text:
Syria: Suicide bombings kill dozens in Homs
Syria: Daesh bomb attack kills at least 60 in Al-Bab
Canadian province passes anti-Islamophobia motion
US: Muslim woman sues after alleged police hijab attack
Turkey: Hijab now permitted in Turkish army
US: Muslims raise $80K to repair Jewish cemetery
Myanmar: 4 Rohingya Muslims found dead in Rakhine state
Palestine: 18 months for Israeli soldier who killed Palestinian
Palestine: Israel rounds up 22 Palestinians in W Bank raids
Iraq: 60,000 troops taking part in anti-Daesh ops in W. Mosul
Afghanistan: Heavy snow kills 25 in Faryab province
Somalia: 22 killed in truck bomb attack on Mogadishu market
Israel: Death of Muslim scientists puts spotlight on Mossad
Germany: Islam is not source of terrorism, says Merkel
Weekly Report: Israeli Human Rights violations in Occupied Palestinian Territory
Turkey: Car bomb in SE Turkey kills 2, wounds 15
Iraq: Massive car bomb kills 47 in western Baghdad
Afghanistan: Religious students among dead in roadside blast
Canada gov't bid for 'anti-Islamophobia' motion stalls
By Chris Carlson International Middle East Media Center: Two Palestinian women have been injured as a result of an Israeli warplane attack, Saturday night. Several locations in the Gaza Strip were targeted. One of the injured is a 15-year-old girl. … Continued
[Car attacked by Israeli settlers: Archive Photo By Land Research Center] IMEMC & Agencies: Saturday, Palestinian medical sources said three Palestinian were moderately wounded after a number of settlers hurled stones at their car while driving near the entrance of Jensafut … Continued
IMEMC & Agencies: Saturday at dawn, as part of the search for three missing Israeli teenage settlers, Israeli soldiers intensified their invasions and strict security measures in the southern West Bank district of Hebron, broke into and searched dozens of homes, … Continued
[File photo: Maan News Agency] IMEMC & Agencies: Israeli soldiers and police officers invaded, late on Friday at night Rahat city, the largest Bedouin community in the Negev, conducted military searches, and kidnapped several residents. The Maan News Agency … Continued
By Chris Carlson International Middle East Media Center: Following Friday prayers in the Old City of Jerusalem, 28 Palestinians were injured and eight detained during fierce clashes which broke out after Israeli troops stormed Al-Aqsa mosque. Worshipers at the mosque … Continued
[Photo by ISM] International Solidarity Movement: Wednesday evening, at approximately 7 pm, Israeli soldiers broke into several Palestinian family's homes in the Qeitun region of al-Khalil (Hebron), following clashes with local youth, which began an hour earlier. The … Continued
By Chris Carlson International Middle East Media Center: Thursday morning, Israeli forces demolished a number of structures in the Naqab village of al-Araqib, to the north of Beersheba. Israeli media has reported that members of the Israeli Committee of … Continued
[File photo: Shasha News Agency] By Saed Bannoura IMEMC & Agencies: Several Israeli military jeeps invaded, on Thursday at dawn the northern West Bank city of Jenin, where they kidnaped two Palestinians and detained one resident … Continued
By Saed Bannoura IMEMC & Agencies: Wednesday evening, Israeli soldiers invaded al-Eesawiyya town, in occupied East Jerusalem, broke into several homes and stores, violently searched them, and kidnapped five Palestinians. The Maan News Agency has reported that the invasion is the fourth … Continued
[Still Image From Palestine TV Video] By Saed Bannoura IMEMC News: Wednesday, nonviolent Palestinian activists held a mock soccer match in front of the Ofer Prison, near the central West Bank city of Ramallah, to express solidarity with hunger striking detainees, … Continued
[File photo: Palestine TV] By Saed Bannoura IMEMC & Agencies: Wednesday night, Palestinian medical sources have reported that one Palestinian was killed, and two were seriously injured, when an Israeli war jet fired a missile targeting a fighter northwest of Gaza city. … Continued
By Chris Carlson International Middle East Media Center: Israeli settlers stormed the park of the Ibrahimi Mosque (Cave of the Patriarchs) in Hebron, on Wednesday, where they attempted to sabotage its content, according to media sources. WAFA correspondence … Continued
Chris Carlson International Middle East Media Center: Israeli forces kidnapped three Palestinians from West Bank cities of Jenin and Hebron, Tuesday, in addition to razing Palestinian-owned lands in Jerusalem. According to Palestinian security sources, the Israeli army raided Jenin refugee … Continued
[File photo: Qudsmedia] IMEMC & Agencies: Tuesday, Israeli soldiers, heavily deployed in occupied East Jerusalem, especially near the al-Aqsa Mosque, attacked dozens of Palestinians, including children, near Bab al-Majlis Gate (The Council Gate), and prevented them from entering the mosque. Sheikh … Continued
[Photo by ISM] International Solidarity Movement: Sunday morning, Israeli police forces raided the village of Iqrit (located on the northern tip of Israel, within eyesight of Lebanon). The police uprooted trees, destroyed and confiscated all the personal belongings of Palestinian … Continued
By Saed Bannoura IMEMC & Agencies: Tuesday morning, several Israeli military jeeps and a number of armored bulldozers invaded Far'oun town, south of the northern West Bank city of Tulkarem, and demolished one home. Head of the Far'oun Town Council, … Continued
[File: Image eshahed.com] IMEMC & Agencies: Monday, Israeli soldiers, stationed across the eastern border of Gaza City, fired dozens of rounds of live ammunition targeting a number of homes, and farmlands, east of the ash-Shujaeyya neighborhood, east of Gaza city. The … Continued
By Saed Bannoura IMEMC & Agencies: Monday, Israeli soldiers kidnapped at least ten Palestinians in different parts of the occupied West Bank, and confiscated around 80 computers from two stores in Tulkarem. Local sources in Hebron, in the southern part of … Continued
By Chris Carlson International Middle East Media Center: A Palestinian fisherman who was recently shot and critically injured by the Israeli navy, off the coast of the northwest part of the Gaza Strip, has died of his wounds Sunday, … Continued
[Image By Raya News] By Saed Bannoura IMEMC & Agencies: Saturday evening, Israeli soldiers invaded Zabbouba town, near the northern West Bank city of Jenin, kidnapped an elderly man and hurled gas bombs, causing several family members to suffer the effects … Continued
Over 120 people attended a landmark conference on the media reporting of Islam and Muslims. It was held jointly by The Muslim News and Society of Editors in London on September 15.
The Muslim News Awards for Excellence 2015 was held on March in London to acknowledge British Muslim and non-Muslim contributions to the society.
The Muslim News Awards for Excellence 2015 was held on March in London to acknowledge British Muslim and non-Muslim contributions to the society.


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We are currently accepting new patients, click the button below for more information.
The appointment lines are open from 8.00am - 6.30pm.
We offer home visits for patient who are housebound or too ill to attend the surgery.
Patients on long-term medication can request repeat prescriptions.
During normal opening times (8.30am - 6.30pm Monday to Friday) please telephone 01773 303591 When we are closed please call 111. (How the 111 service operates is explained here)
Our fax number is (01773) 513470.
Please note ALL calls to and from the practice are recorded.
This website has been designed to tell you about the practice and the services that we offer. Please read it carefully. We trust that you will find it helpful and informative.
Ripley Medical Centre was built in 1993 to provide modern facilities for a comprehensive range of primary health care and is designed for use by the physically disabled.
Although the surgery is sited in a purpose-built centre, it is the oldest practice in Ripley, being over 100 years old and was previously situated on Cromford Road which was known as Scarsdale Surgery.
Parking is available for your use when visiting the surgery.
We aim to provide a caring, punctual and professional service of the highest standard. We have your health and well being at heart and will do all that we can to ensure that you receive the best support available. If you have any suggestions to improve our service then please drop a note in the suggestion box.
There is a large car park for the use of patients whilst visiting the surgery. Disabled access is available to the surgery.
If you are contacted directly by a manufacturer or other orgainsaion to try using a different insulin therapy system, please discuss this ...
With effect from 5th September 2016, the "Sit and Wait "appointment service will ...
See our new page on Accessible Information. The practices is improving its process of communicating with patients who are blind, d/Deaf, ...
The practice would like to thank the various advertisers who have helped to
produce this Website. However, it must be pointed out that the accuracy of any
statements cannot be warranted, nor any products or services advertised, be
guaranteed or endorsed.

