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LocationThurmont, MD
11109 Angleberger Rd.
Thurmont, MD 21788
New 2017 Little Guy Worldwide Little Guy 5 Wide Basic
Inside you will find cabinets for your things, and lights. Each door features a slider window with screen. There is a large roof vent for adequate air flow, and aluminum alloy wheels, plus aluminum fenders as well.
Depending on the package you choose, there is the Basic and Max, you will enjoy additional features and/or options. Some of the items in these packages include a sink, stove, or combo in the rear hatch, a stargazer window, 19" entertainment center, spare tire undermount, plus so much more! Check each package for more details.
We've owned our RV for 3 months & we've traveled 5 times already with 3 boys. This is an adventurous way to spend quality family time together, create amazing long lasting memories, and meet great people along the way. On the scare of 10 of satisfaction, we rate it as 11. - Brandon from FINKSBURG, MD
Stock# 34632
|12 ft 9 in
|Ext Width
|6 ft 6 in
|Ext Height
|5 ft 8 in
|Int Height
|3 ft 6 in
|Hitch Weight
|90 lbs
|Dry Weight
|900 lbs
Teardrop camper trailers evolved after World War II, and were originally constructed with materials obtained from surplus markets. The chassis were made of steel U channel or from round steel tubing, and the wheels came fromJeeps that were salvaged from sunken ships. The exterior skins were usually made from the wings of World War II bombers.While a lot has changed since the introduction of teardrop trailers on the American highway, the reasons for owning a teardrop trailer are the same today as they were when your father or grandfather built his teardrop in the family garage.Teardrop trailers are sleek, aerodynamic, and practical. Depending on the model, your teardrop trailer can sleep up to three adults or two adults and several children. Little Guy teardrop trailers cab be pulled by any vehicle with a hitch—even the smallest of vehicles such as the Mini Cooper or the VW Beetle can easily tow a Little Guy teardrop trailer.Little Guy teardrop trailers are the best selling and most popular teardrop trailers in the world, a reflection of our company's commitment to producing a high-quality, innovative line of products.Whether you're a weekend camper who enjoys spending time with the family, or an avid outdoorsman who hunts, fishes, or rides a four wheeler—we have the Little Guy trailer that will suit your needs. We even sell special trailers just for motorcycles. Need a customized trailer for a particular purpose? We can assist you in your customization efforts, too.Discover the world of teardrop trailers, and you'll find that less really is more. Our trailers come equipped with double, queen, or king size mattresses. Little Guy teardrop trailers come with a galley and can even be equipped with a side mounted gas grill and beverage dispenser—the Little Guy Tailgating Package.You'll find ample overhead storage cabinets inside your little guy teardrop trailer, and you can add a room with our accessory Little Guy snap-on tent. Need to carry bikes, coolers, or other supplies? Not a problem with the platform model, which has a metal platform on the front of the trailer. The Sport models even come with ramps and a platform for loading and transporting your all terrain vehicle.Little Guy Worldwide carries a teardrop trailer for just about every application. Bands, sports teams, outdoor enthusiasts, and animal lovers alike are discovering the wonderful world of teardrop trailers. Please take a little bit of time to explore our website. Should you have any questions, please do not hesitate to pickup the telephone, request an online chat or an immediate callback, or use one of our online forms to submit a question.
Beckleys RVs is not responsible for any misprints, typos, or errors found in our website pages. Any price listed excludes sales tax, registration tags, and delivery fees. All prices may not include Beckley's Camping Center's 145 point pre-delivery inspection. Manufacturer-provided pictures, specifications and features may be used as needed. Inventory shown may be only a partial listing of the entire inventory. Please contact us at 800-615-6171 for availability as our inventory changes rapidly. All calculated payments are an estimate only and do not constitute a commitment that financing or a specific interest rate or term is available.


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Does this restaurant offer takeout or food to go?
Does this restaurant have waiters and waitresses?
Is this an Asian restaurant?
Is this a Chinese restaurant?
Does this restaurant have tables with seating?
Is this restaurant appropriate for Kids?
Does this restaurant specialize in Cantonese food?
Does this restaurant serve alcohol?
Does this restaurant offer delivery?
Does this restaurant accept reservations?
Thanks for helping!
Go for lunch, the all you can eat buffet is super.
Everything is fresh and very tasty - crab rangoon, chicken wings, dumplings, beef and chicken dishes etc,
Also love the egg drop soup.
