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Valley Insight Meditation Society (VIMS) is a community of individuals who practice primarily in the Insight Meditation lineage within the Theravadan Buddhist tradition. Our teachers and formal practice are closely aligned with Joseph Goldstein, Jack Kornfield, Sharon Salzberg, and other lay teachers connected with IMS and BCBS in Barre, MA. We are also informed by the Western Monastic Order of Thai Forest Monks, centered at Amaravati Monastery in England, whose monks and nuns teach world-wide.
Our intention is to provide opportunities and support for exploring the teachings of Buddhism and practice of vipassana, also known as insight meditation. Wise and careful training of attention can lead to a more peaceful, steady mind and kind heart. This path helps us find the way to wisdom and compassion in the midst of our complex, stressful lives. We welcome all who are interested.
New: Daylong Retreats upcoming. Details will be posted when available.
Ajahn Jayanto, co-abbot of Jetavana Temple Forest Monastery – Saturday, September 10
Andrew Olendzki – Saturday, November 19
New: Predatory Loan Laws under review by the Consumer Financial Protection Bureau (CFPB). Loans of 300 to 400% exploit the poorest in our community. The public comment period for the proposed new regulations is now open and UVIP is collecting comments. Please help! Info and Comment Cards are at all sits and here. There's also a url to send your comment electronically. See also calendar for upcoming demonstrations in support of the Moral Economy.
Mark your calendars! Author Michael Pollen will speak at Dartmouth
Monday, October 24, 7 pm
This will be the annual George Link Jr. Environmental Awareness lecture.
Monday Sits: The usual pattern is for Doreen Schweizer to lead the second and fourth Mondays of every month, with Peg Meyer leading the first and third Mondays. Doreen will lead on July 25, August 8 and 22. Peg will lead on August 1 and 15. details
Advisory Board: Gina Sonne is the VIMS president; Geri DeLuca is our treasurer. Doris Hampton, Penelope Prendergast, Suzanne Serat, Mindy Schorr, and Deb Steele complete the board. Brief bios are here.
Woodstock Meditation Group
Doreen Schweizer, our Guiding Teacher, and Lay Buddhist Chaplain Dorie Porcelli, a Woodstock resident, is offering an Insight Meditation instruction and discussion group from 5:00 to 6:30 p.m. on the first Monday of each month at Simmons House at the Ottauquechee Health Center in Woodstock. The group will meet monthly, but regular attendance is not required. The group will meet August 1 . Contact email@example.com for more information.
A VIMS Lending Library is open in the Friends Meetinghouse in Hanover. The library has over 150 books, CDs, and pamphlets. Details
Diversity Policy Valley Insight Meditation Society is committed to offering a safe, welcoming environment for people of all races, cultures, ethnicities, sexual orientations, classes, religions, abilities and ages in which to practice meditation and to explore the ideas put forth in the Buddha's teachings. We recognize that all people hold unexamined beliefs, assumptions, fears and prejudices about others, and especially about those who seem different from themselves, and we acknowledge that these beliefs are a great source of suffering for individuals and societies. We strive for inclusivity, kindness and generosity toward all, in our programs and in our hearts.
VIMS is now a member of the United Valley Interfaith Project and other Advisory Board news.
Prison Sangha – Consider joining us for our regular visits with the prison sangha at the New Hampshire Department of Corrections in Berlin or volunteer to be a letter-writing correspondent with an inmate. View photos of the prison sangha. Consider becoming a member of Valley Insight. Learn about other ways to be involved in Valley Insight. Read reflections on Dhamma practice and Buddhism by Doreen and a Valley News interview of Landon about volunteering at the prison.
In May, 2015, 125 Buddhist leaders met with White House and State Department officials at the first-ever White House-US Buddhist Leadership Conference. Their statements on racial justice and climate change are here.


text: AdChoices Terms of Use Privacy Policy Hide TOP 10 Top 10 Dating Signs It's Time To Break Up Share Tweet 0 Shares 7. Fighting Dirty The two of you used to have silly little fights that were only slightly more mature than a schoolyard spat. Now you're engaged in full-on war games in which anything goes. You know what her weak spots are -- her weight, her body image and her mother -- and you use them just to rile her up. You're not alone, as the rules of engagement clearly state that she can do exactly the same thing back -- she laughs at your sexual prowess and tells her girlfriends how small your manhood is. Next Page TOP 10 9 8 7 6 5 4 3 2 1 Comments Share your opinion Your name More Like This Top 10 Dating Are You About To Be Single Again? Yes, If These 10 Signs Are Present Read More Dating Tips 22 Reasons She's Probably Happy You Broke Up Read More Sex and Dating How To Get Dumped: Hang Out With Her Friends Read More Dating Tips Thinking Of Breaking Up Because Of The Sex? Try This First Read More Dating News Trending News: Why It's A Good Sign If You're Not Friends With Your Ex Read More Dating Tips The Important Lessons Your First Real Relationship Should Teach You Read More Around the Web Special Features Guy Gear The Best Watches Under $500 Read More Special Feature The Ultimate Guide To Hooking Up Online Read More

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Real news. Real funny.
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|Business Type:
|Manufacturer, Trading Company
|Guangdong, China (Mainland)
|Main Products:
|LED Bicycle Light,LED Diving Flashlight,LED Headlamp,sport camera,Waterproof Camera
|Total Employees:
|51 - 100 People
|Total Annual Revenue:
|US$2.5 Million - US$5 Million
|Year Established:
|Top 3 Markets:
|Northern Europe 15.00% Eastern Europe 15.00% Western Europe 15.00%
|Product Certifications:
|CE,RoHS,Germany SGS Certificate
INTON Technology Co., Ltd. is a market-leading innovator and professional LED & HID manufacturer...
INTON Technology Co., Ltd. is a market-leading innovator and professional LED & HID manufacturer that engaged in R&D, manufacturing and sales of LED light series and HID ballasts products, Inton has a well-trained team ,including experienced technicians and salesman, .Continuous product innovation and reliable cooperation with our customers are the foundation of our development. We have the principle as catering for the needs of the customers .More and more customers are continually choosing INTON for our excellent quality and preeminent service, moreover, we could supply the one to one designed &researched unique products service .
LED series
We specialize in Led tube,Led bulb,Led spotlight, Led flashlight, Led miner light, Protable explosion-proof working light , all of which are widely used in commercial lighting, decoration, indoor and outdoor lighting and project infrastructure,etc. Under the ISO9001:2000 Quality Management System, most of our products have CE, ROHS,FCC , and UL certificates, all of them can prove that Inton can supply excellent quality and good service to you. Your OEM or ODM projects are welcomed. As a globally operating Manufacturer, we would very much like to strive for the energy-saving & environmental protection with you around the world - as well as constructing the green great wall at home and abroad!
