit, and he's added a kick to his game now as well, so I think he will do well - hopefully not too well this weekend.''


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The film is unabashedly supportive of Father Hartley, presenting him as a stubborn saint, and depicts the wealthy owners as soulless villains. Presumably they have a different story to tell, but we wouldn't know: When the camera's on, none can be found.
While the political implications of the film are provocative, "Sugar" also happens to be an impressive cinematic achievement. This picture has a visual sweep that many docu films lack; the plantations and nearby towns are vividly evoked. show more
Explores an unheralded but emotionally affecting issue in a straight-forward and engaging manner. show more


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Who are we?
The Dutch Caribbean Business Club is a professional networking organization whose main purpose is to help establish and improve business contacts between Dutch and Caribbean-based companies, (semi) governmental institutions and private persons doing business in the Caribbean and/or The Netherlands. The club consists primarily of Dutch and Caribbean business people with professional backgrounds.
Examples of our members are:
Events are held 5 times a year in the Netherlands and special events are held each year on one or more of the Caribbean islands.
The five network events are held at sponsored locations with drinks/refreshments and finger food. Entrance is free for members, while aspiring members can attend a network event (one time only) by paying 29 euro's excl. VAT (when registering in advance). Normally a short 15 to 20 minute presentation or guided tour on location is given by the host followed by plenty of time to greet familiar colleagues and meet new people in the club.


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WYDaily.com is your source for free news and information in Williamsburg, James City & York Counties.
Game played at Steward.
Halftime Score: WA 1 Steward 0
FINAL Score: WA 3 Steward 2 in OT
Records: WA 7-0-3 Steward 3-2-1.
Goals:WA Solvi Bjornsson 3 Steward-Jack Caldwell, Jonathan Stringfellow
Assists: WA Joshua Addo
Saves: WA Bryce Behm 8 Steward 6
The Trojans managed to strike first despite arriving late due to mechanical issues with their bus. Solvi Bjornsson scored to put the Trojans up at half 1-0.
Steward struck quickly in the second half to even the score, only to have Bjornsson score again midway through the frame to give the Trojans the 2-1 advantage. The home team though wouldn't go quietly as they sent a long ball in, with less than 2 minutes left, that Jonathan Stringfellow was able to bury to again tie the score. The game went into OT and in the first OT, Solvi Bjornsson was clipped in the box after receiving a pass from junior Max Rinaldi. The foul resulted in a PK that Solvi Bjornsson calmly slotted in, for the eventual game winner, and his third goal of the game. Bryce Behm was outstanding in the net for The Trojans with 8 saves, a couple of which were point blank.
Next game for WA, home against Woodberry Forest at 430pm.
Submitted by Richard Plank
This content was provided by a community member via Local Shout, a new initiative at WYDaily. WYDaily cannot guarantee the accuracy of information presented in this story. If you have additional information or would like to submit a story, please contact firstname.lastname@example.com.


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ED Magedson – Founder
Ripoff Report
Clearwire Corporation4400 Carillon Point Kirkland, Washington United States of America
Does your business have a bad reputation?
Fix it the right way.
Corporate Advocacy Program™
This report was posted on Ripoff Report on 10/10/2011 08:41 PM and is a permanent record located here: http://www.ripoffreport.com/reports/clearwire-corporation/kirkland-washington-98033/clearwire-corporation-clear-clearwire-refused-to-issue-a-credit-after-free-modem-was-ret-787261. The posting time indicated is Arizona local time. Arizona does not observe daylight savings so the post time may be Mountain or Pacific depending on the time of year.
Ripoff Report has an exclusive license to this report. It may not be copied without the written permission of Ripoff Report. READ: Foreign websites steal our content
Click Here to read other Ripoff Reports on Clearwire Corporation
If you would like to see more Rip-off Reports on this company/individual, search here:Search Tips
In order to assure the best results in your search:
Advertisers above have met our
strict standards for business conduct.


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It looks like you're new here. If you want to get involved, click one of these buttons!
I am after some leggings/compression shorts, but ideally I would also like some with sewn in over shorts if possible. I have been looking at the Ashmei 2-in-1 shorts which would be perfect if they went past the knee.Does anyone know what my options are?
i have seen one company that doe it, i have a feeling it was underarmour, but i could be wrong, dont see the point personally, why not just wear shorts over the top?
Yes it is better to have separate
Skully14 wrote (see)
Because you won`t look like such a dipstick!! .......
Not sure there are any 2 in 1 shorts with inners long enough to pass the knee Alasdair, most finish 2" above the knee..
How about these from ronhill.
Surely the initial question is a contradiction. Shorts don't go below the knee; if "trousers" go below the knee but are not full length then they are 3/4 or capris. Shorts stop above (ideally way above) the knee. Also begs the question why oh why wear short over tights
Craft - Under Armour - Nike - Adidas - Asics - Salomon etc .. all sell running shorts with some sort of compression lining sewn in ..
Prices range from £25 to £130
May I ask why you wish your knees covered Alasdair


text: ×Close Product Image 1 of 1 Will It Freeze? See image Will It Freeze? By Joan Hood × Added to Cart List Price $14.95 Your Cart ( items) Cart total Continue Shopping CHECKOUT NOW Choose a format: Trade Paperback Format: Trade Paperback Scribner | 196 pages | ISBN 9780743237666 | February 2002 Add to Cart Buy Simon & Schuster Add to Cart Amazon Barnes & Noble Books-a-Million IndieBound Walmart List Price $14.95 In Stock: Usually ships within 1 business day Browse Related Books Cooking > General Buy from another retailer Amazon Barnes & Noble Books-a-Million IndieBound Walmart Get a FREE eBook when you join our mailing list. Plus, receive updates on new releases, recommended reads and more from Simon & Schuster. Email Address Free eBook available to NEW subscribers only. Offer redeemable at Simon & Schuster's ebook fulfillment partner.Offer expires in three months, unless otherwise indicated.See full terms and conditions and this month's choices. About the Author Joan Hood BECOME A FAN BOOKS


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Recently I started looking around for cheaper car insurance. I looked several different ways and got quotes from several companies. I found a company that had cheaper rates and lots of their customers had great things to say about them. Here is how I found cheap car insurance rates when I was getting auto insurance quotes.
First I started by calling a local company my friend recommended to me. He said they were the cheapest out of everywhere he looked. He even looked online, but this company had the lowest rates. I called them and gave them all my information. They called me back a few minutes later to give me a quote on my insurance. I thought maybe I could get it cheaper elsewhere so I told them I would call back.
I went online and started looking for auto insurance comparison websites. I found several companies that had cheap car insurance rates. I entered my information and within minutes had quotes from several companies at once. I found a company that had really cheap rates, so I started learning more about them. I found out that many of their customers said they are a great company to use for car insurance. They also said they work quickly and respond fast too. I decided to get set up with insurance through their company. I was really happy with the rate they gave me.
I had to do a little searching to find cheap car insurance rates. I am glad I took the time to shop around for lower rates. I was able to save $500 a year on my car insurance and I wouldn't have been able to do that without putting in a little effort. My new insurance company has been great so far.


text: Hi. I am completely new to this forum, just joined. Anyways, I have a problem. My laptop {NV53 Gateway} quit searching for networks, and eventually displayed no connections are available. I have looked for solutions all over the internet but I can't find any. The troubleshooter doesn't work for me. It says the driver is experiencing hardware issues, but I know it doesn't, because I looked in the device manager and it doesn't have any issue. I am trying to figure out what the problem is, I've tried to connect to a network, that doesn't work & I've also tried setting up a new network but that doesn't work either. My Wifi button on my computer is on, and I do have a card installed on it, and it still doesn't show up with any networks. Any ideas? EDIT: i did ipconfig/all and my Ethernet Adapter Local Area Connection says media disconnected only because I dont have it connected to ethernet... On the other hand, it says: Tunnel adapter isatap.{45E92676-7CB114F99-8B8C-2BE1DA2A0FEA} and it is a Microsoft ISATAP Adapter (which i didnt know i had) and the other is a Tunnel adapter Teredo Tunnneling Pseudo-Interface both of them are media disconnected. Broadcom NetLink {Tm} Gigabit Ethernet is my ethernet connection. I usually go wirelessely.