subset: commoncrawl


text:
TCM Archive Materials VIEW ALL ARCHIVES (0)
Recent DVDs
Western Classics ... Includes: The Rifleman - Day of the Hunter, Outlaw's Inheritance, Mail Order... more info $5.95was $6.95 Buy Now
The Great American Western:... Starring Olivia de Havilland, William Shatner, Helen Hunt. more info $5.95was $3.99 Buy Now
|Also Known As:
|Robert Patrick Brady, Darol Rice, Bud Dooley, George Bamby, Michael Barton, Pat Brady, Jimmy Bryant
|February 27, 1972
|December 31, 1914
|Cause of Death:
|Birth Place:
|Toledo, Ohio, USA
|actor, musician
Biography CLOSE THE FULL BIOGRAPHY
No biography information exists for this person. Be the FIRST to contribute by CLICKING HERE >
Filmographyclose complete filmography
CAST: (feature film)
Milestones close milestones
Please support TCMDB by adding to this information.Click here to contribute


text:
Tropical Fish Keeping - Aquarium fish care and resources (http://www.tropicalfishkeeping.com/forum.php)
- Ancient Fish (http://www.tropicalfishkeeping.com/ancient-fish/)
- - african butterfly fish (http://www.tropicalfishkeeping.com/ancient-fish/african-butterfly-fish-22676/)
african butterfly fish
i hope this is a good place to post this.... how much and often should i be feeding my butterfly fish? it seems to be a bit of a pig if i let it will eat 5 crickets a day or a worm a day + blood worms is this too much?
|All times are GMT -5. The time now is 09:30 PM.
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text:
Protect-a-Bed Premium Mattress Protector
This premium protector features a cotton terry towel surface for superior comfort and absorbency.Full Details
Product Actions
Additional Information
Whether you are looking to provide a barrier to allergens and dust mites or are looking to keep your mattress and pillows sanitary and protected from spills and accidents, the Protect-a-Bed Premium Mattress Protector is a good solution.
The Premium Mattress Protector features an absorbent cotton terry towel surface that is cool and comfortable to sleep on. In addition, the Premium Mattress Protector contains Protect-a-Bed's exclusive Miracle Membrane backing. The Miracle Membrane is waterproof to keep your bed sanitary and it provides a barrier from allergens and dust mites. It is also air vapor porous, allowing body heat to easily pass through to keep your mattress cool to sleep on.
A fitted sheet-style stretches to fit mattress up to 22-inches deep in a queen and king size. It will not alert the feel of the mattress and can be easily removed for washing. The Premium Mattress Protector can be machine washed and dried.
You MightAlso Like
$1,599.00 - $3,198.00Tempur-Pedic Flex Prima Hybrid Mattress$1,599.00 - $3,198.00Description
The Tempur-Flex Prima Mattress by Tempur-Pedic® features faster adapting comfort and pressure relief with precision coil support. Sleep easier with the instant comfort you'll get from the Tempur-Flex Prima's medium-firm feel. Not too soft and not too firm.
Price is for MATTRESS ONLY. Bed set and foundation in images are sold separately.
$1,329.00 - $1,899.00$699.00 - $999.00Lux Living Memory & Latex Foam Mattress$1,329.00 - $1,899.00$699.00 - $999.00Description
The Lux Living mattress features both Latex Foam and Memory Foam, and is designed for superior comfort and support. This mattress in a box is roll-packed into one-third of a normal mattress size, making it easy to delivery straight to your doorstep.
Price is for MATTRESS ONLY. Bed set and foundation in images are sold separately.
Special Shipping Policies Apply Details
Available Online Only - Ships Separately From Other Items


text:
Federal regulations on emergency medical services helicopters are long overdue, according to blogger Paul Bertorelli. EMS helicopters often fly in risky conditions, and can endanger patients. "They fly at low altitude, often without benefit of the instrument flight plans that help keep airline travel safe and many landings are at urban LZs, not airports," writes Bertorelli.
Full Story:AVweb


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Is Zalora incorrect or missing key business information? Update your Hotfrog listing today.
Contact details
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Hotfrog Philippines provides information regarding Zalora in Makati Metro Manila. Zalora is located at 50 Jupiter Street, Makati Metro Manila 1209 and provides Retail services. Contact them on (02) 858 0777 or by visiting them on their website http://www.zalora.com.ph.
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Select Country
Some products only show off their real performance when they are configured to meet your individual requirements. Our software makes using and configuration easier when it comes to operation, configuration and monitoring.
Product-neutral software
The Weidmüller App provides information and news about all our products.
To the app
The Weidmüller Configurator is a powerful engineering software for the configuration of terminal rails and complete assemblies of Weidmüller products.
To the configurator
Remote I/O
The software supports you in the use of u-remote and provides fieldbus configuration files, firmware, release notes, language files and USB drivers for the web server.
To the software
The software generates EDS files for the configuration of an SAI Active PRO Gateway with SAI Active PRO sub-bus modules.
To the software
GSD and EDS files are recommended for the correct integration of bus participants into PROFIBUS and DeviceNet networks.
To the device drivers
Measuring and monitoring systems
The software allows simple configuration and read-out of data from devices of the Energy Monitoring series.
Marking system
M-Print® PRO is software for printing labels and markers using our PrintJet, MCP and THP print systems.
The appropriate printer driver, manuals and installation instructions for all printing systems.
To the drivers and manuals
Analogue signal converter
For configuration and monitoring of the WAVERSERIES and the ACT20 series analog signal converter, there is a wide range of different software up to the FDT2 frame application called Wi-Manager and its corresponding ACT20 device type managers.
Industrial Ethernet
Software and firmware tool support the use of Industrial Ethernet active components.
To the software and firmware


text:
MD Anderson Events
National Healthcare Decisions Day
Employees, patients, caregivers and visitors are invited to learn more about advance directives for health care.
Date: 4/15/14, 11am to 1pm
Time: 4/15/14, 11am to 1pm
Location: Mid Campus Building 1, Floor 3, Lobby
Format: Community Event
Sponsor: Social Work
Contact: Stephen Collazo - (713) 792-0765 - email@example.com
Related Events
Contact Us
Questions? Need help?
Want to request an appointment?
Call askMDAnderson


text:
Energy snacks that actually taste good. Made with wholesome natural ingredients, created by "Powergirl" Nutritionist Em
Designed for all sports nutrition fuelling needs, especially before exercise and during longer sessions, and general healthy snacking for busy people
No added whey, soy or other protein additives. The modest protein content is naturally occurring, and appropriate for energy snacks
Cardio-protective and natural – coconut, real dark chocolate, sunflower oil, peanuts and almond
From low GI oats and a mixture of natural sugars (rice syrup, honey, dried fruits) and a kiss of brown sugar
Sustained or "low glycaemic index" (Low GI) natural energy
Latest News From Em's Blog
The Loop MTB TourIt started with a casual visit to Golden Bay to see our friends Martin and Marie who own the Quiet Revolution Bike Shop in Takaka, when Martin told us that on the 23rd April at exactly 10am, he and Andy were leaving on a little MTB tour from the Wholemeal Café in... read more
How to Open Em's Power Cookies
Do you find that opening Em's Power Cookies can be a bit challenging? The reason is seals are pretty burly which is important for food safety. You need to open them like a chippie packet. So without further ado, here is a short video to demonstrate!read more


text:
The more you do something, the more comfortable you become while doing it. So it follows that when you spend a lot of time skydiving, you'll dedicate less of your brain bandwidth to shrieking fear and more to testing out just what you can do with yourself during those blessed moments of freefall.
Jokke Sommer has more than a few jumps on his resume, and he's launched a "Freefly the World" tour to skydive in amazing places and hone his skills in the process. His first stop? Lauterbrunnen, Switzerland, with Petter Jönsson and Alex Aimard.
Watch incredible footage of their flights below:
Jumping out of a helicopter at over 13,000 feet of altitude, the team enjoyed 45 seconds of freefall before pulling their chutes to descend safely to the valley floor below. The sport they're practicing is known as "dynamic freeflying," in which the skydiver exerts extreme control over their body while it's moving at terminal velocity.
It was easy for Sommer to pick Lauterbrunnen as the first stop on his "Freefly the World" tour. "Lauterbrunnen is great because of the diverse mountain range – there are so many lines to fly, the weather is good and stable, and it's stunningly gorgeous," he says. "Easy access BASE jumps, heli jumps — it's a human flight mecca."
Crave more Red Bull? Sign up for RedBull.com's newsletter, a one-stop shop of our top stories, and like Red Bull Adventure on Facebook.