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text: Home > About the CIA > News and MediaPast News StoriesFor the MediaYouTube Channel Share This "Supercool" Restaurant Companies Have CIA Grads at the Helm Posted: August 15, 2013 Magazine Recognizes Most Innovative Multiconcept GroupsMedia Contact:Jeff Levine Communications Manager firstname.lastname@example.com Park, NY – Restaurant Hospitality magazine recently released its list of the Top 25 Multiconcept Companies in America. Five of the companies honored by the magazine were founded by a CIA graduate and two more have alumni in the top culinary job.The editors of Restaurant Hospitality referred to these 25 organizations as "supercool multiconcept restaurant companies that are changing the face of dining in America."Those with CIA alumni in top positions are:Charlie Palmer Group—Charlie Palmer '79, founderRichard Sandoval Restaurants—Richard Sandoval '91, chef, founder, and CEOBesh Restaurant Group—John Besh '92, founder and executive chefDavid Burke Restaurants—David Burke '82, founder and executive chefBonanno Concepts—Frank Bonanno '96, chef and proprietorCapital Restaurant Concepts—Bryan Yealy '88, corporate chefSage Restaurant Group—Michael Carr-Turnbough '82, senior vice president of culinary"The many CIA graduates on this list are even more proof that, in addition to becoming successful and celebrated chefs, the college's alumni are well-prepared to assume roles as the top leaders, businesspeople, and entrepreneurs in the food world," said CIA President Dr. Tim Ryan. "The entire CIA community joins in congratulating these culinary innovators on this honor.""Everyone knows how difficult it is to run a restaurant. Imagine how taxing it must be to run multiple restaurants, each with its own personality," wrote the editors of Restaurant Hospitality, explaining why they chose to recognize those who create not just one successful concept, but several. "This breed of entrepreneur has piqued our interest...so we set out to spotlight the coolest, most influential multiconcept companies in the country."While running their respective restaurant groups, Charlie Palmer and Richard Sandoval also serve on the Board of Trustees of The Culinary Institute of America. Palmer is the chairman of the board. Photo Captions and Hi-Res ImagesPhoto 1: Charlie Palmer '79 with students on a visit to The Culinary Institute of America's Hyde Park, NY campus in 2013. Palmer is founder of the Charlie Palmer Group of restaurants and the chairman of the board of the CIA. (Photo credits: CIA/Phil Mansfield)View hi-res image >Photo 2: Richard Sandoval '91, founder and CEO of Richard Sandoval Restaurants and a member of The Culinary Institute of America's Board of Trustees. (Photo credits: CIA/Phil Mansfield)View hi-res image > Founded in 1946, The Culinary Institute of America is an independent, not-for-profit college offering associate and bachelor's degrees with majors in culinary arts, baking and pastry arts, and culinary science, as well as certificate programs in culinary arts and wine and beverage studies. As the world's premier culinary college, the CIA provides thought leadership in the areas of health & wellness, sustainability, and world cuisines & cultures through research and conferences. The CIA has a network of 45,000 alumni that includes industry leaders such as Grant Achatz, Anthony Bourdain, Roy Choi, Cat Cora, Dan Coudreaut, Steve Ells, Johnny Iuzzini, Charlie Palmer, and Roy Yamaguchi. The CIA also offers courses for professionals and enthusiasts, as well as consulting services in support of innovation for the foodservice and hospitality industry. The college has campuses in Hyde Park, NY; St. Helena, CA; San Antonio, TX; and Singapore.# # # Media Relations1946 Campus DriveHyde Park, NY 12538-1499845-905-4288845-452-9430 Related StoriesJul 28, 2016 - Future CIA Students Win SkillsUSA Culinary and Baking CompetitionsJul 19, 2016 - The Food Business School Announces Fall 2016 CoursesJul 12, 2016 - F&B Exec for Some of America's Best Known Restaurants and Resorts Addresses CIA GraduatesJul 05, 2016 - The Food Business School FOODBIZ+ Series is Hosting an Interactive Online ConversationJun 30, 2016 - Vive la France! Bastille Day in The Bocuse Restaurant at the CIA on July 14More News


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Herbert convenes summit to map 50-year water strategy for Utah
Jeffrey D. Allred, Deseret News
PROVO — With Utah's urban population expected to double in the next 45 years, water managers across the state are staring at a future that demands smart management of a resource that is likely to become more scarce with each passing decade.
To that end, Gov. Gary Herbert convened his first water summit Wednesday at the Utah Valley Convention Center, the next step in an ambitious statewide effort to map out a 50-year water strategy for Utah.
"We are at a crossroads for our future here," Herbert said, citing the challenge of ensuring adequate water supplies in the face of demand brought by population growth, the outdoor economy and environmental concerns.
The benefits of action, he stressed, "will be for our children and our grandchildren."
The summit included Herbert's announcement of a 38-member team made up of a diverse group of representatives such as elected officials, water district managers, community activists, special interest groups and federal and state policymakers. The task, Herbert said, is to evaluate water management strategies, their potential ramifications and what may fit with a 50-year plan going into the future.
Herbert's summit Wednesday drew on work forged over the summer through eight listening sessions held across the state. During those sessions, more than 800 comments were submitted to six experts Herbert picked to represent conservancy districts, agricultural interests, water rights law, and recreational and environmental issues.
That team gathered input and submitted white papers to Herbert, detailing the challenges in managing Utah's water supplies into the future, as well as summaries of the some of the more dominant themes that emerged in feedback from the public.
Alan Matheson, Herbert's environmental adviser, said comments compiled do not necessarily represent the views of the state or the team participants, and papers submitted by Herbert's team are not meant to be comprehensive but rather an initial blueprint.
Some of the highlights from the papers include:
Most of the Wasatch Front urban infrastructure was built starting in the 1940s and is now experiencing the first period of large-scale replacement needs.
"In fact, water purveyors are now dealing with the reality that over the next 50 years, replacement costs will rival new capacity construction costs," wrote Tage Flint, general manager of the Weber Basin Water Conservancy District. "Statewide costs for repair and replacement of existing infrastructure will exceed $16 billion between now and 2060."
The governor's goal to conserve water by 25 percent by 2025 should be mandatory.
"From an angler's perspective, a reduced stream flow and fewer fish is one thing. A dry stream bed with no fish is something else entirely," wrote Timothy Hawkes, executive director of the Utah Water Project of Trout Unlimited. "Water clearly touches almost a spiritual chord in people and helps define a sense of place and one's attachment to a place," evidenced by comments made by people detailing memories of certain water bodies.
Multiple water development projects, such as the Lake Powell pipeline, the proposed Gooseberry Narrows Dam and the Southern Nevada Water Authority's groundwater pumping plan, elicited strong public reaction, as did the proposed diversion of 53,600 acre-feet of water for a twin reactor nuclear power plant.
Develop a joint operating agreement for the reservoirs in the Weber River watershed.
Explore additional freshwater storage options in or around the Great Salt Lake.
- Man accused of killing UTA worker dies in prison
- Women underrepresented across Utah's...
- Mike Lee, US Senate to hold monument meeting...
- 7 tips for summer travel while pregnant
- Utah Democrats headed to 'historic' national...
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Where Are They Now? The Joel Huff Edition
Ex-Azul chef heading back to California. Joel, we hardly knew ye.
A few shockwaves went through the Miami dining community when they heard Azul chef Joel Huff had high tailed it outta there. Rumors had it that a big name chef was headed to the Mandarin in his place. Not so. But where is Huff anyway? Reports by some very misinformed folks saying Huff is headed to Geoffrey Zakarian's Tudor House weren't just grossly exaggerated, but completely wrong according to Zakarian's publicist Jaret Keller, who, along with his client, was baffled to say the least. "It is absolutely not true," Keller told us. We also know that the team behind Route 9, Jeremy and Paola Goldberg, were eyeing Huff as a candidate to head up their new Key Biscayne restaurant, but that didn't work out.
So where is he now? Going back to Cali, he tells us, to his hometown of Santa Barbara, where he is helping out a good friend who happens to own a restaurant. "I'll help her for a litle and see what I can do after that," Huff told us. "I was planning on going to The Viceroy and then realized I needed a little break. The Mandarin burned the heck out of me. I need to recharge my batteries." And recharge he will, on the other coast. We wish him luck.
Locally Sourced Miami
- Where to find artisanal chocolate in South Florida
- These are the most popular Indian restaurants in Miami
- How to get gourmet clean eating in South Beach
- RIP Randazzo's Little Italy in Coral Gables
- Who has Miami's favorite acai bowls? Yelp! weighs in
- Tap 42 opening second Miami location in ex-Aperio space in Midtown
- Nibble + Squeak dining club for "parents with pipsqueaks" launches in Miami August 24 at Mignonette
- The Rumor Mill: Murmurs of more Miami restaurant, market closings and chef firings . . .
- 305 Brews beer and coffee bar opening in August in Midtown Miami
- Alex Chang leaves The Vagabond, which will re-debut with new chef, new name


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At Accenture, we are passionate about improving the way the world works and lives. Our approximately 48,000 people across the United States bring the same commitment and rigor to our corporate citizenship initiatives as they do to client service by leveraging digital technologies and delivering measurable outcomes.
Click the image to watch a video from the U.S. Chamber of Commerce Foundation Corporate Citizenship Center featuring Accenture as a finalist for the Citizens Awards for our commitment to education.
Together with KIPP, a national network of open-enrollment, college-preparatory public charter schools, we created Future Focus, a college and career readiness program for KIPP students, and supported the rollout of this program in nine US cities. To date, nearly 7,000 students have received career skilling through Future Focus. Since 2011, Accenture has awarded KIPP nearly $6.8 million in funding and pro bono resources.