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Welcome to WebmasterWorld Guest from 184.108.40.206
Forum Moderators: phranque
What browser do you currently use? Four years ago, more than half of our site visitors were using Internet Explorer, but today that figure is less than a quarter.
The rise and fall of various browsers is well documented, but it's always interesting to analyse the market from a particular point of view - say, that of a UK-based technology website. Luckily, we have the stats to do just that.
The rise of Chrome is the obvious standout, but there's more to the market changes than Google's browser.
Right now, the top three browsers are all pretty even among PC Pro visitors - at the last count there was less than 3% between them. Firefox and Internet Explorer have snaked around one another as they both decline, and Chrome finally edged ahead of them in our stats in April this year.
joined:Mar 3, 2003
votes: 12


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APA Formatting
In the most cases when writing academic papers students have to follow APA guidelines and rules. Sometimes if your writing does not meet American Psychological Association standards it can not be accepted for future checking. If your professor asked you to write whatever paper in APA citation style - you would better learn all the list of requirements first. Headings, punctuation, citations and references need to be formatted in accordance to APA style. Writing papers in APA format usually takes more time because of the great number of rules one should keep in his mind while writing. If you were assigned to write an APA format papers or you just need to reformat already written paper - you will probably need professional APA style paper writing help. It is much more easier to create a usual paper that also has a pretty long list of rules and reformat it than with the help of professionals. EffectivePapers can provide you with APA format papers written from scratch and in accordance to modern standards and rules.
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Find us on Twitter
Get email updates
Contact us
The Conservative Foreign and Commonwealth Council meets regularly in Westminster, for small-group talks with Ambassadors from around the world, as well as MPs, MEPs and others. If you'd like to get involved, we'd love to hear from you.
Our key people
The Patrons of the Conservative Foreign and Commonwealth Council include William Hague, Sir John Major, Baroness Chalker of Wallasey, Lord Howell of Guildford, Lord Hamilton of Epsom and Lord Hurd. Our President is Sir Ronald Halstead CBE and Vice Chairman Rehman Chishti MP.


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Need a Cat Allergy Test in Warrensburg, MO? Simply order online 24/7 and get a fast, affordable Cat Allergy Test at a lab near you (Find More Locations):
Why would I get a Cat Allergy Test?
A Cat Allergy Test checks the blood for evidence of a cat allergy. People in Warrensburg, MO order the Cat Allergy Test for general screening or in the setting of symptoms.
How much does a Cat Allergy Test cost?
Our Cat Allergy Test prices include the doctor's order that you need to get tested in Warrensburg, MO, all lab fees and taxes, and a pdf copy of your results. For current prices, please CLICK HERE.
We are pleased to offer the Cat Allergy Test in Warrensburg, MO.
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Incipio Faxion Black for iPhone 5 Slim Flexible Hard-Shell Case
The FAXION iPhone 5 Case by Incipio features a hybrid design that is composed of a rigid plextonium frame and impact resistant core. A co-molded 1-part design makes installation of the FAXION a breeze without sacrificing iPhone protection.
Rigid Plextonium shell with impact resistant core
Excellent shock absorption
Innovative hybrid design
Superior Protection
|Color of product
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|iPhone 5/5S
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|For Boys, For Girls, For Men, For Women
Write Your Own Review
Refund/ Return Policy
Any product purchased through us can be returned for a full refund or exchange within 30 days from the date you made your order. Please read the below steps explaining how to exercise this.
1. Request a return number by e-mailing us or using on our website contact form before returning the product. Please include your name, order number, the reason for the return, and if you want to return for a full refund or exchange for another product. Within 1 to 2 business days after submitting the request you will be contacted via email with an RMA number and product return instructions.
2. Returned items must be complete condition and not missing necessary parts. When returning a product, please write your RMA number on the package.
3. We will not pay the return shipping costs for non-defective returns. It is also the customer's responsibility to ensure that the returned goods are packed properly.
4. Refunds will be issued back to the original payment method. The refund will be issued the same day as we receive your returned product. If the product is not required to be returned, the refund will be issued on the same date as we specify to you by email.
If you wish to get a replacement of the same product because of a defective product after 30 days from the date you made the order, all products we sell come with our own 6 month replacement warranty from the order date. Please email us with your order number and description of the defect for a replacement. We can send the same product or an equivalent product of similar value that you specify.


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Midland HH50 Handheld Noaa Weather Alert Radio - Black - $18.88
This compact handheld unit is a must for anyone venturing into the woods for any period of time. Knowing about sudden weather changes in advance can save your life. PLL drift-free weather broadcast reception keeps you tuned to the nearest NOAA transmitter and gives you access to the latest weather bulletins and other emergencies that activate the area emergency alert system. There are seven weather channels that are automatically scanned when the power is turned on. Convenient telescoping 6-inch antenna and belt clip. Power is supplied by three AAA batteries (not included). Case color is Black. 1-3/4 L x 1 W x 5-1/2 H. Color: Black. - $18.88


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CALLIDE mine is up for sale, with owners Anglo America stating the mine near Biloela no longer fits the company's growth strategy.
Chief Executive Officer of Anglo American's Metallurgical Coal business, Seamus
French today announced the company had commenced a divestment process for the thermal coal mine.
Mr French said Anglo American's Metallurgical Coal business unit's strategy was focused on growing its metallurgical coal business.
"Callide mine is a thermal coal operation primarily supplying domestic power stations in Biloela and Gladstone," Mr French said.
"The Callide mine does not align with our growth strategy and the divestment process will lead to the sale of the operation as a going concern.
"Callide mine is a low cost coal producer with a significant resource base of more than one billion tonnes, a stable and well run operation in an established mining area and has a strong future underpinned by long term coal supply contracts.
"Anglo American sees an exciting opportunity for investors to expand the Callide business," Mr French said.
"The process will be undertaken with care and respect for our employees and all other stakeholders with our priority during this period being the safety of our employees and stability of the operation," Mr French said.
"Anglo American will work closely with Government, its customers, suppliers and the local community to ensure they are consulted throughout this process," Mr French said.
Member for Flynn, Ken O'Dowd has requested an urgent meeting with Anglo management.
"Anglo has said they wish to focus on expanding their metallurgical coal business and that thermal coal did not align with the company's current strategy.
I need to know what this decision means to the workers and the community of Biloela and district," Mr O'Dowd said.
"We've seen it all before, these companies come to town with all the hoop-la and hype and when they see a 'better' opportunity they move on.
This should sound a warning bell to the State Government in their dealings with the LNG Industry," he said.
"Governments and local councils should be demanding that these companies commit to social infrastructure before they are granted their valuable operating licences, because when they are finished they will pack up and move on, with little consideration for the communities that have bent over backwards to accommodate them.
"I need to know from Anglo just how committed they are to Central Queensland and who they might be considering as a prospective purchaser of these assets.
"I don't like the sound of it and I need to be reassured that the Callide mine has a strong and viable future." Mr O'Dowd said
Read Friday's Central Telegraph for the latest update.
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What Thanksgiving Fail? UberEATS Tackles Turkey Troubles
Cooking the perfect Thanksgiving turkey is a lot of pressure. And despite valiant efforts and age-old recipes, many end up with a frozen, crispy, or dropped #ThanksgivingFail. And a dining room of (politely) disappointed friends and family.
Every year, the bravest of the brave share their not-so-successful turkeys online. And this year, knowing "take two" turkeys are hard to find, we wanted to be ready to respond. You know, just in case…
Three UberEATS delivery partners—Kevin, Daemon, and Lyndon—kind enough to give up their Thanksgiving mornings, and two market partners—Goddess and Grocer and The Market—kind enough to prep beautiful turkeys, joined forces to fix the fails. Here's a taste of what happened:
Thursday morning, Sandra's turkey breast wasn't quite turning out as she'd hoped. She tweeted us this shot from her Chicago kitchen:
It wasn't long before Kevin and Goddess and Grocer came to the rescue with a ready-to eat bird, stuffed with all the goods.
Kevin also stopped by the home of sisters, Kendra and Nykila. With a house bustling with family and rambunctious little ones, a turkey shortage was not an option. So the sisters welcomed Kevin and their Goddess and Grocer bird with open arms. It just so happens that Kevin kept it all in the family–Kendra is a driver partner herself.
On the opposite coast, a great friend made Jenn and Charlie's day turkey-ful after all. Fresh off of a turkey "mishap" last year, the Seattle couple was settling for bird-less Thanksgiving when a good friend caught wind of our turkey adventures. That friend asked us to bring them a bird and Daemon delivered.
Meanwhile in San Francisco, Lyndon brought Kathleen, Anna, and Brenna a turkey spread from The Market. With families on the East Coast, the pals were hosting a Friendsgiving and making do with a chicken substitute. But as the afternoon wore on it became clear that Thanksgiving just didn't feel like Thanksgiving without the turkey. It sure felt like Thanksgiving once Lyndon came through.
After a morning of making turkey worries disappear, Goddess and Grocer and The Market made sure that Kevin, Daemon, and Lyndon had turkeys to bring home for their families to enjoy. No word on whether the birds were used to rescue fails of their own :)
This year we're thankful for all of our partners and the Thanksgiving brave who embraced a whole new kind of turkey delivery. Cook on, you brave turkey tacklers. UberEATS has your back.
To see what UberEATS is cooking up in your city this week, head to UberEATS.com
Featured articles
Uber for Business now makes it easy to give your customers a ride
We're excited to expand the Uber for Business platform beyond business travel, to include a world-class customer transportation solution, Uber Central. With Uber Central, organizations of all shapes and sizes can now easily provide on-demand, door-to-door transportation for their customers, clients, and guests.
UberEATS: Using technology to build a reliable food delivery experience
A little over a year ago, we set out to put a new spin on an old classic–make reliable food delivery available at the tap of a button. Back then, we started by offering food in the UberEATS app from 1,000 pioneering restaurant partners in four cities. And today, more than 40,000 restaurants globally–from poke shops to pasta spots–are sharing food with customers through UberEATS. With a growing restaurant community comes more choices and more complexity. So we're cooking up features to continue to make UberEATS easy and reliable. Here is a taste–
Let's take care of each other
This flu season, we're delivering free flu-fighting care packages across the country. When you receive a pack, you'll have the option to request a free flu shot from a registered nurse–for up to 5 people.