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Okay, if I can't find pro mix in my area, what do I mix together to
make my own potting soil for plants that will be going into pots.
I have quite a few that I want to do.
Vicky Cahanes
New Richmond, WI
zone 4a
To sign-off this list, send email to email@example.com with the
message text UNSUBSCRIBE HOSTA-OPEN


text:
Please sign me up to receive personalized deals in Sydney, shopping and travel.
By Signing up, I agree to the Terms of Use and Privacy Statement
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Enjoy yoghurts with flavours such as cookie dough, coconut, natural, matcha green tea, vegan chocolate, or choose from a range of smoothies
Multiple Locations


text:
September 28 2010 Tuesday at 08:51 AM
See Spot Stitched
before - boo hoo!
We all know what it's like: you're at home, drinking wine alone, spilling it all over yourself, trying to think up something constructive and conscientiously cool to craft. Well, why not embroider over that stain you just made? Or er...I made. Like so:
after - yay!
This project of mine and 999 others (not mine) can be found in Garth Johnson's fantastic, rapid-fire book of inspiration:


text: Land of Nod Robot Chair $47 Shipped $37.95 $129 + $8.95 Shipping See It! About this Deal This chair includes a cover and an insert. The matching ottoman also falls from $49 to $13.95. Shipping starts at $8.95 Flag this deal Edit deal What's the matter? Choose a reason (4) Dead deal Duplicate Bad link Spam Inaccurate No value Alive again 4 Comments


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Thermostat Project using an Arduino
This project was awarded to yhaat for $80 USD.Get free quotes for a project like this
Project BudgetN/A
Total Bids5
Project Description
Simple Arduino project creating a thermostat utilizing a thermistor. The link will give you an idea of what I'm looking for. I would like to replicate the project the link illustrates. I have all the materials needed for this project (ie thermistor, Arduino, breadboards, switches, resistors, relays. I just need someone to draw up a schematic for me to reference, as well as a step by step instruction on how to assemble the thermostat, including how to calibrate the thermistor if needed.
Here is a lay out of what I'm hiring for:
1. Schematic of thermostat. (Can be hand drawn, so long as it's readable).
2. Step by step assembly (simple terms like "place short wire from arduino analog slot # to bread board letter and number") This goes for every step including thermistor, resistors, switches, anything included. Basically write this out for someone who has little experience. Also, step by step instructions on calibrating the thermistor if needed. The instuction should go all the way up to uploading the code, and testing procedure.
From this I should be able to read your instructions and assemble the thermostat and test it myself.
The link below is a wonderful illustration of what I want, plus it gives the code for me to copy and paste, assuming the code works.
[url removed, login to view]
Awarded to:
Looking to make some money?
- Set your budget and the timeframe
- Outline your proposal
- Get paid for your work
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- Forbes
- The New York Times
- Time
- Wall Street Journal
- Times Online


text:
Lenox 810G 5pk 8" x 10 TPI ARC Style Gold Reciprocating Saw Blades
Lenox 810G 5pk 8" x 10 TPI ARC Style Gold Reciprocating Saw Blades
The 810G includes:
- 5x Lenox 810g 8" x 10 TPI ARC Style Gold Reciprocating Saw Blades
- Power arc curved blade delivers up to 2X longer life vs. traditional straight LENOX recip blades
- Aggressive blade design optimizes the blade's angle of attack for faster cutting in a variety of materials
- T2 technology and precision applied titanium coating help dissipate heat making blades last longer
- Titanium coating makes teeth more wear-resistant so they stay sharp for quicker cuts
- 8" length
- 10 teeth per inch
- Pack of 5 blades
The Lenox 810G Reciprocating Saw Blade is designed to be not only the fastest, but also the cleanest cutting blade on the market. Its power arc curve offers up 2x longer blade life than a traditional design. It has a titanium coating which makes teeth both more wear resistant and faster cutting, providing an all around more efficient blade.
- Specially formulated Titanium edge which improves the wear resistance of the teeth for faster cuts through the blade's life
- Bi-metal technology flexes, resists breaking and also extends the life of the blade
- Reinforced tooth design and Titanium coating combine to create the longest lasting blade, ever
Tech Specs:
- Brand: Bosch
- Model: 810G
- Type: Reciprocating Saw Blade
- Length: 8"
- Teeth Per Inch: 10
- Package Qty.: 5
Overall Rating for Lenox 810G 5pk 8" x 10 TPI ARC Style Gold Reciprocating Saw Blades
These blades last the longest of any blades we have tried. We use them to cut wood embedded with nails off pallets. Would not use any thing else.
Lenox Gold tip saw blades
By Bonnie Crouse from Delta pennsylvania on February 17, 2015
I would recommend this item to a friend.
Check out the rest of our...
Our Customers Are Saying...


text:
Department of Statistics Event Calendar
Department of Statistics Event Calendar
advanced search
Event Detail Information
Gene regulation network inference with joint sparse Gaussian graphical models (Purdue-UIUC Lecture Exchange)
Abstract: Revealing networks of biological components is one key objective in systems biology. With microarrays, researchers now routinely measure expression profiles at the genome level under various conditions, and, such data may be used to statistically infer gene regulation networks. Gaussian graphical models (GGMs) have proven useful for this purpose by modeling the Markovian dependence among genes. However, a single GGM may not be adequate to describe the potentially differing networks across these conditions, and hence it is more natural to infer multiple GGMs from such data. In the present study, we propose a class of nonconvex penalty functions aiming for the joint estimation of multiple GGMs. Our proposed penalty functions impose a joint sparsity constraint in a flexible way, which becomes effective regularization when all networks are sparse. We illustrate the property of our proposed nonconvex penalty functions by simulation study. We then apply the method to a gene expression data set from the GenCord Project, and show that our method can identify prominent pathways for different conditions.


text:
WORDPRESS site that need a basketballl STATISTICS function/plugin
This project received 21 bids from talented freelancers with an average bid price of $188 USD.Get free quotes for a project like this
Project BudgetN/A
Total Bids21
Project Description
I need a professional that is able to go into my WordPress admin back end and manage/finish/update a basketball league statistics plugin that records manages, and updates basketball statistic. I can assist during the way with help,direction, and advice. I have not been able to find a plugin that can effectivly do what I need.
I always pay on time. Check my profile.
Skills Required
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- Forbes
- The New York Times
- Time
- Wall Street Journal
- Times Online