Click the image above to watch a video about our support of Girls Who Code's efforts to close the gender gap in technology and inspire, educate and equip young women with computing and professional skills that enable them to pursue technology careers.
We are helping Girls Who Code expand across the United States by hosting Summer Immersion Programs and Clubs in new locations, offering skills training and exposure to real-world business and mentorship opportunities. We're supporting Girls Who Code alumnae transition to higher education and their first jobs through an online community group as well as hiring them as Accenture interns.
Corporate Citizenship
Accenture Community
Inclusion & Diversity
Learn how we foster a culture of inclusion and diversity.
Search our open positions or join our Talent Connection.


text: Visited Washington area for two nights. Hyatt Place was under $65 per night on AARP. Wondered what might be wrong with it at that rate but took the chance. NOTHING was wrong and SO MUCH was right. Great room with separate sitting and sleeping area, free wireless WiFi and 42 in TV. Great staff. Good complimentatary continental breakfast with hot... More


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Pokémon GO has been released for Android and iOS in Australia, New Zealand, the United States, most of Europe, Canada, and Japan with more countries on the way soon! Need help learning Pokémon GO? Take a look at our Pokémon GO Player's Guides
Check BNN and Bulbanews for up-to-date Pokémon news and discuss it on the forums or in our IRC channel #bulbagarden on irc.systemnet.info.
K-Zone is a children's magazine published in Australia, New Zealand, the Philippines and Malaysia. It is best known in the Pokémon fandom for an article released in April 2008 that listed plots for episodes beyond the point the Japanese airings were currently at. While the article scans have been criticized for their poor spelling and grammar, each point turned out to be true.
The article revealed that:
- Dawn's Piplup would defeat Kyle's Crawdaunt in the Wallace Cup. (DP079)
- Dawn would then go on to win the Wallace Cup. (DP079)
- Pastoria City would feature a Croagunk Festival that Brock competed in. (DP082)
- Ash would then win the Badge at the Gym. (DP083)
- Dawn would capture a Swinub at Mr. Backlot's mansion. (DP084)
- The group would meet a Lopunny that is a super model in Hearthome City. (DP086)
- Aaron would appear around Celestic Town. (While the article said it would be before Celestic, the episode actually aired shortly after the events there.) (DP099)
"Lava" Badge
Notably, K-Zone mentioned that Ash would receive the "Lava" Badge as his fourth Badge. Because the fourth Badge in Sinnoh in Diamond and Pearl is the Fen Badge, many discarded all of the assertions made by the magazine. As it began to become apparent that K-Zone was in fact, correct, some fans took a look at the evidence again. The Japanese words "fen" (フェン) and "fuen" (フエン, lava) are very similar, and therefore it is widely believed now that the translators made a small mistake.
A similar issue occurred with assertions that Dawn would compete in the "Mikli Cup" and win against another coordinator, "Kite", improperly romanized from ミクリ (Mikuri) and カイト (Kaito). These errors likely stem from a lack of knowledge on the translators' part of many Pokémon characters and items.
Another unrelated mistake was made, as the article incorrectly sorted some events, such as it stating that Ash and Dawn would not trade their Pokémon until after Ash's battle with Maylene.
External links
- K-Zone on Wikipedia
- Pokémon GO Plus delayed to September: No longer set to be released in July
- Unova Classic Wi-Fi competition announced: Participants will receive Zen Mode Darmanitan
- Pokémon Ranger: Guardian Signs to be released on Wii U Virtual Console in Japan: To be ...
- Pokémon GO panel at SDCC reveals team leaders, more: Hanke teases undiscovered Easter eggs
- Pokémon GO now available in Hong Kong: Available for iOS, Android


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Top StoriesNewswire
Rams Well Represented on Winter-Spring Academic All-UMAC List Winter and Spring sports contributed to all-time high number of NCU athletes on the UMAC Academic All-Conference Award... Full Story
Martin Selected to NCCAA All-American Award List Cody Martin was selected to the NCCAA Division II All-America award list during last week's national tournament... Full Story
Rams Finish Fourth at NCCAA National Tournament Men's Basketball lost 106-96 to Manhattan Christian College in the NCCAA national tournament third place game on... Full Story
Saints Knock off Rams in NCCAA National Semi-final Men's Basketball was defeated by Hillsdale Freewill Baptist College in the NCCAA National Semi-final on Friday night. Full Story
Rams Complete Epic Comeback to advance to NCCAA Final Four Men's basketball got a historic individual effort and finished off a rally for the ages to advance to the NCCAA Final... Full Story
March 9, 2016 Men's Hoops Primed for NCCAA National Tournament Run The 2016 NCCAA Division II National Tournament is set to tip tomorrow with North Central taking on Arlington Baptist...
March 3, 2016 Rams Trio Selected to UMAC All-Conference List Men's Basketball had three players selected to the UMAC Honorable Mention team announced this week.
February 29, 2016 High, Martin and Cline Named to NCCAA All-Region Team Men's Basketball had three players selected to the NCCAA All-Region team as announced this weekend during the region...
February 27, 2016 Rams Claim NCCAA Region Title with Strong Win over Crossroads Men's Basketball responded to a strong challenge from Crossroads College by outpacing the Knights in the second half on...
February 26, 2016 Rams Race Past Eagles in NCCAA Region Semi-final Men's Basketball posted a wire to wire, 105-75, victory over Faith Baptist on Friday night.
February 24, 2016 Rams Fall in UMAC Semi-Final Men's Basketball was defeated by the University of Northwestern in the conference playoff semi-finals in St. Paul on...
Sat 12 Mar
Fri 11 Mar
Thu 10 Mar
Sat 27 Feb
Fri 26 Feb
Tue 09 Feb
Final Men's Basketball
73 North Central
77 at Crossroads
Tue 24 Jan
6:30 PM Men's Basketball
Faith Bapt. Bible
vs. North Central
Tue 31 Jan
7:00 PM Men's Basketball
Crossroads College
vs. North Central


text:
Few purchases are as emotional as fine jewelry. But shop with your head and you'll be less likely to overpay.
Amanda Walker at Consumer Reports Money Adviser can tell you how to get a great deal on jewelry and avoid costly rip-offs.
Amanda Walker with Consumer Reports found, "Fine jewelry is generally one of a kind, so you can't shop for the best deal like you can with a vacuum or a TV. So you need to know what to look for, especially if you shop online."
How pure is that gold bracelet? Maybe it isn't even real!
Emeralds can often be enhanced by filling them with oil. But over time, the oil can leak out.
And a diamond can appear more brilliant through what's called
fracture-filling. But this also makes diamonds more likely to shatter, so you have to be careful when they're repaired and cleaned.
Amanda says, "You should ask if a gemstone or a diamond is imitation, treated, or synthetic. And if the jeweler can't or won't tell you, that's your cue to shop somewhere else."
Buying gold? Look for a karat mark, which indicates quality, and a manufacturer's trademark, so you know whose work it is.
But Money Adviser's top tip for protecting yourself is to deal with a reputable company. Members of the American Gem Society must abide by a strict code of ethics.
Amanda points out, "You go to the American Gem Society dot Org to find a reputable jeweler in your area. Not just here where I am, but anywhere in the country."
And whatever you buy, make sure you get the details in writing.
Consumer Reports also says to consider an appraisal from a certified jewelry appraiser. And be sure the return policy gives you at least enough time to return something if it isn't what you expected.


text:
Sexual-abuse Crisis
Letters | Teaching prisoners, seminarians & ourselves
Shortsighted Kudos to Robert Cowan for saying that the objections that scuttled Governor Andrew Cuomo's plan to pay for college classes for prison inmates...
May 4, 2015The Editors0 comments
Held to Account
Kansas City Catholics have been wondering whether Robert Finn would be replaced with a bishop who would put the safety of children first. They now have their answer.
April 28, 2015The EditorsReligion6 comments
Letter from Rome
Controversy over the canonization of California's founding father continues; Bishop Finn is finally gone; and Pope Francis will make visit to U.S. Seminary in Rome.