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Smoke Signals
If she was able to send out smoke signals, she would. She wouldn't hesitate to do so. She'd shoot a flare sky high if she had one. Hell, she'd use her pager or a phone or send an email if she could.
As it is, she is stuck aiming House pointed looks and tapping out Morse code with her index finger on her arm. S.O.S. Tap. Tap. Tap. Tap-tap-tap. Tap. Tap. Tap.
He raised an eyebrow the first time she did it and then lazily drew his eyes to the man beside her. The man is in his sixties with greying hair and a bald patch and startling green eyes. Also, he is annoying the hell out of her.
She hasn't been so irritated by a hospital donor in years. And never like this. Every little thing she's done as she showed him around has not escaped his attention and he keeps questioning her about it, about her.
It feels more like an awkward first date than an official meeting.
She sighs to herself and tries once more to get House's attention. If he can ask for a consult, anything to free her of this man without losing the donation he wishes to give the hospital, she swears she'll give him five hours off clinic duty. Maybe more.
Perhaps he can read her mind because in about ten seconds, he abruptly starts walking toward them. He is wearing an expression she can't get a read on.
"Need you," he says simply. "Now."
She tries not to sigh in relief. She turns to the donor. "Excuse me, Mr Cole. I have to take this. Can I call you later about the donation?"
The man smiles thinly and nods. He is clearly disappointed that their time has been cut short.
She shudders involuntarily.
Fortunately, Mr Cole is too busy sizing up House to notice.
"I'll call you," she says again. "Thank you for your time."
Mr Cole turns his gaze on her. "No problem at all, Dr Cuddy. Give me a call anytime."
Oh, god.
She ignores House's smirk.
"I will," she forces out. She fakes a smile as Mr Cole leaves. The moment he is out of sight, she sighs in relief. Loudly.
House leers at her.
"That man," she says frowning, "is insufferable."
"'I'll call you'," House says pointedly.
"I had to tell him that and you know it. What he can give this hospital – "
"Is worth getting hit on by someone old enough to be your dad?"
"Shut up." Her tone is tired, weak. He is right of course. She allowed Mr Cole the benefit of the doubt and for what? His money? How pathetic is that? She doesn't get long to wonder.
"So what do I get for rescuing from him?" Mocking.
She tries not to smile but biology wins out.
"You," she says deciding not to go with her previous choice to cut his clinic hours, "get the warm and fuzzy feeling inside that a person gets once they've done good."
He stares at her for a moment. "I think I'll pass on that. What's option number two?"
Shaking her head she slowly starts walking away from him. She quirks an eyebrow when he follows and offers him the same teasing look. "You know what? I think I'm starting to miss Mr Cole."


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George Noory on WAEB
Monday thru Friday 1am-5am
Saturdays: 1am-6am
Sundays: 12am - 5am
On his first official night in the "captain's chair," George Noory offered a special message about his connection with Art Bell, telling the listeners that Art had a major role in his hiring and eventual "grooming" to take his place. George described his early interactions with Art. "My connection with Art Bell was almost like…talking to your idol baseball player when you're a kid. Because he truly is a living legend. I've got to tell you he was so supportive, informative and helpful. He pointed out things that he thought I was doing wrong and he pointed out things he thought I was doing right. And he really told me one major thing: 'George, put your own mark on this program. You're going to have lots of people who'll support you, millions of people, and you're going to have some that won't. It doesn't matter. You do what you think is best!'
Speaking directly to Art via the airwaves, George said: "I will do you proud, the best I can, Art. There comes a time in this life wherever you might be that you have an opportunity to work with someone who is literally…like a god in this business. Someone you can look up to and respect. I've had the opportunity and so now, no matter what happens in this broadcasting business for me, I had a chance to work with Art Bell."
For more on Coast to Coast with George Noory CLICK HERE!


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`The Voice` loses Shakira, gets Christina Aguilera back
Los Angeles: Colombian singer Shakira will leave NBC`s "The Voice" and Christina Aguilera will return to take her place next season, the Web site E! News said.
"It was a hard decision for me to make, because I`m really fond of the coaches, the people in production and everybody around me," the 36-year-old superstar told E! News.
"And I really enjoyed `The Voice`, but I also have a musical career and I`m also a mother now and my poor baby`s so tired flying such long distances. He already has more miles than any pilot. So I think I have to give him a little break, and we`ll see what happens in the future," she said.
Shakira and Spanish soccer international Gerard Pique celebrated the birth of their baby boy Milan last January in Barcelona, where the couple has settled.
Shakira`s absence will be filled by Aguilera, one of the coaches in previous years who will be back for Season 5.