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By Cristina Arreola | 10/24/2013 - 14:51
It was recently announced that Catalina Sandino Moreno will replace Rosario Dawson as the lead in the upcoming film Incarnate, a "micro-budget film" about an exorcist who meets his match in a 9-year-old boy under the influence of a demon.
Read more
By Cristina Arreola | 10/07/2013 - 15:44
George Lopez will be entering into an exciting new partnership with Pantelion Studios -- the company behind box office hit Instructions Not Included!
According to the Hollywood Reporter, Lopez signed a major deal with the company, and will be producting and starring in a series of upcoming movies.
Read more
By Cristina Arreola | 09/25/2013 - 11:10
The surprise hit of the summer? It's not a superhero flick or even a Holllywood production with a big name star.
Nope. It's Mexican comedy-drama Instructions Not Included that has taken the box office by storm.
Read more
By Jesus Trivino | 08/23/2013 - 13:13
Take a break from binge-watching Orange is the New Black and click on these flicks featuring Latino leads on Netflix. Thank us later!
Read more
By Damarys Ocaña Perez | 10/13/2011 - 15:53
Fall doesn't just mean changing leaves and chillier weather. It also marks the season for big, Oscar-contending movies. This year we have a variety of films, from comedies to epic dramas, starring Latinos. Here are some of the movies that we'll be checking out this fall!
Fall TV Calendar: Premiere Dates for 30 Shows Starring Latinos!
Read more