April 22, 2015Robert MickensReligion5 commentsPremium
Letter from Rome
In his general audience, Francis listed ways that children undergo their own "passion" (suffering), which he said was almost always caused by the "errors of adults."
April 8, 2015Robert MickensReligion1 commentPremium
Letter from Rome
If you believe media reports, you'd be convinced that Pope Francis strengthened the hand of Australian Cardinal George Pell. But you'd be wrong.
March 4, 2015Robert MickensReligion0 commentsPremium
Clerical Errors
Amid what the media has presented as a general feeling of optimism about the papacy of Francis, there are some matters that remain causes of concern among American...
February 19, 2015The EditorsReligion, Reading Lists3 comments
Tall Orders
Seminaries have four to five years of post-college priestly formation to train men to be leaders of the small "corporations" that parishes have become.
February 17, 2015Mary GautierReligion14 comments
'Spiritual Assault'
In the trenches of parish life, I am aware of how an ecclesiastical environment that produces wounded people can lead to a community's unraveling.
February 17, 2015Barbara ParsonsReligion22 comments
Inside the Seminary
Any college-aged man entering a Catholic seminary during the ongoing crisis of the priestly sexual-abuse scandals does so with a certain amount of self-...
February 17, 2015Paul BlaschkoReligion56 comments
Letter from Rome
The Holy See has publicly dealt with four bishops for committing abuse or trying to cover it up. But there has been no transparency on their status or whereabouts.
February 11, 2015Robert MickensReligion8 commentsPremium
An Interview With Archbishop Blase Cupich
Archbishop Cupich talks about immigration, abuse and accountability, what happened at the synod on the family, and meeting the needs of Chicago Catholics.
January 22, 2015Grant GallichoReligion7 comments
The Pope's American Messenger
The appointment of Blase Cupich will have an impact beyond the Catholic Church because it tells us about the role Francis wants the church to play in American life.
September 25, 2014E. J. Dionne Jr.Religion2 comments
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text:
Bone marrow-derived mesenchymal stem cells are capable of mediating hepatitis B virus infection in injured tissues
Version of Record online: 27 MAY 2008
DOI: 10.1111/j.1365-2893.2008.00978.x
© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd
Journal of Viral Hepatitis
Volume 15, Issue 8, pages 607–614, August 2008
Additional Information
How to Cite
Rong, Q., Zhang, L., Su, E., Li, J., Li, J., Liu, Z., Huang, Z., Ma, W., Cao, K. and Huang, J. (2008), Bone marrow-derived mesenchymal stem cells are capable of mediating hepatitis B virus infection in injured tissues. Journal of Viral Hepatitis, 15: 607–614. doi: 10.1111/j.1365-2893.2008.00978.x
Publication History
- Issue online: 8 JUL 2008
- Version of Record online: 27 MAY 2008
- Received October 2007; accepted for publication January 2008
- Abstract
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- Cited By
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text:
Book your room at Smile Hotel Tokyo Asagaya
Get an overview of this hotelTokyo hotel in Suginami with restaurant and bar/lounge
Located in Suginami, this hotel is within 1 mi (2 km) of Mabashi Park, Hikawa Shrine, and Za-Koenji Public Theatre. Suginami Science Museum and Renkoji Temple are also within 2 mi (3 km).
Hotel Features
A restaurant, a bar/lounge, and a porter/bellhop are available at this hotel. WiFi in public areas is free.
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All 112 rooms feature thoughtful touches like bathrobes and slippers, plus free WiFi, free wired Internet, and TVs with satellite channels. Refrigerators, coffee makers, and in-room massages are among the other amenities available to guests.Would you like to give us feedback? Share it! Opens in a new window
Smile Hotel Tokyo Asagaya3-37-11 Asagaya Minami Suginami-ku Tokyo Tokyo-to 166 0004
Hotel Amenities
Hotel Amenities
The hotel offers a restaurant. A bar/lounge is on site where guests can unwind with a drink. High-speed wireless Internet access is complimentary.
- Bar/lounge
- Breakfast available (surcharge)
- Free WiFi
- Number of buildings/towers - 1
- Number of floors - 5
- Porter/bellhop
- Restaurant
- Total number of rooms - 112
Available in all rooms: Free WiFi , Free wired high-speed Internet
Available in some public areas: Free WiFi
Room Amenities
- Air conditioning
- Bathrobes
- In-room climate control (air conditioning)
- Coffee/tea maker
- Free toiletries
- Desk
- Free WiFi
- Free wired high-speed Internet
- Hair dryer
- In-room massage available
- Iron/ironing board (on request)
- Pay movies
- Phone
- Refrigerator
- Satellite TV service
- Slippers
- Television
Where to Eat
Cooked-to-order breakfasts are available for a surcharge and are served each morning between 7 AM and 9:30 AM.
BISTRO LA BAULE - This restaurant serves breakfast only.
Start your morning with a cooked-to-order breakfast, available each day from 7 AM to 9:30 AM for a surcharge.
Hotel Policies
Check-in time ends at 1 AM
Check-in time starts at 3 PM
Check-out time is 10 AM
Payment types
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Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
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- Fee for cooked-to-order breakfast: JPY 850 per person (approximately)
Hotel Name
- Asagaya Hotel
- Asagaya Smile Hotel
- Smile Asagaya
- Smile Asagaya Hotel Tokyo
- Smile Asagaya Tokyo
- Smile Hotel Asagaya
- Smile Hotel Asagaya Tokyo
- Smile Hotel Tokyo Asagaya
- Smile Tokyo Asagaya
- Tokyo Asagaya
- Sky Court Asagaya Suginami
- Smile Hotel Tokyo Asagaya Japan - Suginami
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text:
Dermacentor reticulatus (Fabricius, 1794) is distributed in Europe and Asia where it infests and transmits disease-causing pathogens to humans, pets and other domestic and wild animals. However, despite its role as a vector of emerging or re-emerging diseases, very little information is available on the genome, transcriptome and proteome of D. reticulatus. Tick larvae are the first developmental stage to infest hosts, acquire infection and transmit pathogens that are transovarially transmitted and are exposed to extremely stressing conditions. In this study, we used a systems biology approach to get an insight into the mechanisms active in D. reticulatus unfed larvae, with special emphasis on stress response.
Principal Findings
The results support the use of paired end RNA sequencing and proteomics informed by transcriptomics (PIT) for the analysis of transcriptomics and proteomics data, particularly for organisms such as D. reticulatus with little sequence information available. The results showed that metabolic and cellular processes involved in protein synthesis were the most active in D. reticulatus unfed larvae, suggesting that ticks are very active during this life stage. The stress response was activated in D. reticulatus unfed larvae and a Rickettsia sp. similar to R. raoultii was identified in these ticks.
The activation of stress responses in D. reticulatus unfed larvae likely counteracts the negative effect of temperature and other stress conditions such as Rickettsia infection and favors tick adaptation to environmental conditions to increase tick survival. These results show mechanisms that have evolved in D. reticulatus ticks to survive under stress conditions and suggest that these mechanisms are conserved across hard tick species. Targeting some of these proteins by vaccination may increase tick susceptibility to natural stress conditions, which in turn reduce tick survival and reproduction, thus reducing tick populations and vector capacity for tick-borne pathogens.
Citation: Villar M, Popara M, Ayllón N, Fernández de Mera IG, Mateos-Hernández L, Galindo RC, et al. (2014) A Systems Biology Approach to the Characterization of Stress Response in Dermacentor reticulatus Tick Unfed Larvae. PLoS ONE 9(2): e89564. doi:10.1371/journal.pone.0089564
Editor: Kelly A. Brayton, Washington State University, United States of America
Received: September 25, 2013; Accepted: January 21, 2014; Published: February 21, 2014
Copyright: © 2014 Villar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by grants BFU2011-23896 and the EU FP7 ANTIGONE project number 278976. M. Popara is an Early Stage Researcher supported by the POSTICK ITN (Post-graduate training network for capacity building to control ticks and tick-borne diseases) within the FP7- PEOPLE – ITN programme (EU Grant No. 238511). N. Ayllón and R.C. Galindo were funded by MEC, Spain. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: Marina Manrique and Raquel Tobes work at Era7 Bioinformatics (www.era7bioinformatics.com) that provides Bioinformatics services. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Ticks are blood-sucking ectoparasites that infest and transmit pathogens to humans and animals. Dermacentor reticulatus (Fabricius, 1794) is a three hosts tick (larvae, nymphs and adults feed on different hosts) distributed in Europe and Asia where it infests humans, pets and other domestic and wild animals. D. reticulatus transmit disease-causing pathogens such as Rickettsia slovaca (tick-borne lymphoadenopathy; TIBOLA), Omsk hemorrhagic fever virus (OHFV; Omsk hemorrhagic fever), tick-borne encephalitis virus (TBEV; tick-borne encephalitis), Francisella tularensis (tularemia) and Babesia canis (canine babesiosis) [1]–[3].