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|Print edition Sports
Page 8C
Barbosa, Ginobili have sixth sense
By David DuPree
When it comes to making things happen for their teams, the spotlight often turns on Leandro Barbosa of the Phoenix Suns and Manu Ginobili of the San Antonio Spurs.
Overshadowed most nights this season by higher-profile All-Stars Steve Nash, Amare Stoudemire and Shawn Marion of the Suns and Tim Duncan and Tony Parker of the Spurs, Barbosa and Ginobili often set the tone with their all-out, frenetic play.
That could be particularly important tonight in one of the biggest games of the season for both teams (9:30 ET, TNT).
The rivals appear headed for a second-round showdown in the playoffs, with the big question being who will have the home-court advantage. The Suns go into the game with a three-game lead over the third-place Spurs in the race for the No. 2 seed in the West behind the Dallas Mavericks. The winner will also capture the season series 2-1.
"They're one of the best in the league," Duncan said of the Suns. "It's a great test time for us, especially coming down toward the end of the season here."
It's also a head-to-head showcase for Barbosa and Ginobili, the two leading candidates for the NBA's Sixth Man Award. Ginobili began the season as San Antonio's starting shooting guard, but when the Spurs bench faltered, coach Gregg Popovich did what he has often done � turned to Ginobili to provide the spark. This time it happened in late January.
"It isn't the first time," Ginobili said. "I understand why it's this way. It's the best way to help the team win, so I'm in favor of it."
To win the Sixth Man Award, a player has to have come in off the bench in more games than he has started. Ginobili has started 36 games and been a reserve in 33. The Spurs have eight games left, so Ginobili will have to come off the bench in four of them without any more starts to be eligible.
Barbosa, although he has started the last two games and 17 overall this season, will end the season having come off the bench 55 times if he remains the starter.
"It doesn't matter to me," Barbosa said. "My role is still the same � to make things happen."
Ben Gordon of the Chicago Bulls was the early leader for the Sixth Man Award, but he has started 44 games, making him ineligible.
The Suns lead the league in scoring and two of the three shooting categories, and the Spurs, third in opponents' field goal percentage and second in opponents' three-point percentage, are one of the league's premier defensive teams.
"We shoot the ball so well that it gets overlooked how well guys are shooting," Suns coach Mike D'Antoni said.
For the Spurs, going into the playoffs with everything clicking has always been their objective. "We haven't really focused on getting the best record in any year," Popovich said. "The focus has always been making sure our priorities are in order, beginning with defense, that we're as healthy as we can possibly be, and that our bench has gotten enough minutes."
Page 8C
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The protein encoded by ZNF335 enhances transcriptional activation by ligand-bound nuclear hormone receptors. Additionally we are shipping ZNF335 Kits (6) and and many more products for this protein.
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Garapaty, Mahajan, Samuels: Components of the CCR4-NOT complex function as nuclear hormone receptor coactivators via association with the NRC-interacting Factor NIF-1. in The Journal of biological chemistry 2008
we performed a meta-analysis of our data with the previous reports, identifying two further loci harbouring the ZNF335 and NIFA (show INA Antibodies) genes which now exceed genome-wide significance, taking the total number of CD susceptibility loci to 42.
NIF-1 expression is associated with tumor grade in bladder cancer.
Study identifies and characterizes a nuclear zinc finger protein, ZNF335/NIF-1, as a causative gene for severe microcephaly, small somatic size, and neonatal death.
CCR4 (show CCR4 Antibodies) plays a role in the regulation of certain endogenous RARalpha (show RARA Antibodies) target genes and RCD1 (show RQCD1 Antibodies) and CCR4 (show CCR4 Antibodies) might mediate their function through their interaction with NIF-1
NRC-interacting factor 1 is a novel cotransducer that interacts with and regulates the activity of the nuclear hormone receptor (show NR0B1 Antibodies) coactivator NRC (show NCOA6 Antibodies)
p35 (show CDK5R1 Antibodies) interacts with NIF-1 and regulates the subcellular localization of the protein independent of Cdk5 (show CDK5 Antibodies)-dependent phosphorylation. ...reveal a new mechanism of p35 (show CDK5R1 Antibodies) in transcriptional regulation that does not require the kinase activity of Cdk5 (show CDK5 Antibodies)
Results suggest that NIF-1 directs neuronal morphogenesis during early developmental stages through modulating activity-dependent gene transcription
Zinc finger protein Zfp335 is required for the formation of the naive T cell compartment.
Znf335 null mice are embryonically lethal, and conditional knockout leads to severely reduced cortical size.
The protein encoded by this gene enhances transcriptional activation by ligand-bound nuclear hormone receptors. However, it does this not by direct interaction with the receptor, but by direct interaction with the nuclear hormone receptor transcriptional coactivator NRC. The encoded protein may function by altering local chromatin structure.
NRC-interacting factor 1
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, zinc-finger/leucine-zipper co-transducer NIF-1


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Bale crowned 'Prince of Goals' after first La Liga treble
By Iain Rogers
MADRID (Reuters) - Real Madrid's record signing Gareth Bale was hailed as the "Prince of Goals" after his hat-trick and assist in Saturday's 4-0 La Liga win at home to Real Valladolid.
With prolific Portuguese Cristiano Ronaldo sidelined by injury, Bale stepped up to net his first treble since his 100 million-euro ($136 million) move from Tottenham Hotspur and coach Carlo Ancelotti suggested the Wales winger's adaptation period was over.
"He is in good shape, he is confident and he is a very important player for us," Ancelotti told a news conference. "He performed very well today."
Bale's pre-season preparations were disrupted by the protracted negotiations over his transfer and he then picked up a minor muscle strain but now appears to be back to full fitness and eager to impress the Real faithful.
Saturday's haul was a so-called "perfect hat-trick" - a header and a goal scored with each foot - and was only the second La Liga treble by a Briton after Gary Lineker's for Barcelona in 1987.
Sports daily As acclaimed Bale on their front page on Sunday as "Prince of Wales" (Principe de Gales), while rival newspaper Marca went for the headline "Prince of Goals" (Principe de Goles).
"It is always a pleasure to score but even more when you are able to score a hat-trick at the Santiago Bernabeu," Bale told reporters after grabbing a match ball and soaking up a post-match ovation from the Real faithful.
"I am very happy here," added the 24-year-old. "I needed time to adapt after not having a proper pre-season but now I am improving. Continued...