text:
- Be the first to comment
Taiwan small- to mid-size LCD panels – 4Q 2016
TAIWAN LCD PANEL | Feb 17, 15:02
The fourth quarter is usually the low season for the small- to medium-size LCD panel industry, but significant demand for feature phone applications boosted Taiwan's shipments in fourth-quarter 2016.
TAIWAN MOBILE DEVICE | Feb 14, 15:13
The notebook market has been showing signs of recovery, with Taiwan makers seeing their first on-year shipment growth since first-quarter 2012 in fourth-quarter 2016.
Taiwan large-size LCD panels - 4Q 2016
TAIWAN LCD PANEL | Feb 8, 15:58
Taiwan-based makers' large-size LCD panel shipments dropped 2.3% in 2016, worse than the average global decline of 1.1%, due to competition from China makers. Challenges from China competitors have been growing.
Global notebook shipment forecast, 2017 and beyond
RESEARCH EXTRAS | Jan 25, 14:34
Digitimes Research estimates that 2016 was another down year for the global notebook market, with shipments dipping below 150 million. While the market will continue shrinking in 2017, the drop will be much less dramatic, as Windows 10 adoption in the enterprise...
Global AP demand forecast, 2017-2020
RESEARCH EXTRAS | Jan 16, 12:01
Digitimes Research expects global application processor (AP) shipments to increase more than 8% in 2017 and surpass the 1.9 billion mark, with smartphones remaining the main application. After slumping in 2015, Qualcomm returned to form in 2016 and will continue...
China smartphone AP shipments to fall 16% in 1Q17
CHINA SMARTPHONE AP | Feb 17, 15:59
January shipments of Wintel notebooks down on month, up on year
RESEARCH EXTRAS | Feb 16, 22:21
LED, optoelectronics focus on automotive applications at Lighting Japan 2017
RESEARCH EXTRAS | Feb 15, 23:00
Taiwan makers ship nearly 313 million small- to medium-size TFT-LCD panels in 4Q16
TAIWAN LCD PANEL | Feb 14, 20:07
IR LED, laser LED widely used in range finding
RESEARCH EXTRAS | Feb 13, 14:26
All-in-one PC shipments to increase in 2017
RESEARCH EXTRAS | Feb 10, 15:38
Taiwan makers ship over 10 million LCD TVs in 4Q16
TAIWAN DISPLAY SYSTEM | Feb 9, 22:54
Taiwan makers ship over 33 million notebooks in 4Q16
TAIWAN MOBILE DEVICE | Feb 9, 22:47
Various robots showcased at RoboDEX 2017
RESEARCH EXTRAS | Feb 8, 11:11
China FTTx, mobile Internet-access user bases increase to 228 million, 941 million at end of 2016
RESEARCH EXTRAS | Feb 7, 14:42
124.2 million smartphones shipped in China market in 4Q16
CHINA SMARTPHONE | Feb 6, 21:00
Panasonic developing smart towns
RESEARCH EXTRAS | Feb 6, 16:01
China makers aggressively expanding AMOLED capacity
CHINA FPD | Feb 3, 16:04
Taiwan makers ship 312.94 million small- to medium-size TFT-LCD panels in 4Q16
TAIWAN LCD PANEL | Feb 3, 15:12
Taiwan makers ship over 61 million large-size TFT-LCD panels in 4Q16
TAIWAN LCD PANEL | Jan 25, 22:45
- Blogs
- Press releases
- China foundries planning FD-SOI process technology
- Foxconn subscribes to Lytro fund-raising project, says paper
- CHT ties up with NTT, Itochu to develop hardware specifications for SDN/NFV equipment
- Digitimes Research: January shipments of Wintel notebooks down on month, up on year
- Samsung unveils a new line of smart appliances at Samsung SEAO Forum 2017
- Equipment maker Foxsemicon to post record EPS for 2016
- Taipei City Government opens PV power-generating station on restored landfill site
- Eight-inch fabs operating at full capacity
- UltraChip posts 2016 EPS of NT$2.28
- Xintec swings to loss for 2016
- Leadtek Research steps into health care devices
- Digitimes Research: ADAS expected to become mainstream car-driving safety system
- Digitimes Research: NTT DoCoMo, KDDI, SoftBank step into power supply
- Philips Lighting boosting CityTouch
- VIS posts record 2016 profit
- AUO opens rooftop PV system in southern Taiwan
- MediaTek to lag in 10nm-chip race, says paper
- Coretronic nets NT$4.52 per share for 2016
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text:
With a smile you once told me, there was naught you could do
You pulled me into your arms, and whispered "I love you"
Ooooo oooooh ooooh you've got me on fire
Ooooo ooooh ooooooh ooooh you've got me on fire, remember when
That night by the river, you looked into my soul
I melted beneath your touch, lost all self control
Ooooh ooooh oooooh, you've got me on fire,
Ooooh oooooh ooooh oooh you've got me on fire, once again
Before this we were strangers, though I think we both knew
How our hearts and souls blurred into one, I cannot deny the truth
Now our lives are cast adrift, as I walk this lonely road
The depth I felt is never lost, I just wander here on my own
All these years I ached to know, to learn of what I'd lost
Now I see you in a window, gasp, my breath is caught
Years gone by without a word, though I hoped and I prayed
Looking back, I think you know, there was no other way
Ooooh oooh ooooh oooh, you've got me on fire,
Ooooh ooooh oooh ooooooh, I burn with desire, just like then
Ooooh oooh oooh oooh, you've got me on fire,
Oooh oooh oooh oooh, don't let me go, at the end
© Copyright 2017 Aerial90. All rights reserved.
Status: Finished
Genre: Romance
Poem / Poetry
Short Story / Humor
Short Story / Humor
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text:
Arabidopsisreplacement histone variant H3.3 occupies promoters of regulated genes
DOI: 10.1186/gb-2014-15-4-r62
© Shu et al.; licensee BioMed Central Ltd. 2014
Received: 8 January 2014
Accepted: 21 March 2014
Published: 21 March 2014
Histone variants establish structural and functional diversity of chromatin by affecting nucleosome stability and histone-protein interactions. H3.3 is an H3 histone variant that is incorporated into chromatin outside of S-phase in various eukaryotes. In animals, H3.3 is associated with active transcription and possibly maintenance of transcriptional memory. Plant H3 variants, which evolved independently of their animal counterparts, are much less well understood.
We profile the H3.3 distribution in Arabidopsis at mono-nucleosomal resolution using native chromatin immunoprecipitation. This results in the precise mapping of H3.3-containing nucleosomes, which are not only enriched in gene bodies as previously reported, but also at a subset of promoter regions and downstream of the 3′ ends of active genes. While H3.3 presence within transcribed regions is strongly associated with transcriptional activity, H3.3 at promoters is often independent of transcription. In particular, promoters with GA motifs carry H3.3 regardless of the gene expression levels. H3.3 on promoters of inactive genes is associated with H3K27me3 at gene bodies. In addition, H3.3-enriched plant promoters often contain RNA Pol II considerably upstream of the transcriptional start site. H3.3 and RNA Pol II are found on active as well as on inactive promoters and are enriched at strongly regulated genes.
In animals and plants, H3.3 organizes chromatin in transcribed regions and in promoters. The results suggest a function of H3.3 in transcriptional regulation and support a model that a single ancestral H3 evolved into H3 variants with similar sub-functionalization patterns in plants and animals.
Histones are abundant in most eukaryotic cells where they package DNA into chromatin. Dimers of histones H2A, H2B, H3 and H4 assemble into the histone octamer core to organize 147 bp of DNA into nucleosomes, the basic building blocks of chromatin. In recent years, much has been learned about how posttranslational modifications of histones affect chromatin, such as modifying inter-nucleosomal contacts or nucleosome stability. It is now also well established that incorporation of histone variants can result in formation of chromatin with particular properties [1–3]. In centromeric chromatin, for example, canonical H3 is replaced by the CenH3 variant forming tetrameric hemisomes instead of the conventional octameric nucleosomes [4]. Another H3 variant that was found in many eukaryotes is the histone replacement variant H3.3 [1]. In contrast to the canonical histone H3.1, incorporation of metazoan histone H3.3 into chromatin is mostly replication-independent [5]. The sequences of metazoan H3.1 and H3.3 differ only at position 31 in the amino-terminal tail and at positions 87 to 90 in the core histone fold [6]. Genome-wide profiling of H3.3 in Drosophila and mammalian cells revealed specific incorporation into the gene body of active genes, into promoter regions of both active and inactive genes, as well as into regulatory elements [7–10], supporting the idea that H3.3 has a role in transcription [11]. Unexpectedly, recent data have revealed H3.3 enrichment also at silent loci in pericentric heterochromatin and in telomeres [9, 12], and have shown a requirement of H3.3 for correct heterochromatin formation in mouse embryos [13]. H3.3 is incorporated into pericentric heterochromatin during S phase when pericentric repeats are transcribed; therefore, it was suggested to have a role in the initial formation of double stranded RNA-dependent heterochromatin [13]. Recently, mutations in an H3 replacement pathway were connected to pathogenesis of glioblastoma multiforme, a lethal brain tumor [14].
Replication-coupled and replication-independent (replacement) H3 histone variants evolved independently in animals, plants, basidiomycetes, and alveolates [15]. Similar to the replacement H3 variant H3.3 in animals, Arabidopsis H3.3 differs from H3.1 at positions 31, 87 and 90 but also at some additional positions [16]. There are three H3.3 genes in Arabidopsis (At4g40030, At4g40040, and At5g10980) [16, 17], which are expressed constitutively in a replication-independent manner [16, 18]. However, it is currently unknown if Arabidopsis H3.3 has the same epigenomic properties as the animal H3.3 variant. Recently, the genome-wide distribution of the Arabidopsis H3.3 protein At4g40040 was reported and, similar to animal H3.3, was preferentially found in the bodies of transcribed genes [19, 20]. In contrast to animal H3.3, however, plant H3.3 was not generally detected outside of gene bodies. Therefore, we decided to re-analyze H3.3 profiles using a protocol with increased sensitivity and mono-nucleosomal resolution. This protocol revealed H3.3 enrichment not only in gene bodies as previously reported but also at a subset of promoter regions and downstream of the 3′ ends of active genes. In particular, promoters containing GA motifs were targeted for H3.3 incorporation regardless of their activity. Our data suggest that the evolutionary constraints behind the evolution of animal and plant H3 histone variants are more general than previously assumed and may contribute to transcriptional regulation.
Histone H3.3 is targeted to euchromatin
To investigate H3.3 distribution at the nucleosome level, we located well-positioned single nucleosomes detected by either H3.3 or histone control ChIP by fitting the ChIP-chip signals to a parabola model (Figure 1C). Total detectable nucleosomes were combined from the two lists, resulting in 138,609 positioned single nucleosomes. To determine a H3.3 enrichment score for each nucleosome, we calculated the median of H3.3 ChIP-chip scores normalized to the control histone ChIP-chip scores in a window of 147 bp around the detected nucleosome centers. Similarly, we calculated the background noise level of each nucleosome using the median of a control IgG ChIP-chip score normalized to the histone ChIP-chip score. We then used two criteria to select H3.3 nucleosomes. First, the nucleosome H3.3 enrichment score had a higher than 75% probability of belonging to the specific component in a two-component mixture model (Figure 1D). Second, the nucleosome H3.3 enrichment score was higher than two times the background noise score. Under these high stringency criteria, we identified 28,220 H3.3 nucleosomes in the genome (Table S1 in Additional file 2), of which the majority (>99.6%) were located on the euchromatic chromosome arms and in close proximity to annotated genes. We associated H3.3 nucleosomes with the nearest genomic feature (that is, protein coding gene, pseudogene, transposable element gene, transposable element, microRNA, tRNA, non-coding RNA) if the distance did not exceed 2,000 bp. Using this criterion, 26,216 (92.9%) H3.3 nucleosomes were associated with genomic features. Out of all 20,381 genomic features that had closely associated H3.3 nucleosomes, 15,378 (75.5%) were protein coding genes, which is a 2.3-fold enrichment over random sampling (P-value = 2.21 × 10-11; two-tailed t-test; Figure 1E). All other genomic features were only rarely associated with H3.3 nucleosomes (Figure 1E).
H3.3 is enriched at promoters and around transcriptional termination sites in a transcription-dependent manner
Together, these results show that H3.3 deposition targets mainly promoters and the region around the TTS, and that the H3.3 levels in both regions positively correlate with transcriptional activity of genes.
H3.3 in promoters co-localizes with RNA Polymerase II
In Drosophila, H3.3-containg nucleosomes repackage DNA following the passage of elongating RNA Polymerase II (Pol II) during the transcription of genes [24], but H3.3-containg nucleosomes were also reported in promoters [10]. We hypothesized that in Arabidopsis the role of H3.3 in nucleosomes would also be coupled to Pol II activity. Indeed, we found that in Arabidopsis H3.3 co-localizes with Pol II in the nucleus (Figure 3C). At the gene level, we expected that only TTS-associated but not promoter-associated H3.3 would reflect Pol II presence. However, we found that H3.3 at promoters as well as around TTSs was associated with substantial Pol II binding (Figure 3D; Pol II data were from [23]), in particular on the subset of genes with H3.3 nucleosomes in their promoters. No considerable Pol II binding to promoter regions was observed for genes without H3.3 nucleosomes upstream of the TSS (Figure 3D). Although Pol II binding outside of transcribed regions is not well documented, a high-resolution study in Saccharomyces cerevisiae had reported a Pol II peak at -100 bp of the TSS for moderately expressed genes [25]. It should be noted that in Arabidopsis, Pol II occupancy at promoters was much lower than occupancy around TTSs. In contrast, H3.3 levels differed much less between these two positions (Figure 3B,D). These observations support the notion that H3.3 nucleosomes, both at promoters and around TTSs, are associated with the presence of Pol II in Arabidopsis.
H3.3 incorporation at promoters reflects strong transcriptional regulation
In budding yeast, Pol II was found upstream of inactive genes that could be rapidly activated upon exit from the stationary phase [27]. We asked whether the Arabidopsis genes with H3.3-enriched nucleosomes and Pol II at their promoters are strongly regulated. To test this hypothesis, we calculated for each gene an expression entropy using collections of Arabidopsis transcript profiling data [26, 28] (Figure 4B; Figure S6 in Additional file 1). Expression entropy is a measure for the extent of transcriptional regulation, with small values indicating a high extent of regulation. Consistent with our hypothesis, genes with H3.3 enriched at their promoters had significantly smaller expression entropies than the genome-wide median (P-value = 8.89 × 10-5, one-tail Wilcoxon test). In contrast, genes with H3.3 enriched only around the TTS had significantly larger expression entropies than the genome-wide median (P-value <2.2e-16, one-tail Wilcoxon test). Genes with H3.3 enriched at both promoters and around TTSs had reduced expression entropies, which were, however, much larger than those of genes with H3.3 enriched only at their promoters. These results support our hypothesis that genes with H3.3 enriched at their promoters are subject to strong transcriptional regulation.
H3.3 at promoters is independent of H2A.Z
In mammalian cells, DNA of active promoters is often bound by nucleosomes containing both H3.3 and H2A.Z, and it was suggested that combined incorporation of both histone variants could affect the access of transcription factors [10, 33]. We asked whether H3.3-enriched nucleosomes at promoters of Arabidopsis genes also coincided with H2A.Z. Contrary to the finding in mammalian cells, plant H2A.Z [34] is mostly enriched downstream of the TSS (Figure S8 in Additional file 1, black line), where H3.3 levels are low (Figure S8 in Additional file 1, red line). This non-overlapping localization of H3.3 and H2A.Z on different sides of the TSS is consistent with earlier observations of H3.3 depletion at sites enriched with H2A.Z [19, 20] and suggests that H3.3-H2A.Z-containing nucleosomes are not highly abundant at Arabidopsis gene promoters.
H3.3 at promoters negatively correlates with DNA methylation
Furthermore, genes with H3.3 enriched at promoters lack almost any mCG, especially in the -800 to -200 bp window where H3.3 is highest (Figure 6C). In contrast, mCG levels are elevated in the same window for promoters without H3.3 enrichment (Figure 6C). The difference in mCG levels in this window between the two groups of promoters is highly significant (genes with only promoter H3.3-enriched nucleosomes compared to genes with only TTS H3.3 nucleosomes, P-value = 1.46 × 10-10, Wilcoxon's rank test, one tail). Therefore, H3.3 and mCG appear to exclude each other at promoters.
In summary, H3.3 enrichment in nucleosomes does not strongly correlate with mCG in gene bodies and is negatively correlated with mCG at promoters.
GA promoters are targeted by H3.3
H3.3 nucleosomal DNA is more accessible
H3.3 is a histone variant that differs only in four or five amino acids from the canonical H3.1 but it can have profound effects on chromatin functionality. Earlier studies had suggested that animal H3.3-containing nucleosomes isolated from native chromatin are less stable in vitro[42] but more recent reports indicate that H3.3 per se does not affect stability of mononucleosomes [33]. Instead, animal H3.3 appears to mainly impair higher-order chromatin folding. Our finding that DNA flanking H3.3-containing nucleosomes in plant chromatin is much more accessible to DNase I than DNA flanking H3.3-free nucleosomes is consistent with the notion that H3.3 interferes with higher-order chromatin folding. In addition, H3.3 deposition, which disrupts chromatin, could directly result in increased accessibility of DNA at H3.3-enriched nucleosomes.
H3.3 incorporation is generally thought to be associated with the transcription initiation and/or elongation activities of Pol II in animals and is highest in gene bodies [7, 9, 11, 43–46]. We have used high-resolution mapping of the Arabidopsis replacement histone variant H3.3 and found that in plants H3.3 is located in gene bodies as well and shows a positive correlation with transcriptional activity, which is consistent with earlier reports [19, 20]. In contrast to the general consent on a positive correlation between transcriptional activity and H3.3 levels in animals, the actual distribution of H3.3 over the gene body remains controversial. Reported patterns of H3.3 distribution range from 5′-biased in Drosophila[7, 8] to 3′-biased in mammals [9, 10]. Although the choice of methods may have contributed to the reported H3.3 patterns in animals, the observed patterns could also reflect different nucleosome turnover rates during transcription elongation in different organisms or different cellular environments [7]. It is noteworthy that Pol II can transcribe through hexasomal nucleosomes in vitro after eviction of a single H2A/H2B dimer while the H3/H4 tetramer remains associated with the DNA [47, 48]. Complete dissociation of histone octamers from the DNA appears to be restricted to highly transcribed genes with multiple elongating Pol II molecules. Thus, transcription per se might not be sufficient to cause H3 replacement. This is consistent with the non-uniform and specific H3.3 patterns along gene bodies. Our results in Arabidopsis revealed a strong 3′ bias along gene bodies when examining plant H3.3 patterns by normalizing either to input or histone density, similar to earlier observations [19, 20]. Our data also show high histone density at the 5′ end and a sharp decrease towards the 3′ end in the gene bodies, demonstrating that low H3.3 levels at 5′ ends were not caused by local loss of nucleosomes during the chromatin preparation. Thus, H3 exchange in plants and animals appears not to be linked to Pol II passage per se but appears to be restricted to specific phases of the transcription process.
In animals, H3.3 levels correlate with transcriptionally active, Ser-5 phosphorylated Pol II, transcription initiation site-related mono- and tri-methylation of histone 3 lysine 4 (H3K4me1, H3K4me3), and transcription elongation-related tri-methylation of histone 3 lysine 36 (H3K36me3) [9]. In Arabidopsis, H3K4me3 is found proximal to TSSs [29] where H3.3 is mostly absent, suggesting that the functional relevance between H3.3 localization and Pol II transcription initiation is different between plants and animals. On the other hand, Arabidopsis H3.3 is highly enriched for H3K36me2 [49], the histone mark thought to be associated with transcription elongation in Arabidopsis[29]. This suggests a connection between H3.3 and elongating Pol II similar to the situation in animals. In addition, our finding in Arabidopsis that H3.3 localization extends considerably beyond the TTS indicates an even stronger connection between H3.3 and Pol II transcription termination.
In addition to gene bodies, we found H3.3 also in plant promoters. H3 replacement at active promoters has been reported for mammals [10], and it is frequent in yeast, where H3 replacement is found more often in promoters than in gene bodies [50–52]. Although yeast does not have separate H3.1 and H3.3 genes, it does have mechanisms for replication-independent H3 replacement [53]. However, H3 replacement at promoters in yeast is not strongly correlated with transcription initiation or Pol II promoter occupation [50–52]. In Arabidopsis, we found H3.3 at promoters of both active and inactive genes. It is possible that H3.3 is incorporated at promoters independently of transcription or that it is a footprint of past transcription activity of the gene. Since we could also find Pol II associated with H3.3-enriched promoters, it is possible that RNA Pol II promoter occupation or transcription caused local H3.3 incorporation. Indeed, transcription of promoter-associated short RNAs is more ubiquitous than initially thought during transcription activation [54, 55]. H3.3 insertion at promoters could also be a consequence of abortive rounds of transcription initiation that occur at repressed promoters [44] and can in turn poise the genes for transcription activation upon future induction [9]. Alternatively, H3.3 might be targeted to promoters by a transcription-independent mechanism as proposed for yeast [50–52] to facilitate binding of inactive Pol II to promoters of strongly regulated genes, such as genes that are activated upon exit of yeast from stationary phase [27]. The increased accessibility of DNA at H3.3-containing nucleosomes, likely reflecting reduced higher-order chromatin folding [33], suggests that the enrichment of H3.3 at promoters could allow easier access of transcription factors or the Pol II transcription initiation complex to the DNA template. Indeed, H3.3 incorporation can promote gene activation [46, 56] or prime genes for subsequent activation [33]. Our data revealed that GA motif-containing promoters are targeted by H3.3 even when repressed and that this preferential targeting coincided with higher expression dynamics of these genes. These observations implicate H3.3 in potentiating transcription activation in plants similar to the binding of inactive Pol II to promoters of regulated genes in yeast.
Animal and plant H3 variants evolved independently [15, 57], but H3.3 incorporation patterns in plants and animals and replication-independent H3 deposition in yeast [51] have many similarities. Replication-independent chromatin assembly is essential for life, but separate H3.1 and H3.3 variants appeared independently in animals and plants. The evolutionary history of histone genes is still a matter of debate [15], but it is likely that the ability to affect higher-order chromatin structure by incorporation of specific histone variants confers major selective advantages that facilitated the repeated diversification of histones. The similarity of Arabidopsis and animal H3.3 incorporation patterns is consistent with a general association of H3.3 with several eukaryotic chromatin remodeling processes. The presence of H3.3 on active as well as on many inactive plant promoters of strongly regulated genes suggests a function of H3.3 in transcriptional regulation.
Materials and methods
Plant material
All experiments used Arabidopsis (Arabidopsis thaliana) accession Columbia-0 plants. To produce 35S:H3.3-YFP lines the cDNA of HTR4 (At4g40030) was fused to the cauliflower mosaic virus (CaMV) 35S promoter at the amino terminus and the YFP cDNA sequence at the carboxyl terminus, and the fusion construct was inserted into the binary vector pCambia1380. Cloning and amplification of the plasmid was done in Escherichia coli DH5α. The plasmid was transformed into Agrobacterium tumefaciens (strain C58C1) and then transformed into Arabidopsis using the floral dip method. Transformants were selected on Murashige and Skoog medium agar plates containing hygromycin. Experimental plants were grown on soil at 21°C in dark (16 h) and 20°C in light (8 h). Plant age was recorded as days after imbibed seeds were sown on soil and transferred to the growth chamber. Leaves (leaf number 6 from about five plants per sample) were harvested after 35 days at zeitgeber time 7 (that is, 7 h after start of the photoperiod), and frozen in liquid nitrogen. Note that cell division and expansion had ceased at this developmental stage in the harvested leaves. The experiment was performed with three independent biological replicates.
RNA expression analysis and protein blots
Expression analysis of the H3.3 transgene was performed as described [22] using gene-specific primers and Universal Probe Libraries (Roche, Basel, Switzerland); Table S2 in Additional file 2) on an ABI Prism 7700 Sequence Detection system (Applied Biosystems, AB, Foster City, CA, USA). The experiment was performed in duplicates. Gene expression levels were normalized to PP2A.
For protein immunoblots, 50 mg of frozen 35S:H3.3-YFP seedlings were ground and the powder was extracted with Buffer M (10 mM Tris-(hydroxymethyl)-aminomethan pH 7.5; 0.5% IGEPAL CA 630; 1% Triton X-100; EDTA free protease inhibitor cocktail (Roche)) plus 150 mM NaCl for 10 minutes at 4°C. The suspension was centrifuged at 16,100 × g at 4°C for 10 minutes. The pellet was subsequently extracted using Buffer M containing 500 mM NaCl, centrifuged again, and extracted once more with Buffer M containing 2 M NaCl. Extracted proteins were separated using SDS-PAGE. Total protein was transferred to PVDF-membrane (Carl-Roth, Karlsruhe, Germany). The H3.3-YFP fusion protein was detected using anti-GFP antibody (mouse monoclonal, #11 814 460 001, Roche) and horseradish peroxidase (HRP)-coupled anti-mouse antibody (#115-035-003, Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK), and was visualized using Immun-Star HRP Substrate (Bio Rad, Berkeley, CA, USA).
Nuclei preparation, immunostaining and confocal microscopy
Seeds of the 35S:H3.3-YFP line were germinated and grown for 3 days in Petri dishes on wet filter paper. For visualizing nuclear DNA in live cells, 1 μM of DRAQ5 (eBioscience, Vienna, Austria) was applied to Arabidopsis roots for 5 to 10 minutes with vacuum to facilitate penetration. DRAQ5 stain and YFP signals in roots were consecutively analyzed using a Zeiss 710 confocal laser scanning microscope.
For immuno-staining, seedlings were fixed for 20 minutes with ice-cold 4% (w/v) paraformaldehyde in MTSB buffer (50 mM PIPES, 5 mM MgSO4, 5 mM EGTA, pH 6.9). Root tips were digested for 10 minutes at 37°C with a PCP enzyme mixture (2.5% pectinase, 2.5% cellulase Onozuka R-10, 2.5% Pectolyase Y-23 (w/v) dissolved in MTSB) and squashed in a drop of MTSB buffer. Immunostaining was performed as described [58]. H3.3-YFP was detected with rabbit anti-GFP (1:100; #A11122, Molecular Probes, Eugene, OR, USA) and donkey anti-rabbit Rhodamine (1:200; #31685, ThermoScientific, Waltham, MA, USA). Pol II was detected using mouse anti-Pol II (1:100; #ab817, Abcam, Cambridge, England) and goat anti-mouse Dylight488 (1:200; #35503, ThermoScientific). For confocal laser scanning microscopy, 35S:H3.3-YFP seedlings were grown on Murashige and Skoog medium for 5 days before YFP signals in roots were analyzed using a Zeiss 710 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).
ChIP-qPCR and ChIP-chip
Native ChIP was performed as described [59] with minor modifications. Crude nuclei extracts were produced by treating 100 mg of frozen leaf powder in Nuclei Extraction Buffer (NEB; 20 mM PIPES-KOH pH 7.6, 1 M hexylene glycol, 10 mM MgCl2, 0.1 mM EGTA, 15 mM NaCl, 60 mM KCl, 0.5% Triton-X, 5 mM β-mercaptoethanol and EDTA-free protease inhibitor cocktail (Roche)) for 15 minutes at 4°C. The homogenate was filtered through Miracloth (Calbiochem, Nottingham, UK), and a nuclei pellet was collected by centrifugation for 10 minutes at 1,500 × g at 4°C. Isolated nuclei were washed once in MNase buffer (50 mM Tris-HCl pH 8, 10 mM NaCl, 5 mM CaCl2, and EDTA-free protease inhibitor cocktail (Roche)), treated with 1.3 μl of RNase A, 30 μg/μl (Sigma-Aldrich, St. Louis, MO) and used for Micrococcal Nuclease (New England BioLabs, Ipswich, MA, USA) digestion for 4 minutes (final concentration 0.2 U/μl) in MNase buffer. The reaction was stopped with 10 mM EDTA. After a centrifugation the supernatant was collected as phase 1 chromatin preparation. The pellet was resuspended in buffer S2 (1 mM Tris-HCl pH 8, 0.2 mM EDTA, and EDTA-free protease inhibitor cocktail (Roche)) for 30 minutes. After centrifugation the supernatant was collected as phase 2 chromatin preparation. The two phases of chromatin preparations were combined and the NaCl concentration was adjusted to 50 mM. The majority of the chromatin was of mononucleosome size (data not shown). Histone H1 was depleted by incubating the chromatin preparation with Sephadex C25-CM resin (Pharmacia, Stockholm, Sweden) for 1 h at 4°C [60]. The Triton-X concentration in the mononucleosomal chromatin was adjusted to 0.1% followed by preclearing using non-immune rabbit IgG (see below) and Dynabeads Protein A (Invitrogen, Carlsbad, CA, USA). One tenth of the precleared mononucleosomal chromatin was kept as input control, and one-quarter was used for each immunoprecipitation with 2.5 μg antibody (MAB3422, monoclonal anti-histone antibody, Upstate/Millipore, Billerica, MA, USA; #A11122, polyclonal anti-GFP antibody, can also recognize YFP, Invitrogen; #I5006 non-immune rabbit IgG, reconstituted in H2O, Sigma-Aldrich) and collected with Dynabeads Protein A (Invitrogen). After washing, beads were re-suspended in TE buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA), and DNA was extracted using phenol-chloroform extraction and ethanol/salt precipitation. Cross-linked ChIP was performed as described [61]. ChIP was performed in biological triplicates.
qPCR was performed using the ChIP-recovered DNA as template using specific primers and probes (Table S1 in Additional file 2). Recovery for H3.3, histone and non-immune IgG was calculated relative to input signals. H3.3 enrichment was calculated using the anti-GFP immunoprecipitation signal normalized to the anti-histone signal.
DNA amplification was performed using the GenomePlex® Single Cell Whole Genome Amplification Kit (Sigma) followed by purification using MinElute PCR Purification kit (QIAGEN, Hilden, Germany).qPCR was performed for six genomic fragments before and after amplification to control for amplification bias (data not shown). Amplified ChIP DNA was fragmented, labeled and hybridized to Affymetrix AGRONOMICS1 Arabidopsis tiling arrays as described [62].
ChIP-chip data analysis
Background correction and normalization were performed as described previously [62]. ChIP-chip data were normalized using MAT [63] implemented in the Aroma.Affymetrix package [64] with the window size parameter set to 100. To detect nucleosomes, data were smoothed using the Savitzky-Golay method [65]. The properties of the Savitzky-Golay filter ensure that the area under each peak, the position of the extrema and the peak widths will not be changed. Numerical derivatives of smoothed ChIP-chip signals were analyzed to identify nucleosomes. Zeros of the first derivative indicate centers of nucleosomes, zeros of the second derivative indicate borders of nucleosomal peaks (Figure S9 in Additional file 1A). After locating the positions of nucleosomal peaks, we estimated peak height and width by least square fitting of each peak to a parabola, as a simplest suitable analytical shape (Figure S9B in Additional file 1). Estimated peak widths had a pronounced maximum at approximately 150 bp, demonstrating that our approach mainly identified signals of nucleosome size (Figure S9C in Additional file 1). The workflow was organized using the Python programming language; all other analysis was performed in R [66]. Deconvolution of the nucleosome H3.3 incorporation scores was done using the MCLUST package [67]. H3.3 enrichment was calculated by normalizing H3.3-YFP ChIP-chip data to histone ChIP-chip data, while H3.3 density was calculated by normalizing H3.3-YFP ChIP-chip data to input data. Visualization of tiling array data was done using the Integrated Genome Browser [68]. H2A.Z data were from [34]. H3K36me2 and H3K27me3 data were from [29]. Pol II data were from [23]. Expression data from leaves were from [22], and expression data from different organs and developmental stages were from [26]. P-values were calculated using Wilcoxon's signed rank test.
Data availability
Supplementary raw data are available in ArrayExpress [69], accession number E-MTAB-1685. Pol II data [23] are available in the Gene Expression Omnibus [70], accession number GSE21673.
- bp:
base pair
- ChIP:
chrommatin immunoprecipitation
- MNase:
micrococcal nuclease
polymerase chain reaction
- Pol II:
RNA Polymerase II
- qPCR:
quantitative PCR
transcription start site
transcription termination site
yellow fluorescence protein.
We thank Yana V Bernatavichute for sharing her native ChIP protocol, Jonathan Seguin and Benjamin Knoerlein for helping with the bioinformatics analysis, and Xiaochun Fan for sharing his experience for analyzing nucleosome density profiling data. We thank the Functional Genomics Centre Zurich for microarray hybridization and scanning. This work was supported by the Sixth Framework Program of the European Commission through the AGRON-OMICS Integrated Project (grant number LSHG-CT-2006-037704), by the Swiss National Science Foundation, and by grants from the Knut and Alice Wallenberg Foundation as well as the Swedish Research Councils VR and FORMAS.
Authors' Affiliations
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text: The Sunday Poll – What's Your Poison? Posted on February 3, 2013 by Zafar Khurshid We all like a drink everyone once in a while, so when you walk up to the bar after a long day, what do you choose? Take Our Poll Advertisements Share this:TwitterFacebookTumblrEmailPinterestGoogleRedditLike this:Like Loading... Related