The food is very good. The staff is attentive and it's an adult atmosphere where you can relax. It's not loud and if Sonny is working you'll enjoy the experience. If you don't go that's fine you won't take my seat.
We have frequented the Chen-du restaurant many times for both take out and dining in. The quality of the food and service has always been excellent and they offer an extensive gluten free menu.
Go and enjoy an excellent lunch or dinner.
After evening events my friends and I usually head here for a drink and apps. The wait staff know us and greet us with big hugs. They know our usual order as well. The service is fast and everything is tasty and hot. Drinks are made well. We will continue to return regularly.
Great drinks and best dry sautéed wings evah!!! Sonny, Adam, & Jade very customer focused. Best mai tai's I've ever had.
We were looking for a local place for lunch on Saturday that we hadn't yet been to. Cheng-Du came up on a search so we thought we'd give it a try. This is a nice store front type place on Main Street in Mansfield MA. It's a little on the smallish side, clean, and appears well maintained.
My family of...
It is a quaint little restaurant and nothing exotic or extravagant about the interiors. Service is decent, nothing to rave about but the food is terrific for a Chinese restaurant. Ordered a hot and sour soup and was fantastic, in USA you rarely get a good hot and sour soup but this restaurant is an exception. Ordered a chicken dish...
They have a little bar. Don't go there for a drink, not unless you are rich because they are expensive as hell. They Chinese food is okay. I know a couple of the Mexican/Guatamalan cooks who work there.
But I have to admit the the bartenders were friendly.
We had gone to Sharon to pick up some Bidding for Good items and one of them was a gift card to Chengdu. So we there on the way home. Everything on the menu seemed to be offered at the all you can eat luncheon buffet so we ordered two buffets. There was plenty to chose from: green beans in...
I used to live in the next town, Norton. Cheng-du represents the best of a local Bar/Restaurant-Chinese of course and a fine restaurant with excellent food. Complete with a warm friendly and accomplished bartender. The Owner 'Sonny' is really hands on when it comes to anything and is very well supported by a very able staff.
The food is exceptional...
Own or manage this property? Claim your listing for free to respond to reviews, update your profile and much more.

subset: commoncrawl


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Your weekly spin through the non-revenue sports at Maryland.
Field hockey
The No. 2-ranked Terrapins improved to 8-0 with two victories last weekend, a 4-1 win over No. 15 Wake Forest on Friday and a 2-0 win over Michigan on Sunday. Jill Witmer, Anna Dessoye, Ali McEvoy and Katie Gerzabek scored against the Demon Deacons, and goalkeeper Natalie Hunter and Maryland's defense kept the Wolverines off the board, even with the Terrapins down a player because of yellow card accumulation.
Witmer was named Terp of the Week. She will miss this weekend's games — Maryland plays at No. 17 Boston College on Friday at 7 p.m., and then at No. 7 Massachusetts on Sunday at 1 p.m. — because she is in Mendoza, Argentina, competing with the U.S. national team at the Pan American Games.
Men's soccer
Senior Patrick Mullins scored two penalty-kick goals as visiting Maryland defeated Clemson, 3-1, on Saturday night. The Terrapins followed that up with a 1-1 home draw with Old Dominion on Tuesday.
Maryland (2-2-3, 2-0-1 ACC), ranked 14th by Soccer America, hosts Pittsburgh on Friday night.
Women's soccer
Maryland opened ACC play on Sept. 19 with a 2-0 loss to Virginia Tech, but the Terps bounced by defeating Wake Forest, 1-0, on Sunday, handing the Demon Deacons their first loss of the season. Senior Hayley Brock scored the lone goal of the Wake Forest match in the fourth minute.
The 22nd-ranked Terrapins travel to No. 3 Notre Dame on Thursday night at 7 and then visit top-ranked Virginia on Sunday at 2.
The Terrapins dropped matches to Ohio, Michigan and Marshall at last weekend's Michigan Invitational in Ann Arbor. Maryland (8-4) hosts Georgia Tech on Friday night at 7 and Clemson on Sunday at 2. The match against the Tigers will be streamed on TerpsTV.


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COCINET Workshop Berlin
Co-op City NETwork (COCINET) Workshop
January 26-28, 2015
TU Berlin, Habitat Unit
Following the three regional COCINET-workshops in Dar es Salaam, Rio and Bengaluru, Berlin is host to three-day international COCINET workshop.