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Antimicrobial peptides (AMPs) are a crucial component of the natural immune system in insects. Five types of AMPs have been identified in the tobacco hornworm Manduca sexta, including attacin, cecropin, moricin, gloverin, and lebocin. Here we report the isolation of lebocin-related cDNA clones and antibacterial activity of their processed protein products. The seventeen cDNA sequences are composed of a constant 5′ end and a variable 3′ region containing 3~16 copies of an 81-nucleotide repeat. The sequence of the corresponding gene isolated from a M. sexta genomic library and Southern blotting results indicated that the gene lacks introns and exists as a single copy in the genome. The genomic sequence contained 13 complete and one partial copy of the 81-nucleotide repeat. Northern blot analysis revealed multiple transcripts with major size differences. The mRNA level of M. sexta lebocin increased substantially in fat body after larvae had been injected with bacteria. The RXXR motifs in the protein sequences led us to postulate that the precursors are processed by an intracellular convertase to form four bioactive peptides. To test this hypothesis, we chemically synthesized the peptides and examined their antibacterial activity. Peptide 1 killed Gram-positive and Gram-negative bacteria. Peptide 2, similar in sequence to a Galleria mellonella AMP, did not affect the bacterial growth. Peptide 3 was inactive but peptide 3 with an extra Arg at the carboxyl terminus was active against E. coli at a high minimum inhibitory concentration. Peptide 4, encoded by the 81-bp repeat, was inactive in the antibacterial tests. The hypothesis that posttranslational processing of the precursor proteins produces multiple bioactive peptides for defense purposes was validated by identification of peptides 1, 2, and 3 from larval hemolymph via liquid chromatography and tandem mass spectrometry. Comparison with the orthologs from other lepidopteran insects indicates that the same mechanism may be used to generate several functional products from a single precursor.
Antimicrobial peptides (AMPs) participate in innate immunity of various organisms ranging from prokaryotes to plants, invertebrates, and vertebrates [1,2]. Most of them are less than 5 kDa, hydrophobic, membrane-active, and carry positive net charge at physiological pH. These peptides are either absent or present at low constitutive levels in naïve insects. Upon microbial infection, association of host recognition molecules and microorganisms triggers extracellular serine proteases to activate a spätzle precursor via limited proteolysis [3]. Spätzle then binds to the Toll receptor to initiate an intracellular pathway that relays the signal into nucleus, where transcription factors of the Rel family induce the AMP gene expression [4]. Identification of orthologous genes in the Anopheles gambiae, Aedes aegypti, Apis mellifera, Tribolium castaneum, and Bombyx mori genomes suggests that similar signaling pathways exist in other insects to induce the production of defense proteins by fat body and hemocytes [5-9]. Fat body, a tissue analogous to liver, synthesizes AMPs and secretes these heat-stable compounds into the plasma to kill the invading microbes.
Insect AMPs can be categorized into the following groups: α-helical peptides (e.g. cecropin, moricin), disulfide-stabilized peptides (e.g. defensin, drosomycin), proline-rich peptides (e.g. lebocin, drosocin), glycine-rich peptides (e.g. gloverin, diptericin), and others [2]. In the tobacco hornworm Manduca sexta, at least five types of AMPs have been identified, including attacin, cecropin, moricin, gloverin, and lebocin [10-13]. As a step towards understanding the role of AMPs in humoral immune responses of M. sexta, we have characterized a lebocin-like protein, its processing products, and corresponding gene in this work.
Lebocins are proline-rich AMPs first purified from the silkworm, Bombyx mori [14]. These 32-residue peptides are glycosylated at Thr15 and this modification is important for the antimicrobial activity against Gram-negative bacteria. cDNA and gene cloning indicates that the active peptide is located near the carboxyl terminus, after a signal peptide and a 102-residue pro-segment [15,16]. Lebocins increase the permeability of liposomes at a low ionic strength and have weak antibacterial activities under physiological conditions. They seem to function as synergists by reducing the minimum inhibitory concentration of cecropin D [17]. Lebocin cDNAs have been isolated from Trichoplusia ni [18] and Pseudoplusia includens [19]. In lebocin homologs of Samia cynthia [20], the pro-segment aligns well with the Bombyx mori sequences, but the part corresponding to the mature lebocin differs significantly. Two peptides, purified from hemolymph of Helicoverpa armigera and Galleria mellonella, are similar to a region in the pro-segment of B. mori lebocins [21,22]. The 42-residue anionic peptide-1 from the greater wax moth G. mellonella is active against Micrococcus luteus, Listeria monocytogenes and filamentous fungi, but neither of these peptides inhibits the growth of Gram-negative bacteria (e.g. Escherichia coli). It is unclear how these peptides are derived from their protein precursors (pro-lebocins). Here we report the cDNA and genomic cloning of lebocin-related proteins from M. sexta, which suggests a conserved mechanism in Lepidoptera to generate structural/functional diversity in products derived from pro-lebocins. This mechanism is validated by identification in hemolymph of three processing products by mass spectrometry. For simplicity, we use "lebocin" in parts of the paper to describe the entire gene, cDNA, and protein that are related but not the same as the mature lebocin peptide.
M. sexta (eggs purchased from Carolina Biological Supply) were reared on an artificial diet [23]. Day 2, fifth instar larvae were injected with formalin-killed E. coli (2×108 cells/larva). Alternatively, day 2, fifth instar larvae or day 1, male adults were injected with 100 μg M. luteus (Sigma) or with sterile water as a control. At various time points after injection, hemocytes and fat body were collected for RNA preparation. Muscle tissues or gut-removed carcasses were used for genomic DNA isolation.
A 280 bp M. sexta lebocin cDNA fragment [11] was labeled with [α-32P]-dCTP using Multiprime DNA Labeling System (GE Healthcare Life Science). A M. sexta induced fat body cDNA library [24] was screened according to Sambrook and Russell [25]. Positive plaques were purified to homogeneity via secondary and tertiary screening. Plasmids, in vivo excised from the positive bacteriophages, were sequenced using a BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems). Sequences were assembled using MacVector Sequence Analysis Software (Oxford Molecular Ltd).
Based on BLAST search of GenBank (http://www.ncbi.nlm.nih.gov/) and ButterflyBase (http://butterflybase.ice.mpg.de/) using M. sexta lebocin as query, homologous protein sequences were retrieved and compared using ClustalX 1.83 (ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/) [26]. A Blosum 30 matrix [27], with a gap penalty of 10 and an extension gap penalty of 0.1 were selected for multiple sequence alignment and unrooted phylogenetic tree was constructed based on neighbor-joining algorithm. Treeview (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) [28] was used to display Phylogram.
The insert from a full-length lebocin cDNA clone (NC2), obtained after digestion with EcoRI and XhoI, was labeled with [α-32P]-dCTP and used as a probe to screen a M. sexta genomic library in λGEM11, kindly provided by Dr. Yucheng Zhu at the Southern Insect Management Research Unit (USDA ARS). Following plaque purification and amplification, phage DNA was isolated using Wizard Lambda Preps DNA Purification System (Promega). To determine its restriction map, the DNA was digested with one, two or three of the enzymes (XhoI, ApaI, EcoRI, HindIII, KpnI, SacI, SalI and XbaI) and separated by 0.8% agarose gel electrophoresis. After transferring onto a GenScreen Plus membrane (NEN Life Science Products), the DNA fragments were hybridized with the full-length lebocin cDNA, labeled by DIG-High Prime DNA Labeling Detection Kit (Roche Applied Science). Fragments of the lebocin gene were subcloned, sequenced, and assembled as described above.
M. sexta genomic DNA was extracted from muscles of a single fifth instar larva using a DNeasy Blood and Tissue Kit (Qiagen). The genomic DNA was also isolated from two larval carcasses according to Bradfield and Wyatt [29]. About 10 μg of DNA was digested with XhoI and ScaI at 37 °C overnight and separated by 1% agarose gel electrophoresis. After capillary transfer onto a nitrocellulose membrane, hybridization was carried out using 32P-labeled XhoI-ScaI fragment of the lebocin cDNA.