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Matt Barkley appeared on the verge of greatness for much of the year but never quite got to that point. Still, the 2010 season has to be deemed a success for the sophomore, who really only had two bad games -- Oregon and Oregon State. Robert Woods also exploded onto the scene and is likely to be the Trojans' top receiver for at least the next two years.
The complete surprise: Marc Tyler. The outright disappointment: Dillon Baxter. Allen Bradford and C.J. Gable fall somewhere in between. Tyler was an afterthought heading into training camp but seized the job when Bradford got hurt and is on the verge of 1000 yards; Bradford had a couple great mid-season games but got only six carries in November.
The offensive line, as Coach Lane Kiffin pointed out early on, was very up and down -- with some horrible games (Oregon State, Virginia) and some terrific games (Cal, Arizona State). On defense, Jurrell Casey was maybe USC's best overall player and Wes Horton was better than expected, but Nick Perry and Armond Armstead have to be considered 2010 disappointments.
The biggest problem with this year's USC team was undoubtedly the defense, which seemed to get it in gear some toward the end of the year but struggled mightily early in the year against teams like Washington. The secondary was expected to be a problem; what really ended up hurting USC was a linebacking corps that badly unperformed.
Special teams coach John Baxter obviously did a lot of good in his first year with the Trojans, but this unit was hurt by not having a reliable kicker early on. The Washington game, in particular, would likely have had a different outcome. But blocked kicks and solid returns from Woods and Ronald Johnson did become USC's specialty.
Kiffin was more different from Pete Carroll than we realized coming in, as he proved entirely unafraid of slamming his players after a crucial mistake. That approach did some good and some bad, but you can positively say this about Kiffin's first year: In large part, his team appeared ready to play every Saturday.