The question of affordable housing for low-income groups has been recognized as one of the key challenges in the global discussion on the Sustainable Development Goals. Many governments have already responded through new mass housing programmes, but due to bureaucratic, managerial, financial or contextual inadequacies, the new housing packages, if delivered, so far often fail to build adequately diverse, adaptable live-work environments which match the demands and resources of the end-users.
Emerging from a belief that the possibility of sustainable affordable housing solutions lies in combining the State's resources and delivery capacities with the end-user's "resources, skills and expertise", we would collectively attempt to imagine a transformative global knowledge network that could promote such user-driven housing solutions.
The aim is to find common ground in our approach to strengthening self-built housing by imagining the structure and workings of a network on this theme, and discussing the aims and objectives of the full funding bid to the ISSC.
Participating experts are:
Gautam Bahn, IIHS Bengaluru
Tile von Damm, MOD Institute
Rüdiger Haum, WBGU
Rainer Hehl, TU Berlin
Marie Huchzermeyer, University of the Witwatersrand Johannesburg
Gerhard Kienast, University Kassel
Rachel Lee, Habitat Unit TU Berlin
Grace Lubaale, Kenya Urban Sector at UN HABITAT
Philipp Misselwitz, Habitat Unit TU Berlin
Matt Nohn, Harvard University
Malin Praktiknjo, Habitat Unit TU Berlin
Oliver Schetter, Habitat Unit TU Berlin
AbdouMaliq Simone, Max Planck Institute for the Study of Religious and Ethnic Diversity
Jörg Stollmann, TU Berlin
Klaus Täschner, Habitat Initiative Germany
Carsten Zehner, Consultant and Trainer for Urban Development, Planning and Management, Berlin
Habitat Unit at TU Berlin in cooperation with MOD Institut have sucessfully gained a seed grant from ISSC in October 2014.
For more informations please contact MOD Institute.
About MOD
We work independently and in collaboration with an extended network of individuals and organisations to visualise urban processes and challenges so as to inform participation and intervention.
The content of this website and all downloads are licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License.
von Damm / Ewald / Fenk (eds.): People's Vision on Future Shanthinagar; 2015
People's Vision on Future Shanthinagar Vision Map; 2015
Fenk: (Not) Affordable Housing; 2015
Fenk / Königstein: Where is the knowledge we have lost in information?; in dérive No 56: Smart Cities, 2014
von Damm / Ewald / Fenk / Lynen: Crowdsourcing the city. Nextbangalore Newspaper; 2014
The Nextbangalore Change Map; 2014
The Nextbangalore Change Map Booklet; 2014
Fenk / von Damm: Nextbangalore; in Revue Magazin, 2013
Lee: Marg Magazine: A Tryst with Architectural Modernity; in ABE Journal, 2013
Fenk / von Damm: Bangalore - Dimensions of a City; in: The Law of the Market, 2012
Fenk / von Damm: Was ist Stadt? Fragen an den indischen Urbanismusdiskurs; in: Gegenworte 26, 2011
Narasimhan / Fenk / Chattapadhyay: Space is the Yantra; in Urban Design 119, 2011


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Astro is the fruit vape you've been looking for. A tantalizing granny smith apple on the inhale with strawberries on the exhale.
Primary Flavors: Granny Smith Apple, Strawberries
Bottle Size: 30ml
Posted by Justin S. on 21st Apr 2015
I liked this juice. I am a apple fanatic and I've tasted most apple e-juices and this one is defenately one of the good ones. This found its way into my all day vape collection. Neither dominant flavor is overpowering, the apple and the strawberry come together to make a single layer, it's very tasty. No medicine flavors like many apple flavors have in them.


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Chat with Bill Williamson
Welcome to SportsNation! On Thursday, ESPN.com AFC West blogger Bill Williamson will stop by to talk about the NFL.
Williamson joined ESPN.com in 2008 after four seasons covering the Denver Broncos for the Denver Post. He previously covered the Minnesota Vikings, Green Bay Packers and Seattle Seahawks.
Send your questions now and join Williamson Thursday at 2 p.m. ET!
More Bill Williamson: Columns | AFC West blog | Chat | NFL index
Caleb (Denver)
Would Tony Dungy be a good fit in Denver?