Despite its role as a vector of emerging or re-emerging diseases, very little information is available on the genome, transcriptome and proteome of D. reticulatus (115 nucleotide sequences of which only 15 were not of rRNA and 9 protein sequences deposited in the GenBank on June 2013).
This research focused on tick larvae because this is the first developmental stage to infest hosts, acquire infection and transmit pathogens that are transovarially transmitted. Additionally, D. reticulatus larvae hatch at a temperature range of 20–34°C and can survive for 83.5 days at 5°C and 100% relative humidity [4]. However, under natural conditions, larvae are active within 16–20 days after hatching and survive about a month before feeding [5]. D. reticulatus larvae feed on small mammals and are active during the summer [6].
All these facts put tick unfed larvae under extremely stressing conditions. For example, under natural conditions only 5–15% D. reticulatus larvae produced from a single clutch are activated [5]. In this study, we characterized the transcriptome and proteome of D. reticulatus unfed larvae to get an insight into the mechanisms active at this developmental stage, with special emphasis on stress response.
Results and Discussion
D. reticulatus Unigenes Identified after Trasncriptomics Analysis of Unfed Larvae
A total of 21,677,414 (∼2.1 Gb) Illumina 101 bp paired-end reads (207 bp average insert size) were subjected to analysis. After read assembly, 18,946 transcripts were obtained and annotated (Table S1). Transcripts were clustered by encoded proteins. If two transcripts were annotated as the same protein, then these transcripts were clustered together in the same protein cluster. We considered each set of transcripts annotated by the same protein as a unigene to identify transcripts from the same locus/gene. This approach identified a set of 3,808 unigenes with 1,231±286 (Ave±S.E) estimated counts per unigene (Table S1).
The analysis of Biological Process (BP) and Molecular Function (MF) gene ontology (GO) showed that the most represented BPs corresponded to unknown process (N = 2,163; 57%), metabolic process (N = 411; 11%) and cellular process (N = 378; 10%) (Fig. 1A) while proteins with unknown function (N = 2,163; 57%), catalytic activity (N = 658; 17%) and binding activity (N = 628; 16%) were the most represented MFs (Fig. 1B). A closer analysis of the most expressed genes showed that translation and structural constituent of the ribosome were the most represented BP and MF in D. reticulatus unfed larvae, respectively (Figs. 2A and 2B). These genes encoded for 80S ribosomal proteins (Table 1). With the exception of yeast, which lacks L28e, eukaryotic cytoplasmic 80S ribosomes contain the same set of 80 core ribosomal proteins [7]. Thus, the transcripts identified in D. reticulatus larvae encoded for 72% (34/47) and 73% (24/33) of the large and small subunit 80S proteins, respectively (Table 1), representing a high coverage for ribosomal proteins. These results showed that metabolic and cellular processes involved in protein synthesis were the most active in D. reticulatus unfed larvae (Figs. 1A, 1B, 2A, 2B), suggesting that tick metabolism is highly active during this life stage.
(A) Transcripts identified in D. reticulatus unfed larvae were functionally annotated and grouped according to the biological process of the encoded proteins. The number of proteins on each category is shown. (B) Transcripts identified in D. reticulatus unfed larvae were functionally annotated and grouped according to the molecular function of the encoded proteins. The number of proteins on each category is shown.
(A) The 500 more represented unigenes (protein clusters) identified in D. reticulatus unfed larvae were functionally annotated and grouped according to the biological process of the encoded proteins. The number of proteins on each category is shown. (B) The 500 more represented unigenes (protein clusters) identified in D. reticulatus unfed larvae were functionally annotated and grouped according to the molecular function of the encoded proteins. The number of proteins on each category is shown.
D. reticulatus Proteins Identified after Proteomics Analysis of Unfed Larvae
Proteomics analysis was replicated using two different experimental approaches to increase the probability of identifying low abundant proteins such as those involved in stress response. In both approached, mass spectra were searched against Ixodida protein database. The first approach used protein concentration and resulted in the identification of 74 proteins while the second approach analyzed proteins separated by SDS-PAGE and resulted in 239 proteins identified (Table S2), suggesting that for non-quantitative analysis protein fractionation provides better resolution. Of 74 proteins identified with the first approach, 26 (35%) were identified by both methods.
A recently described technique named proteomics informed by transcriptomics (PIT) [8] was used against data generated by the first proteomics approach to validate this method in ticks. This approach uses a database created from transcriptomics data to search mass spectra and has been reported to increase the number of identified proteins [8]. PIT approach resulted in 104 proteins identified in unfed tick larvae (Table S2), representing a 40% increase with respect to the search against Ixodida protein database. The analysis of de novo sequences increased the number of identified proteins using both approaches for proteomics data analysis (Table S2). However, while de novo protein sequences represented 4% (N = 3) of the identified proteins searching against Ixodida protein database, the number of identified proteins increased in 47% (N = 49) using PIT (Table S2). These results support the use of PIT for the analysis of proteomics data, particularly for organisms such as D. reticulatus with little sequence information available.
After removing proteins with unknown BP and MF, transcriptomics and proteomics data correlated well with respect to the most represented BPs (Figs. 3A–3C) and MFs (Figs. 4A–4C). These results were similar for both proteomics approaches, showing a good correlation in the proteomics analysis and providing additional support for the identified mechanisms active in D. reticulatus unfed larvae.
(A) Transcripts identified in D. reticulatus unfed larvae were functionally annotated and grouped according to the biological process of the encoded proteins after removing transcripts with unknown function. (B) Proteins identified in D. reticulatus unfed larvae after searching against Ixodida database were functionally annotated and grouped according to their biological process. (C) Proteins identified in D. reticulatus unfed larvae after searching against transcripts database (PIT) were functionally annotated and grouped according to their biological process. The number of proteins on each category is shown.
(A) Transcripts identified in D. reticulatus unfed larvae were functionally annotated and grouped according to the molecular function of the encoded proteins after removing transcripts with unknown function. (B) Proteins identified in D. reticulatus unfed larvae after searching against Ixodida database were functionally annotated and grouped according to their molecular function. (C) Proteins identified in D. reticulatus unfed larvae after searching against transcripts database (PIT) were functionally annotated and grouped according to their molecular function. The number of proteins on each category is shown.
Rickettsia sp. Identified in D. reticulatus Unfed Larvae
Although these ticks were obtained from a colony considered to be free of tick-borne rickettsiae, some reads matching Rickettsia spp. were identified in D. reticulatus unfed larvae resulting in 16 unigenes (Table S3). These transcripts were probably wrongly annotated as I. scapularis proteins in Uniprot when they are likely Rickettsia proteins. In these cases, the Uniref representative protein of the cluster to which belongs the I. scapularis protein is a Rickecttsia protein and the rest of the members of the Uniref90 cluster are also from Rickettsia. Proteomics analysis corroborated the presence of Rickettsia proteins in D. reticulatus unfed larvae with the identification of 14 proteins searching against Rickettsiae database (Table S3).
The Rickettsia sp. identified in unfed larvae could be a commensal bacterium that has been described in Dermacentor and other tick species, but not in D. reticulatus [9]–[12] or a pathogen [13]. The Rickettsia proteins identified in D. reticulatus unfed larvae are highly conserved among Rickettsia spp. and thus not suitable to characterize these organisms at the species level.