Fat body RNA samples were prepared from the individual insects using Micro-to-Midi Total RNA Purification System (Invitrogen) or the method described by Chinzei et al [30]. Denatured total RNA samples were separated on a 1% agarose gel containing 2.2 M formaldehyde. After electrophoresis, RNA was transferred to a membrane and hybridized with 32P-labeled XhoI-ScaI fragment of the cDNA. Similarly, the time course of lebocin expression in larvae and adults were examined. For RT-PCR analysis, hemocyte and fat body total RNA samples were prepared from naïve and injected larvae. In each reaction, RNA (2~4 μg), oligo(dT) (0.5 μg) and dNTPs (1 μl, 10 mM each) were mixed with DEPC-treated H2O in a final volume of 12 μl and denatured at 65 °C for 5 min. M-MLV reverse transcriptase (1 μl, 200 U, Invitrogen), 5×buffer (4 μl), 0.1 M dithiothreitol (2 μl), and RNase OUT (1 μl, 40 U) were added to the RNA for cDNA synthesis at 37 °C for 50 min. The M. sexta ribosomal protein S3 mRNA was used as an internal control to normalize the cDNA samples in a PCR using primers j501 (5′-GCCGTTCTTGCCCTGTT-3′) and j504 (5′-CGCGAGTTGACTTCGGT-3′). The lebocin cDNA fragment was amplified using forward (5′-CTGATTTTGGGCGTTGCGCTG-3′) and reverse (5′-GCGCGTATCTTCTATCTGGA-3′) primers under conditions empirically chosen to avoid saturation: 30 cycles of 94 °C, 30 s; 50 °C, 30 s; 72 °C, 30 s. The relative levels of lebocin mRNA in the normalized samples were determined by 1.5% agarose gel electrophoresis.
Eight lebocin-related peptides (~10 mg each) (LP1, QRFSQPTFKLPQGRLTLSRKFR; LP1A, QRFSQPTFKLPQGRLTLSRKF; LP2, ESGNEPLWLYQGDNIPKAPSTAEHPFLPSIIDDVKFNPDRRYAR; LP2A, ESGNEPLWLYQGDNIPKAPSTAEHPFLPSIIDDVKFNPDRRYA; LP3, SLGTPDHYHGGRHSISRGSQSTGPTHPGYNRRNAR; LP3A, SLGTPDHYHGGRHSISRGSQSTGPTHPGYNRRNA; LP4, SVETLASQEHLSSLPMDSQETLLRGTR; LP4A, SVETLASQEHLSSLPMDSQETLLRGT) were prepared by stepwise solid-phase synthesis using Fmoc-amino acid derivatives (Bio-Synthesis Inc). Following deprotection and cleavage, the peptides were purified by reversed-phase HPLC to >95% purity and analyzed by mass spectrometry.
The synthetic peptides were separately tested against pathogenic strains of Salmonella typhimurium, E. coli O157:H7, Klebsiella pneumoniae, S. typhimurium DT104, L. monocytogenes, Staphylococcus aureus, and two strains of methicillin-resistant S. aureus, kindly provided by Dr. Guolong Zhang in the Department of Animal Science at Oklahoma State University. The minimum inhibitory concentrations (MICs) were determined in a broth micro-dilution assay [31]. Briefly, overnight bacterial cultures were subcultured into 4 ml of Trypticase Soy Broth for 3-5 h until the bacteria reached mid-log phase. After centrifugation at 1000 × g at 4 °C and washing with 10 mM Tris-HCl (pH 7.4), the cells were suspended in 5% Trypticase Soy Broth (5×105 cfu/ml). Aliquots of the diluted cultures (90 μl) were mixed with 10 μl of the synthetic peptide at 1000, 500, 250, 125, 62.5 μg/ml. All bacteria were cultured at 37 °C for overnight in a 96-well cell culture plate, and the lowest concentration of peptide that caused no visible growth was recorded. The experiment was performed at least three times for each strain to obtain MICs of the M. sexta lebocin-related peptides against the bacterial strains.
Synthetic peptides and biological samples were characterized using a hybrid LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific) coupled to a New Objectives PV-550 nanoelectrospray ion source and an Eksigent NanoLC-2D chromatography system. MS and MS/MS spectra of LP1A through LP4A were first collected by infusion using 1-micron infusion tips (New Objective). These peptides were further analyzed by trapping on a 2.5 cm ProteoPrepII pre-column (New Objective), followed by analytical separation on a 75 μm ID fused silica column packed in house with10 cm of Magic C18 AQ, terminated with an integral fused silica emitter pulled in house. The peptides were eluted using a 3-70% acetonitrile (AcCN)/0.1% formic acid gradient performed over 33 min at a flow rate of 300 nL/min. During each one-second full-range FT-MS scan (nominal resolution of 60,000 FWHM, 300 to 2000 m/z), the three most intense ions were analyzed via MS/MS in the linear ion trap. MS/MS spectra were collected using a trigger threshold of 1000 counts and monoisotopic precursor selection. Parent ions were rejected for MS/MS if their charge states were unassignable, if they were previously identified as contaminants on blank gradient runs, or if they had already been twice selected for MS/MS (data dependent acquisition using a dynamic exclusion for 150% of the typical chromatographic peak width). Column performance was monitored using the peptide standards, and via blank injections between samples to assay for contamination. A 100 μl aliquot of pooled hemolymph from bacteria-injected M. sexta larvae was heated at 95 °C for 5 min and centrifuged at 4 °C for 10 min at 12,000g. Polypeptides in the supernatant (40 μl) was cleaned up on a PepClean™ C-18 Spin Column (Pierce) according to the manufacturer's instructions. Peptide samples eluted with 5-40% AcCN and 40-70% AcCN were lyophilized, reconstituted in 0.1% formic acid, and combined for LC-MS/MS analysis as described above. The resultant LC-MS/MS files were inspected manually, using the synthetic peptide's retention time, m/z, charge states, isotopic distributions, and characteristic MS/MS decay spectra as identifying criteria. For use as a standard to show that the natural LPA1 has a pyroGlu at the amino terminus, the first Gln in the synthetic peptide was partly converted to pyroGlu by incubating LPA1 (1 μl, 1 mg/ml) with 50 μl, 100 mM NH4HCO3 (pH 8.2) overnight at 37 °C [32]. The peptide mixture was enriched on a ZipTipC18 (Millipore) and analyzed by LC and tandem mass spectrometry as described above.
During the cDNA cloning of M. sexta hemolin [33], we isolated a false positive clone (pP4-7) which encodes a region 48% identical to B. mori lebocin in the amino-terminal pro-segment followed by twelve copies of a 27-residue repeated amino acid sequence. In a separate project to identify bacteria-induced genes by suppression subtractive hybridization and molecular cloning, Zhu et al. [11] identified 5 cDNA fragments (BI262584, BI262626, BI262686, BI262688, and BI262708) encoding sequences similar to pP4-7 and the proregion of silkworm lebocins. Intrigued by the unique structure of pP4-7 and similarity of the PCR-derived clones to lebocin, we screened an induced fat body cDNA library using the 5′ unrepeated region as a probe and isolated fifteen positive clones.
Sequencing from both ends of the positive clones indicated that all of the cDNAs contained a 5′ region identical to that found in pP4-7, which includes a 5′ untranslated region (5′-UTR) and a 363-nucleotide sequence encoding a 20-residue signal peptide and a 101-residue segment similar to the proregion of B. mori lebocin (Fig. 1A). Following this region, an 81-nucleotide sequence was repeated 3~16 times in different clones. Four clones (NC2, NC3, NC10 and NC11) contain thirteen repeats and the other twelve (NC7, G6, G1, NC6, NC9, NC13, NC1, NC5, G2, NC8, pP4-7 and C5) have 3, 3, 5, 7, 8, 8, 9, 10, 11, 12, 12 and 16 repeats, respectively. We have identified in DQ115324 an open reading frame of the pro-lebocin with the constant region and 6.3 repeats, out-of-frame fused with M. sexta HP23 cDNA as an artifact of library construction [34]. The repeats in these seventeen clones are nearly identical at the nucleotide level and they encode peptide sequences that are highly conserved. Following these complete repeats is a constant region consisting of a 33 bp partial repeat, a stop codon, and a 3′-UTR.
Amino acid sequence comparison shows that the 101-residue pro-segment in M. sexta lebocin is similar to its counterpart in T. ni (45%), P. includens (49%), Papilio dardanus (68%), Spodoptera frugiperda (66% for 1, 64% for 2, 70% for 3), B. mori (64%), Heliothis virescens (72%), S. cynthia (78%), Antheraea mylitta (67% for 1 and 75% for 2), Antheraea pernyi (70% for 1 and 73% for 2), Lonomia obliqua (81%) (Fig. 2). In the remaining carboxyl-terminal region, the T. ni and P. includens sequences are 70% identical to each other, the identity between A. mylitta-1 and A. pernyi-1 is 76%, and the S. cynthia, A. mylitta-2 and A. pernyi-2 sequences are ~80% identical. However, this high similarity in the carboxyl terminus is not conserved in lepidopterans from different families. It appears that rapid evolution has given rise to major variations in length and sequence of the M. sexta repeat, B. mori mature lebocin, and their counterparts in the above three groups. In fact, this region in S. cynthia, A. mylitta-2 and A. pernyi-2 are Ser/Thr, Gly, His-rich (instead of Pro-rich), whereas the 27-residue repeat in M. sexta lebocin contains eight Ser or Thr, five Leu, and only one Pro.
Three mechanisms that might account for variable numbers of the 81-nucleotide repeats in M. sexta sequences are: 1) multiple lebocin genes in the M. sexta genome, 2) allelic differences in the colony of insects used for cDNA library construction, or 3) alternative splicing of exons coding for the repeats. To explore these possibilities, we screened a M. sexta genomic library and obtained one positive clone, L45. Southern blot analysis of L45 using the full-length cDNA probe identified two hybridizing bands (Fig. 3A). The 2.9 kb XhoI-HindIII and 2.5 kb XhoI-XhoI fragments were subcloned and completely sequenced. Comparison of the cDNA and genomic sequences indicated that the M. sexta lebocin gene did not contain any intron or alternative splicing site flanked by GT and AG. The B. mori lebocin gene is also intron-free [16].