text:
I smiled at Slobby. Slobby was my ex- boyfriend. I called him Slobby because when we use to live together he was a slob. I enjoyed the mess. It was funny to see the mess. I miss him. I don't remember why we broke up. Slobby didn't smile back though, he just turned and walked away.
"Slobby wait!" I shouted.
"Don't call me that." Slobby said.
"Slob...uh...Michael what did I ever do to you?"
"You should remember because it was your fault."
"I know, but that's the only thing I do remember."
"Come on Betty, don't lie."
"Just please tell me."
"Fine, it was at the end of last year. You were saying how much you loved college. I was smiling and listening, even though I didn't really care. I nodded and you asked me something. You asked me if I was cheating on you. I thought what a dumb question. I would never cheat, and then you accused me of doing such a thing. That's cold. That's why I broke up with you."
"Oh. I made a mistake. Can't we just forget it?"
"I don't think I will ever forget. I pretty much say it every night."
"I'm sorry! Please. I still love you."
"You should have thought about that before you accused me of cheating on you. Anyway, why would I cheat on my girlfriend?"
"I don't know, I can't remember what I was thinking."
"No I am talking about now! Why would I cheat on Melissa?"
"My girlfriend!"
"Just forget about me and go out with someone else."
"Sorry for bothering you. I guess I won't talk about this ever again."
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The controversial judge James Pickles has died aged 85, his son said yesterday.
Judge Pickles, from Halifax, rose to fame in 1985 when he criticised what he saw as government pressure on the judiciary to keep people out of prison.
He also once described a Lord Chief Justice, Lord Lane, as an "ancient dinosaur" and jailed a woman said to be too frightened to give evidence against a man accused of assaulting her.
He retired as a judge in 1990 and became a tabloid columnist.
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Dealers and Distributors
Our weekly roundup of dealers and distributors includes news from Tallon Marine and CWR Electronics. Tallon Marine, the New Zealand-based manufacturer of the integrated accessory systems, appointed Tides Marine of Deerfield Beach, Fla., to distribute its receiver platforms and accessories across the United States. Tides Marine is showcasing Tallon's products this week at the Marine…more