To gain further information on this Rickettsia sp., the PCR amplification and sequencing of gene markers that have been previously used for species classification was conducted [14]–[16]. The results showed >99% pairwise nucleotide sequence identity to Rickettsia sp. sequences, especially to R. raoultii (Table 2). As previously shown [15], the in silico PstI and RsaI restriction analysis of ompA sequences was highly informative and corroborated that the Rickettsia sp. identified in this study is similar to R. raoultii. These results suggested, as in previous studies in tick cell culture [17], that the Rickettsia sp. identified in D. reticulatus unfed larvae is closely related to the tick-borne pathogen, R. raoultii. However, until this Rickettsia sp. is fully characterized, we cannot exclude the possibility of a symbiont closely related to R. raoultii. These results suggested that the pathogen could be an additional stress factor in D. reticulatus unfed larvae, which correlated with the activation of immune response in these ticks (Figs. 1A, 3A and 3C). Rickettsia sequences were deposited in the GenBank with accession numbers [GenBank: KF478838, KF478839].
Stress Response in D. reticulatus Unfed Larvae
The results showed that metabolic processes and translation in particular were highly represented at the transcriptional level by genes encoding 80S ribosomal proteins in D. reticulatus unfed larvae (Table 1). Stress regulates ribosomal protein expression in other organisms, but no information is available in ticks [18]–[21]. Furthermore, a growing body of evidence suggests that the ribosome serves as a hub for co-translational folding, chaperone interaction, degradation, and stress response [22]. These results suggested a connection between transcription of ribosomal protein genes and stress response in ticks that deserves further investigation.
Transcripts and proteins mapped to stress response BP in D. reticulatus unfed larvae were selected for further analysis. Transcriptomics results showed that heat shock, cold shock and other stress responses were active in unfed larvae, represented by 39 unigenes (1% of all identified unigenes) and 27,937 counts (Table 3). Of them, the most represented functions corresponded to heat shock response (Figs. 5A and 5B). In general, protein identification has a lower resolution when compared to transcriptomics, a limitation that is more evident when working with species such as D. reticulatus for which sequence information is very scarce in the databases [23]. The search of MS data against the Ixodida database resulted in 8 stress response proteins identified (Table 4). However, when a database of transcripts identified as encoding for stress response proteins was generated and used for targeted PIT analysis, the results showed that 16 new stress response proteins were identified (Table 4). Additionally, while only 1% of the unigenes corresponded to stress response proteins, over 7% of the identified proteins were involved in this BP, supporting that stress response is active in tick unfed larvae. Furthermore, in agreement with transcriptomics data, the most represented function corresponded to heat shock response (Figs. 5C and 5D).
(A) Stress response transcripts identified in D. reticulatus unfed larvae were grouped according to the function of their encoded protein. The number of proteins and percent in each category is shown. (B) Number of counts per protein (Ave+S.E.) in stress response proteins identified by transcriptomics analysis in D. reticulatus unfed larvae. (C) Stress response proteins identified in D. reticulatus unfed larvae were grouped according to the function of their encoded protein. The number of proteins and percent in each category is shown. (D) Number of peptides per protein (Ave+S.D.) in stress response proteins identified by proteomics analysis in D. reticulatus unfed larvae.
Some transcripts mapped to stress response BP were selected for the characterization of mRNA levels in D. reticulatus tick unfed larvae and guts and salivary glands from adult ticks incubated at 4, 37 or 19°C by real-time RT-PCR (Figs. 6A–6E). The results showed that all selected genes encoding for stress response proteins were more expressed in unfed larvae than in adult tissues, thus reinforcing the significance of this BP in D. reticulatus tick unfed larvae (Fig. 6A). In adult ticks, some genes were differentially expressed in response to temperature changes in guts or salivary glands (Figs. 6B–6E). The differential expression of selected genes encoding for stress response proteins was more evident in female salivary glands than in female guts and male tissues (Fig. 6E), suggesting differences between female and male ticks and between tissues in stress response to temperature changes. Additionally, at least for the genes characterized in this experiment, differential expression was more pronounced at 4°C than at 37°C (Fig. 6E), suggesting that D. reticulatus ticks respond differently to different temperatures. The sequences of D. reticulatus genes encoding for stress response proteins were deposited in the GenBank with accession numbers [GenBank: SRR950367; Bioproject: PRJNA214849].
(A) The mRNA levels were characterized by real-time RT-PCR in D. reticulatus unfed larvae and adult female and male guts and salivary glands (N = 3), normalized against tick ribosomal protein S4 and shown as Ave+S.D. in arbitrary units. Normalized Ct values were compared between larvae and adult samples by Student's t-test with unequal variance (*P≤0.05). (B–D) The mRNA levels were characterized by real-time RT-PCR in D. reticulatus guts and salivary glands from adult female and male ticks incubated at 4, 19 and 37°C for 4.5 h prior to RNA extraction (N = 3), normalized against tick ribosomal protein S4 and shown as Ave+S.D. in arbitrary units. Normalized Ct values were compared between samples from ticks incubated at 4 or 37°C and 19°C by Student's t-test with unequal variance (*P≤0.05). (E) For genes with significant differences between samples from ticks incubated at 4°C or 37°C and 19°C, the log2 4/19°C or 37/19°C normalized Ct values ratio was calculated to show differential expression in response to temperature. Abbreviations: FG, female gusts; FSG, female salivary glands; MG, male guts; MSG, male salivary glands.
Ticks are very sensitive to temperature and their life cycle is dependent on a complex combination of climate variables for development and survival [24]. In particular, D. reticulatus tick unfed larvae are exposed to extremely stressing conditions that affect their survival and development [5]. The heat-shock and other stress responses are a conserved reaction of cells and organisms to different stress conditions such as extreme temperatures, toxicity and pathogen infection [25]. Crucial to cell survival is the sensitivity of proteins and enzymes to heat inactivation and denaturation. Therefore, adaptive mechanisms exist that protect cells from the proteotoxic effects of stress. The heat-shock proteins and other stress response proteins protect cells and organisms from damage, providing higher levels of tolerance to environmental stress. Recent studies demonstrated that the stress response is activated in ticks and cultured tick cells in response to Anaplasma spp. infection and heat shock [26], [27]. These results showed that at high temperatures and during blood feeding, when hsp20, hsp70 and subolesin are overexpressed, Ixodes scapularis ticks are protected from stress and pathogen infection and have a higher questing speed. Herein, genes encoding for stress response proteins were also differentially expressed in D. reticulatus in response to cold or heat shock. These results suggested a connection between tick stress response, questing behavior and pathogen infection [26], [27], which may be present also in D. reticulatus tick unfed larvae. Experiments characterizing the mRNA and protein levels of genes identified in this study in D. reticulatus ticks exposed to blood feeding and pathogen infection would add additional support to the importance of these proteins in tick stress response.
The characterization of the transcriptome and proteome of D. reticulatus unfed larvae showed that stress response was active in this developmental stage. Although descriptive in its nature, these results showed that combination of transcriptomics and proteomics approaches provide strong support for the characterization of biologically relevant pathways in ticks. The activation of stress responses in D. reticulatus unfed larvae likely counteracts the negative effect of temperature and other stress conditions such as Rickettsia infection and favors tick adaptation to environmental conditions to increase tick survival. These results are relevant to understand how D. reticulatus ticks have evolved mechanisms to survive under stress conditions and suggest that these mechanisms are conserved across hard tick species. Targeting some of these proteins by vaccination may increase tick susceptibility to natural stress conditions, which in turn reduce tick survival and reproduction, thus reducing tick populations and vector capacity for tick-borne pathogens [28].
Materials and Methods
Experimental Design and Rationale
In this research, we completed the analysis of the transcripts and proteins present in D. reticulatus unfed larvae, which are described in Tables S1 and S2. This information, which was not available for this species, was then used to characterize stress response by focusing on the relevant genes and proteins. Individual variability, which certainly exists in ticks as in other organisms, was considered by pooling a large number of larvae for transcriptomics (N = 500) and proteomics (N = 200) studies. As in previous studies [29]–[31], we did not use biological replicates for RNA-Seq but the algorithm used to quantitate transcriptomics data allows the use of non-replicated samples [32]. Proteomics analysis, although also used for a non-comparative study that does not require replicates [31], was replicated using a different experimental approach to increase the probability of identifying low abundant proteins such as those involved in stress response. The statistical significance of reads and peptide assignments is supported by the application of eXpress and SEQUEST (FDR<0.01) algorithms described bellow for the analysis of transcriptomics and proteomics data, respectively.