The M. sexta lebocin gene includes a 1,452 bp coding sequence ranging from nucleotides 2581-4032. The open reading frame encodes a 483-residue polypeptide, which includes the putative signal peptide, pro-segment, and 13.4 repeats (Fig. 1B). Repeats 1-4, 6-10, 12 and 13 contain 1-8 synonymous substitutions while repeats 7, 8 and 10 also contain 3 nonsynonymous changes (ProCCG to SerTCG; GlnCAA to HisCAC). In the partial repeat, four nucleotide substitutions do not alter the amino acid residues, while five other substitutions change HisCAT to ValGTA and SerAGC to a stop codon (TGA). A polyadenylation signal (AATAAA) is located at 89 bp after the TGA. Except for the repeat numbers, there is no major difference between the gene and cDNA sequences. For instance, clone NC6 contains 7.4 (instead of 13.4) repeats (Fig. 1A).
Computer analysis of the 5′ flanking sequence allows us to identify potential regulatory elements in the lebocin gene. Two imperfect NF-κB motifs (9/10 match) [35] are present at nucleotides 634 (minus strand) and 764 (plus strand). One GATA box is located on the plus strand at position 1308 and two are on the minus strand at positions 1523 and 2305. There is an interferon-stimulated response element at nucleotide 115, matching twelve of the thirteen positions in the consensus motif. The CCAGT sequence at position 1406 closely resembles the consensus (TCAGT) which typically resides within ten nucleotides before or after the transcription initiation site in arthropod genes [36]. At 38 bp upstream of CCAGT, there is a TATAAT sequence, reminiscent of the TATA or Goldberg-Hogness box.
In Southern blot analysis of the genomic DNA isolated from multiple insects (Fig. 3B, lane 0), digestion with XhoI-ScaI released a 1.1 kb fragment hybridizing with a cDNA probe that only recognized the repeats. Since no other hybridization signal was found, this result suggests a single lebocin gene in the genome, which contains an XhoI-ScaI fragment identical in size to the genomic clone (Fig. 3C) and to the cDNAs (NC2, NC3, NC10, and NC11) with 13.4 repeats. We repeated the experiment using DNA samples isolated from six different insects randomly chosen from the colony. Four of them (A2-A5) showed the 1.1 kb band that corresponded to 13.4 repeats, but the other two (A1 and A6) exhibited a single band at 2.5 kb (Fig. 3B, lanes 1-6). Further analyses indicated that the 2.5 kb band resulted from XhoI cleavage (Fig. 3C), rather than XhoI-ScaI double digestion. Sequencing of the PCR products of genomic DNA from A1 and A6 revealed 13.4 repeats and a point mutation that abolished the ScaI site (data not shown).
Northern blot analysis of fat body RNA from insects A1, A4, A5, and A6 showed three bands at 1.9, 2.5 and 4.2 kb positions (Fig. 4A). The 1.9 kb band represented the major transcript, consistent in size with the cDNAs containing 13.4 repeats. The other two bands, due to their large sizes, may result from transcription at an alternative initiation or termination site rather than containing more than twenty repeats. There was no significant variation in the transcript sizes among these four insects. Neither was there a ladder of mRNA with different numbers of repeats, although we cannot rule out that smaller transcripts were present at a low level. We carried out RT-PCR to test whether lebocin transcription occurred in fat body and hemocytes in response to the bacterial injection. The lebocin mRNA in hemocytes or fat body of naïve larvae was below the detection limit, and a PCR product at the expected size was amplified from fat body but not hemocytes of the insects after injection of E. coli (Fig. 4B). This is consistent with the results from Northern analysis of fat body RNA from M. luteus-injected larvae and adults, indicating that lebocin gene expression is highly induced upon immune challenges (Fig. 4C). The mRNA level and its increase in larvae seem to be higher than those in adults.
Although its transcription pattern suggests a role in defense, we do not know the exact function of M. sexta lebocin, especially those repeats which correspond to the silkworm mature lebocins in position but not sequence. Do these repeats function in a whole or separately as a processed 27-residue peptide? If processing does occur, where is the scissile bond located? Does the conserved pro-segment carry any function? To address these questions, we synthesized the peptide (RSVETLASQEHLSSLPMDSQETLLRGT). The synthetic peptide did not kill any bacteria (data not shown). Recombinant expression of the pro-lebocin was unsuccessful using E. coli or insect cells. These negative results stimulated us to closely examine the precursor sequences and propose that processing of the repeats yields 27-residue peptides ending with RGTR. This prediction is based on an RXXR motif recognized by intracellular processing enzymes [37,38], often followed by a hydrophilic residue (e.g. Ser). We have identified three RXXR motifs in the pro-segment of M. sexta lebocin: R19KFR22*E, R63YAR66*S and R98NAR101*S (Fig. 1). These Arg residues at the -4 and -1 positions are 100% conserved in the lebocin-related proteins from other insects (Fig. 2A). A convertase in the secretory pathway may cleave the lebocin precursors to form five peptides: LP1, Q1-R22 (22 mer); LP2, E23-R66 (44 mer); LP3, S67-R101 (35 mer); LP4, S102-R128 (27 mer); LP5, SVETLASQEVL (11-residue partial repeat). LP4 (SVETLASQEHLSSLPMDSQETLLRGTR) would be produced in multiple copies since each polypeptide contains 3~16 of the 27-residue repeats.
To test their biological functions, we chemically synthesized and purified four of the peptides. Their experimental masses were 2,691.95 (LP1), 5,056.39 (LP2), 3,814.12 (LP3) and 2,988.89 (LP4), nearly identical to the theoretical values (2,691.19, 5,054.64, 3,814.09 and 2,985.33 Da). LP1 is highly cationic with a calculated isoelectric point of 12.9. LP2 (pI = 4.8) is similar in sequence to the anionic AMP isolated from G. mellonella [22] (Fig. 2B). LP3 has a calculated pI of 11.8, whereas LP4, the repetitive sequence of 27 residues (SVET…RGTR), is anionic (pI = 4.6). We tested these peptides for antibacterial activity against Gram-negative and Gram-positive bacterial strains using a broth micro-dilution assay [31]. LP1 was active against both Gram-positive and Gram-negative bacteria. Its MIC was 25 μg/ml against all the bacteria except for S. aureus, whose growth was completely blocked by LP1 at 100 μg/ml (Table 1). LP2 did not exhibit antimicrobial activity against the bacterial strains tested, differing from its homolog in G. mellonella [22]. LP3 had a low activity against E. coli O157:H7 with a high MIC (200 μg/ml). LP4 showed no activity against the bacteria. After realizing that the carboxyl-terminal Arg in these four peptides is likely removed by a carboxyl peptidase in vivo, we synthesized another set of peptides LP1A, LP2A, LP3A, and LP4A, one residue shorter than the corresponding LPs, and tested antimicrobial activities against the same panel of bacteria strains. The only difference we observed was between LP3A and LP3: missing the terminal Arg rendered LP3A inactive against the E. coli strain (Table 1). The extra positively charged residue in LP3 may have facilitated the interaction between the peptide and negatively charged bacterial cell surface.
To identify lebocin-related peptides in larval plasma, we raised a polyclonal antiserum against a mixture of the four synthetic peptides, which only detected one immunoreactive band with mobility similar to that of synthetic LP2 (data not shown). Concrete evidence for the existence of LP2 and other peptides came from analysis liquid chromatography (LC) and tandem mass spectrometry. We first assayed synthetic LP1A through LP4A to obtain chromatographic retention times, peptide m/z's, and MS/MS fragmentation fingerprints (Table 2, left columns). For instance, LP2A eluted at 22 min and the peak was composed primarily of +5 and +6 ions with monoisotopic m/z of 980.489 and 817.242, respectively. These ions corresponded to a neutral molecular mass of 4897.45 Da, a value close to the calculated LP2A mass of 4897.42 Da. Upon MS/MS fragmentation, each ion of synthetic LP2A ion yielded a characteristic fingerprint identical to that of a peptide in the induced plasma (Table 2, right columns). This peptide, with the same retention time of 22 min, represented a major peptide constituent of the hemolymph (data not shown). While LP3A was found in the same way (Table 2), we did not detect LP4A in the induced plasma. The identification of LP1A in the induced plasma was complicated by a posttranslational modification. Synthetic LP1A eluted at 19 min and yielded +4 and +5 ions that correspond to a neutral mass of 2534.43 Da (Table 2). When the biological sample was analyzed, the 19-min major peak was dominated by ions corresponding to a neutral mass of 2517.42 Da. The mass decrease of 17.01 Da suggested that the amino-terminal Gln of LP1A was cyclized in vivo to form pyroGlu. Consistent with this interpretation, the predominant MS/MS fragments generated by the biological ions were nearly identical with those observed for synthetic LP1A (Table 2). We converted the first Gln of synthetic LP1A to pyroGln and analyzed the peptide mixture by NanoLC-MS/MS, which yielded ions corresponding to 2534.43 Da (original) and 2517.42 Da (converted) peptides (data not shown). Fragmented +4 and +5 ions (630.35 and 504.47 Da) from the modified LP1A were identical in mass fingerprints to those from the 19-min fraction of the biological sample.