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dream teamnoun [ C usually singular ]uk /ˈdriːm ˌtiːm/ us /ˈdriːm ˌtiːm/
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(Definition of "dream team" from the Cambridge Advanced Learner's Dictionary & Thesaurus © Cambridge University Press)
(Definition of "dream team" from the Cambridge Business English Dictionary © Cambridge University Press)
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to (cause someone to) be in a position in which the lower part of the body is resting on a seat or other type of support, with the upper part of the body vertical


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Distances are calculated in a straight line and may not reflect actual travel distance.
Please welcome to the market this brilliant 4 bedroom detached bungalow set within a highly sought after location.
The home itself comprises of an entrance hall, 4 well proportioned bedrooms, living room, modern fitted kitchen and orangery.
Externally the property benefits from a generous plot providing ample off road parking to the front and landscaped garden to the rear.
Internal inspection is a must!
Contact branch for relevant Energy Performance Certificate
Radiator, patio doors leading to Orangery
Double glazed window, modern fitted kitchen, range of base and wall units with complementary work surfaces and built in appliances
Double glazed windows, patio doors
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Below is a sample of the current (4-2014) version of the standard San Francisco Association of Realtors Contract for the Purchase and Sale of Real Property. The contract is, of course, the foundation of the transaction between buyer and seller, and how it is written – how the price and terms are delineated – is of critical importance. There are a number of decisions regarding conditions of sale, due diligence, arbitration, liquidated damages, close of escrow date and so on that will have to be made by the buyer in their offer and ultimately agreed to by the seller, though the final agreement may include changes made in counter offers.
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Locksmith Catonsville
Locks & Locksmith
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