Ticks and Sample Preparation
D. reticulatus unfed larvae were obtained from a single female from a Dutch colony maintained at the Utrecht Centre for Tick-borne Diseases (UCTD), Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands. Total RNA and DNA were extracted from approximately 500 D. reticulatus larvae kept off-host for 7 days using the AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Valencia, CA, USA) according to manufacturer instructions. RNA was purified with the RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA, USA) and characterized using the Agilent 2100 Bioanalyzer (Santa Clara, CA, USA) in order to evaluate the quality and integrity of RNA preparations. RNA concentration was determined using the Nanodrop ND-1000 (NanoDrop Technologies Wilmington, Delaware USA). For protein extraction, approximately 200 D. reticulatus larvae were pulverized in liquid nitrogen and homogenized with a glass homogenizer (20 strokes) in 4 ml buffer (0.25 M sucrose, 1 mM MgCl2, 10 mM Tris-HCl, pH 7.4) supplemented with 4% SDS and complete mini protease inhibitor cocktail (Roche, Basel, Switzerland). Sample was sonicated for 1 min in an ultrasonic cooled bath followed by 10 sec vortex. After 3 cycles of sonication-vortex, the homogenate was centrifuged at 20×g for 5 min at room temperature to remove cellular debris. The supernatant was collected and protein concentration was determined using the BCA Protein Assay (Thermo Scientific, San Jose, CA, USA) using BSA as standard.
D. reticulatus unfed female and male adults were obtained from a tick colony originally collected in southern Slovakia and maintained at the Biology Centre of the ASCR, Parasitology Institute, České Budějovice, Czech Republic.
Transcriptomics Data Acquisition
The RNA purified from unfed tick larvae was used for library preparation using the TruSeq RNA sample preparation kit v.1 and the standard low throughput procedure (Illumina, San Diego, CA, USA). Briefly, 0.7 µg total RNA was used as starting material for library preparation. Messenger RNA was captured using poly-dT magnetic beads and purified polyA+ RNA was chemically fragmented and reverse-transcribed. Remaining RNA was enzymatically removed and the second strand generated following an end repair procedure and preparation of double-stranded cDNA for adaptor ligation. Adaptor oligonucleotides containing the signals for subsequent amplification and sequencing were ligated to both ends and the cDNA was washed using AMPure SPRI-based magnetic beads (Beckman Coulter, IZASA, Barcelona, Spain). Adaptors contained identifiers, which allow multiplexing in the sequencing run. An enrichment procedure based on PCR was then performed to ensure that all molecules in the library conserved the adapters at both ends. The number of PCR cycles was adjusted to 15. The final amplified library was checked again on a BioAnalyzer 2100 (Agilent, Santa Clara, CA, USA) and titrated by quantitative PCR using a reference standard to characterize molecules concentration in the library (12.44 nM). The library was denatured and seeded on the lane of the flowcell at a final concentration after re-naturalization of 10–14 pM. After binding, clusters were formed in the flowcell by local amplification using a Cluster Station apparatus (Illumina). Following sequencing primer annealing, flowcell was loaded into a GAIIx equipment (Illumina) to perform sequencing using the TruSeq® system (Illumina). The sample was run under a pair-end 2×100 bp protocol for de novo sequencing. After sequencing and quality filtering, reads were split according to their different identifiers and fastq files were generated to proceed to quality analysis and de novo transcript assembly and gene expression analysis.
Bioinformatics for Transcriptomics Data
Sequence reads were trimmed at the error probability higher than 0.05 and assembled only when two members of the pair remained after filtering at trimming. Oases [33] was used for read assembly in the mode of single (not merged) assembly because results were better in this mode. A K value of 79 was chosen, which was very close to the total length of the read (∼100 bp) to avoid misassemblies since the higher the overlapping required the more accurate the transcript is. Final assembly was explored in detail using Tablet (http://bioinf.scri.ac.uk/tablet/download.shtml) [34].
Functional annotations were inferred by similarity to Uniprot reference proteins using Blast E values <10E-10. We selected a set of 34,095 reference proteins downloaded from Uniprot on March 7, 2013, including all proteins that were representative of Uniref90 clusters belonging to the taxonomic node Chelicerata, which are 8 levels above D. reticulatus taxon. In the Uniref90 clusters, each protein belongs to only one cluster with a 90% similarity to the representative protein for all members of the cluster. It provides a more homogeneous and uniform distance between reference proteins. Reference proteins were used for transcript clusterization to obtain a protein-centred analysis of gene expression that is more useful for functional analysis in a de novo transcriptome.
The eXpress algorithm was used for mapping reads to multiple targets to quantify gene expression levels [32]. The eXpress algorithm [32] for quantifying the abundances of the transcripts addresses multi-mapping based on an on-line expectation–maximization algorithm (online-EM) [35] that is used to estimate transcript abundances in multi-isoform genes and gene families, and that does not require a reference genome. The underlying model is based on previously described probabilistic models developed for RNA-seq and allows the use of parameters for fragment length distributions, errors in reads, and sequence-specific fragment bias [36]. The algorithm alternates between assigning fragments to targets with a probability according to abundance parameters (expectation step) and updating abundances to the maximum-likelihood solution on the basis of the expectation-step assignments (maximization step). At the beginning the abundances are set to a uniform initial value. Then, for the fragments that map to multiple sites, eXpress calculates probabilities for each site, considering previous estimates of target-sequence abundances. As fragments are processed, they are assigned increasing 'mass' to improve the estimation of abundance according to the assignment probability. Parameters for fragment-length (L) distribution, sequence bias and sequence read errors are updated and used in the next round of assignment. While relative abundance and count estimates are updated, uncertainties in assignment are propagated so that posterior count distributions can be estimated. The probabilistic model is described in detail in the online methods section in Roberts and Pachter [32].
eXpress was also used to analyze the read mapping results. The mapper tool used was Bowtie setting the mapping parameters following the eXpress recommendations. Bowtie is an ultrafast, memory-efficient short read aligner that indexes the reference with a Burrows-Wheeler index to have low memory requirements [37]. Bowtie indexes the reference genome using a scheme based on the Burrows-Wheeler transform (BWT) [38], that is a reversible permutation of the characters in a text developed for data compression and the Ferragina and Manzini (FM) index [39]. Bowtie adopts the exact-matching algorithm of Ferragina and Manzini for searching in the FM index but introduces a quality-aware backtracking algorithm that allows mismatches and 'double indexing', to avoid excessive backtracking.
The script used for mapping the reads to the transcripts with Bowtie and for the final quantification with eXpress (File S1) was performed using cloud computing (Amazon Web Services). The process took 100 minutes in an Amazon EC2 m2.4xlarge instance. This kind of instances has 8 virtual CPUs and 68.4 GiB of RAM.
Proteomics Data Acquisition
Proteins concentrated by SDS-PAGE.
The protein extract (150 µg) was precipitated following the methanol/chloroform procedure [40], resuspended in 100 µl Laemmli sample buffer and applied onto 1.2-cm wide wells on a 12% SDS-PAGE gel. The electrophoretic run was stopped as soon as the front entered 3 mm into the resolving gel, so that the whole proteome became concentrated in the stacking/resolving gel interface. The unseparated protein band was visualized by staining with GelCode Blue Stain Reagent (Thermo Scientific), excised, cut into 2×2 mm cubes and digested overnight at 37°C with 60 ng/µl sequencing grade trypsin (Promega, Madison, WI, USA) at 5∶1 protein:trypsin (w/w) ratio in 50 mM ammonium bicarbonate, pH 8.8 containing 10% (v/v) acetonitrile [41]. The resulting tryptic peptides from the gel band were extracted by 30 min-incubation in 12 mM ammonium bicarbonate, pH 8.8. Trifluoroacetic acid was added to a final concentration of 1% and the peptides were finally desalted onto OMIX Pipette tips C18 (Agilent Technologies, Santa Clara, CA, USA), dried-down and stored at −20°C until mass spectrometry analysis.