Insects and other multicellular organisms rely on a network of host defense mechanisms to survive and prosper in microbe-rich environments [4]. One such mechanism involves the synthesis and secretion into hemolymph of AMPs that are effective against a broad spectrum of pathogens including bacteria, fungi, and protozoa [1,39]. Gene duplication and sequence divergence has given rise to a sizable repertoire of AMPs in various insect groups. For instance, analysis of the silkworm genome has revealed over thirty AMP genes in the cecropin, moricin, gloverin, attacin, lebocin, and enbocin families [40]. X-tox genes in lepidopteran species encode proteins with multiple defensin-like domains that are not processed or active as AMPs [41-44].
M. sexta lebocin was first reported as a differentially expressed gene in response to bacterial injection in a subtractive suppression hybridization experiment [11]. In this study, we confirmed its inducible transcription and discovered an interesting feature of the cDNAs: 3~16 copies of an 81 bp repeat between the 5′ and 3′ constant regions (Fig. 1). Gene sequencing and Southern blotting (Fig. 3 and Fig. 4) ruled out three potential mechanisms (i.e. multiple genes, allelic variations, and alternative splicing) for generating these variations. The negative results led us to suspect that the variation in copy numbers was an artifact caused by the tandem repeats when these cDNAs were first synthesized. No change was observed during DNA subcloning or propagation.
Regardless of the number of repeats in the transcript, five different peptides can be derived from the protein precursors via proteolytic processing after RXXR (Fig. 2). LP1 and LP3 were both active against bacteria, although the latter had a high MIC. These two peptides, as well as probably also G. mellonella anionic peptide 1, result via proteolytic processing from the amino-terminal region previously thought to be a pro-segment in lebocin-related proteins. In the initial biochemical study, B. mori lebocin-1/2 and -3 were names for 32-residue polypeptides active against Gram-negative bacteria [14], which are now known to be processed from the carboxyl-terminal end of a larger precursor. However, the carboxyl-terminal region in their counterparts in the M. sexta, T. ni, P. includens, S. cynthia, A. mylitta, A. pernyi, S. frugiperda, P. dardanus, H. virescens, and L. obliqua homologs differ significantly from the B. mori lebocins in amino acid sequence (Fig. 2). We propose to expand the original definition of lebocin to include the entire precursor proteins. Then, the processed products can be sequentially named from amino to carboxyl terminus as lebocin peptide 1 (LP1) through LPn. For instance, the M. sexta peptides are named LP1 through LP5: LP1, LP2 and LP3 come from the "pro-segment"; LP4 (27 mer) and LP5 (11 mer) correspond to the complete and partial repeats (Fig. 1A). B. mori LP4 is the same as lebocin by the original definition and LP5 represents the 25-residue fragment at the carboxyl terminus. While B. mori LP5 results from processing at R151YRR154*H, there is no LP5 counterpart in T. ni, P. includens, S. cynthia, A. mylitta, A. pernyi, S. frugiperda, H. virescens, or L. obliqua lebocins.
B. mori LP4 variants were thought to be encoded by members of a gene family in the genome [15,16]. Since the genome analysis only revealed a single gene [40], the minor differences in the cDNA and gene sequences may reflect allelic variations in the silkworms used for protein purification and library construction. No evidence was available for the existence of such multigene family in T. ni [18], H. virescens, L. obliqua, or P. dardanus. On the other hand, A. pernyi and A. mylitta have two genes: lebocin-2's are most similar to the S. cynthia lebocin [20] and lebocin-1's to the B. mori variants (Fig. 2C). Lebocin gene duplication must have occurred in the evolution of Saturniidae and, in another species from the same family (S. cynthia), a separate lebocin gene may exist to encode a precursor protein more similar to A. pernal-1, A. mylitta-1 and B. mori lebocins. In one lineage of the superfamily Noctuoidea, gene duplication has generated two to three lebocins in S. frugiperda, which form a clade with their homologs of P. includens and T. ni (Fig. 2C). In the pyrosequence analysis of immunity-related cDNAs from M. sexta [44], we identified four contigs (#5813, #6639, #6760 and #6851) coding for polypeptides similar to a part of the prosegment (LP1-LP2 region). Sequence comparison suggested two additional lebocin-like genes in the M. sexta genome (data not shown). One or both of them may be more similar to silkworm lebocins than the one reported herein. Taken together, the sequence-based phylogenetic relationships are consistent with those derived from morphological characteristics.
B. mori, T. ni, P. includens and S. cynthia lebocin genes were mainly expressed in fat body of immune challenged insects [15,18-20]. Their mRNA levels were much lower in hemocytes even after bacterial injection. Consistent with the presence of immune regulatory elements in its gene (Fig. 1B), M. sexta lebocin expression was up-regulated during antimicrobial responses in larvae and adults (Fig. 4C). Similarly, cecropin B of M. sexta was mainly produced in fat body [10]. These results are consistent with the observation that lebocin and cecropin expression was up-regulated by injecting B. mori spätzle into M. sexta larvae [45].
Although direct evidence is limited, it seems to be a common mechanism in lepidopteran insects to generate several active peptides from a lebocin polypeptide precursor. This is supported by the purification of LP2 from G. mellonella and H. armigera hemolymph [21,22] and by the detection of antibacterial activities of M. sexta LP1, LP3, and G. mellonella LP2 (i.e. anionic peptide 1) (Fig. 2B). The purification and functional characterization of B. mori LP4 variants (i.e. lebocin-1/2 and 3) further supported this hypothesis [14]. In this study, our antiserum prepared toward a mixture of the four synthetic lebocin peptides recognized synthetic LP2 efficiently and labeled a polypeptide in the induced plasma, which is similar to synthetic LP2 in apparent Mr (data not shown). Direct evidence for the existence of LP2A (as well as LP1A and LP3A) at a high level was obtained by LC-MS/MS analysis of the larval hemolymph (Table 2): 1) two plasma polypeptides were identical to synthetic LP2A and LP3A in all molecular properties; 2) a peptide with MS/MS fragments identical to those of synthetic LP1A had its amino-terminal Gln converted to a pyroGlu in vivo. Surprisingly, we did not detect any LP4A, which should be approximately 13 times as abundant as the other peptides. There are three possible reasons for this negative result: 1) the repeated region is resistant to processing, 2) LP4A attaches to hemocytes or proteins that precipitate during sample preparation; 3) LP4A is rapidly degraded in the plasma. The existence of the perfect repeats in multiple copies, which is not found in the other lepidopteran species, suggests that LP4A plays a unique role in the M. sexta defense system. On the other hand, the high sequence similarity of the LP1, LP2 and LP3 regions in other species suggest that these predicted processing products are functionally conserved. A very recent peptidomics study of AMPs in hemolymph of G. mellonella [46] has revealed the same mechanism discovered in this investigation.
Processing of large precursors is a strategy commonly used by neuroendocrine systems of vertebrates and invertebrates to generate biologically active peptides. Rules have been proposed to predict the processing sites in insect neuropeptide precursors [37,38]. This study provides initial evidence that the insect immune system may use the same tactic to produce functional products by processing protein precursors. This hypothesis would support the notion that parts of the nervous, hormonal, and immune systems have a common evolutionary origin.
We thank Dr. Kent Shelby at USDA ARS for sharing the unpublished sequence of H. virescens lebocin and Drs. Steffi Gebauer-Jung, Hendrik Tilger, and Alexie Papanicolaou at Max Planck Institute for Chemical Ecology in Germany for developing and maintaining ButterflyBase. This work was supported by National Institutes of Health Grants GM58634 (to H.J.) and AI31084 (to M.K), as well as National Science Foundation Award 0722494 (to S.H.) for the LTQ mass spectrometer. This article was approved for publication by the Director of Oklahoma Agricultural Experimental Station and supported in part under project OKLO2450.
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