The desalted protein digest was resuspended in 0.1% formic acid and analyzed by RP-LC-MS/MS using an Easy-nLC II system coupled to an ion trap LCQ Fleet mass spectrometer (Thermo Scientific). The peptides were concentrated (on-line) by reverse phase chromatography using a 0.1×20 mm C18 RP precolumn (Thermo Scientific), and then separated using a 0.075×100 mm C18 RP column (Thermo Scientific) operating at 0.3 µl/min. Peptides were eluted using a 180-min gradient from 5 to 35% solvent B (Solvent A: 0,1% formic acid in water, solvent B: 0,1% formic acid in acetonitrile). ESI ionization was done using a Fused-silica PicoTip Emitter ID 10 µm (New Objective, Woburn, MA, USA) interface. Peptides were detected in survey scans from 400 to 1600 amu (1 µscan), followed by three data dependent MS/MS scans (Top 3), using an isolation width of 2 mass-to-charge ratio units, normalized collision energy of 35%, and dynamic exclusion applied during 30 sec periods.
Proteins separated by SDS-PAGE.
The protein extract (150 µg) was precipitated following the methanol/chloroform procedure [40], resuspended in 100 µl Laemmli sample buffer and applied onto 1.2-cm wide wells on a 12% SDS-PAGE gel. The protein bands were visualized by staining with GelCode Blue Stain Reagent (Thermo Scientific) and sliced each gel lane into 25 slices as previously described [42]. Protein digestion and RP-LC-MS/MS analysis was performed as described before for proteins concentrated by SDS-PAGE.
Bioinformatics for Proteomics Data
The MS/MS raw files were searched against Ixodida (40,849 entries in June 2013) and Rickettsieae (58,899 entries in June 2013) Uniprot databases and against a database created from transcriptomics data (PIT) [8] using the SEQUEST algorithm (Proteome Discoverer 1.3, Thermo Scientific) with the following constraints: tryptic cleavage after Arg and Lys, up to two missed cleavage sites, and tolerances of 1 Da for precursor ions and 0.8 Da for MS/MS fragment ions and the searches were performed allowing optional Met oxidation and Cys carbamidomethylation. For peptide validation, the Percolator node present in the Proteome Discoverer 1.3 software was used. Percolator is a machine-learning supplement of the Sequest algorithm that uses a decoy database search strategy to learn to distinguish between correct and incorrect peptide identifications increasing the sensitivity and specificity of peptide identification [43], [44]. The filtering criteria applied in this case are based on the q-value generated by Percolator that is defined as the minimal false discovery rate at which the identification is deemed correct [43]. These q-values are estimated using the distribution of scores from the decoy database search. A false discovery rate (FDR) <0.01 was considered as condition for successful peptide assignments, including only peptides with q-values ≤0.01 and delta Cn >0.05. De novo peptide sequencing was conducted with Peaks Studio 6.0 software (Bioinformatics Solutions Inc., Waterloo, ON Canada).
Gene and Protein Ontology Assignments
Functional data for each protein were obtained from Uniprot and included GO annotations, EC number and Interpro motifs. Assignment of GO terms to identified proteins was done by Blast2GO software (version 2.6.6; http://www.blast2go.org/) in three main steps: blasting to find homologous sequences, mapping to collect GO-terms associated to blast hits and annotation to assign functional terms to query sequences from the pool of GO terms collected in the mapping step [45]. Sequence data of identified proteins were uploaded as FASTA file to the Blast2GO software and the function assignment was based on GO database. The blast step was performed against NCBI public databases through blastp. Other parameters were kept at default values: e-value threshold of 1e-3, recovery of 20 hits per sequence, minimal alignment length (hsp filter) 33 (to avoid hits with matching region smaller than 100 nucleotides) and Blast mode was set to QBlast-NCBI. Configuration for annotation was an e-value-Hit-filter of 1.0E-6, annotation cut off of 55 and GO weight of 5. For visualizing the functional information (GO categories: Molecular Function and Biological process), the analysis tool of the Blast2GO software was used.
The GO analysis for the 500 more represented unigenes was based on the GO annotations included in the Uniprot entry of the representative protein of each cluster. The GO analysis was done using Bio4j Go Tools developed by Era7 Bioinformatics and available at http://gotools.bio4j.com:8080/Bio4jTestServer/Bio4jGoToolsWeb.html. Bio4j Go Tools is a set of GO related Web Services using the open source graph bioinformatics platform Bio4j as back-end. Bio4j is a graph-based database including most data available in UniProt KB (SwissProt+Trembl), Gene Ontology (GO), UniRef (50,90,100), RefSeq, NCBI taxonomy, and Expasy Enzyme (http://bio4j.com/). Specifically designed java programs were used for the generation of the GO frequency chart data.
Effect of Temperature on Gene Expression
Three groups of 9 D. reticulatus unfed female or male adults each were incubated for 4.5 h at 4°C, 19°C or 37°C and 20% relative humidity. After incubation, ticks were dissected and salivary glands and guts were separated, pooled in groups of three (3 groups for each temperature and sex) and immediately stored in TriReagent (Sigma, St. Louis, MO, USA) for RNA extraction.
Analysis of mRNA Levels by Real-time RT-PCR
For real-time RT-PCR, tick larvae (three pools of 100 larvae each), unfed female and male adult ticks (3 ticks each) and guts and salivary glands from unfed female and adult adults incubated at different temperatures (3 groups for each temperature and sex) were used for RNA extraction using TriReagent (Sigma, St. Louis, MO, USA) following manufacturer's recommendations. Real-time RT-PCR was performed on tick RNA samples (5 ng) with gene specific primers (20 pmol each) and conditions (Table 5) using the iScript One-Step RT-PCR Kit with SYBR Green and the iQ5 thermal cycler (Bio-Rad, Hercules, CA, USA) following manufacturer's recommendations. A dissociation curve was run at the end of the reaction to ensure that only one amplicon was formed and that the amplicons denatured consistently in the same temperature range for every sample [46]. The mRNA levels were normalized against tick ribosomal protein S4 using the genNorm method (ddCT method as implemented by Bio-Rad iQ5 Standard Edition, Version 2.0) [47], [48]. Normalized Ct vales were compared between larvae and adult samples and between samples from ticks incubated at 4 or 37°C and 19°C by Student's t-test with unequal variance (P = 0.05).
PCR and Sequence Analysis of Rickettsia Amplicons
Rickettsia sp. DNA was characterized by PCR, cloning and sequence analysis of the amplicons. At least three clones were sequenced for each amplicon. Genes targeted by PCR included fragments of ATP synthase alpha subunit (atpA), heat-shock protein 70 (dnaK), outer membrane protein A (ompA), outer membrane protein B (ompB), 16S rRNA, and recA [14]–[16]. Nucleotide sequence identity to reference strains and in silico PstI and RsaI restriction analysis of ompA sequences was used to characterize Rickettsia sp. [15], [16].
Supporting Information
Table S1.
Tick transcripts and encoded proteins identified in transcriptomics analysis of D. reticulatus unfed larvae.
Table S2.
Tick proteins identified in proteomics analysis of D. reticulatus unfed larvae.
Table S3.
Rickettsia sp. transcripts and proteins identified in D. reticulatus unfed larvae.
File S1.
Script used for mapping the reads to the transcripts with Bowtie and for the final quantification with eXpress.
We thank F. Jongejan (Utrecht Centre for Tick-borne Diseases) and J. Erhart and L. Grubhoffer (Biology Centre of the ASCR, Parasitology Institute, České Budějovice, Czech Republic) for providing tick samples.
Author Contributions
Conceived and designed the experiments: JF. Performed the experiments: MV MP NA IGFM LM-H RCG. Analyzed the data: MV MM RT JF. Wrote the paper: MV MP MM RT JF.
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I'm looking for a daemon that I can install on my Debian server, and can be controlled through PHP (preferably with included libraries). It must have a basic key-value cache, BUT it must never ever delete any entry in the cache until I tell it to do so. Is there anything like this?
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I suppose Redis should fit the requirements. It's packaged in Debian and has several PHP client libraries of which one appears to be packaged, though only in Sid at the moment (but I suppose it should be trivial to backport).