Bill Williamson (2:03 PM)
Too much money and I don't think he'd want the job.
Jake (Scottdale, PA)
Jason Campbell or Bruce Gradkowski? Michael Bush Or Darren McFadden?
Bill Williamson (2:03 PM)
Jury is out on Campbell and I like the McFadden-Bush combo. It was brilliant at San Diego.
Ryan (NJ)
Hey BW, do you think the Broncos will try to hire a coach who won't blow up their current offense?
Bill Williamson (2:04 PM)
You mean like the last guy did?
Keith (New Jersey)
could you see the raiders going after somebody in the offseason like Vince Young for quarterback? would he even be a good fit in the raiders offense?
Bill Williamson (2:05 PM)
They could very well be in the market for a QB and Young very well could be available ... so, yes, I could see it. I don't love the idea, though.
Chris (Lees Summit, MO)
What do you think the Chiefs chances of winning this weekend are with the possibility of Brodie Croyle starting at QB?
Bill Williamson (2:07 PM)
If that's the case, the Chiefs need to run very well. It's dififuclt to expect a QB to win after not playign for mroe than year. So, the run game better in gear.
Chris (Lees Summit, MO)
What do you think the chances are that the Chiefs make the playoffs?
Bill Williamson (2:08 PM)
Right now, they are very strong. They are in good shape. Just have to keep running.
Lars (KC)
If the Chiefs make the playoffs, what do you think of thier chances? They have the best running game in the NFL seems to me that would bode well for the cold playoffs.
Bill Williamson (2:10 PM)
They way they run and the way win at home, I'd like their chances.
Keith (New Jersey)
what do you see with the progress of bruce campbell? has he just not gotten chance to play or is he still working out the kinks?
Bill Williamson (2:12 PM)
I think he's a fourth-round pick who is still developing. He's taking some time, but that OK.
Chuck (Kansas City)
What is your take on Matt Cassel playing this weekend?
Bill Williamson (2:14 PM)
The Kansas City Star talked to an expert in the medical field, who said he may be able to play. I think if he doesn't play, he'll know by Friday, meaning, if he's not feeling well they'll know early. This is just a guess, but I think he'll try to play.
Scott (Denver)
True/False: Jammal Charles will be a top 4 RB next year. Moreno will be a top 15.
Jared L (Eugene)
How does everything happening in Denver right now effect Champ Bailey's contract? Hard to pay him all that money when they know they're gonna be rebuilding, wouldn't you think?
Bill Williamson (2:18 PM)
Sure, why not ... I think it affects everything. Clearly, the team won't want to sign him until they get new leadership on baord and Bailey's patience may run out. I could see him leaving, which would be the end of an era.
Bruce (Beaumont)
Do you think an AFC west team could get into the conference title? With the Pats, Jets, Ravens, and Steelers being the best teams in the AFC currently.
Bill Williamson (2:22 PM)
It will be tough. It would likely mean a big road win in the Divisional round. But we'll 0ahve to wait to see the matchups before delving into the chances.
Scott (Denver)
When you're 3-10, I don't think the offense is untouchable.
Bill Williamson (2:22 PM)
Can't disagree.
Alexander (Hayward)
What happened to all of your talk about Gradkowski being a "spark"? Other than that ONE Rams game I have not noticed him being much of anything, your thoughts?
Bill Williamson (2:24 PM)
You can't deny he gave the tema a spark. But I'm not a big Gradkowski guy in the big picture and I think he is a guy you always try to replace. He's too limited and too injury prone to be a long-term starter. But you can't say, he wasn't a sparkplug at times. He's a solid bakcup.
Richard (Encinitas, CA)
Do you think Matt Cassel will play this week? any up dates on his status?
Bill Williamson (2:26 PM)
No updates yet. Adam Schefter ust said on SportsCenter that it may be a stretch for him to play. I think we'll know more tomorrow. I'd be surprised if we had an answer today.
Clifford (Mission)
In your opinion who do you think the Broncos should hire as GM or Coach?...Would you hire a GM first and let him hire the coach?
Bill Williamson (2:27 PM)
I say hire a GM and then a coach. That's the way the league is working these days.
Dave S (Kingfisher)
What is your opinion on Coach Stoops out of OU as the new guy in Denver?
Bill Williamson (2:28 PM)
Bowlen is a OU guy and Stoops was an early candidate two years ago ... But in the end, I'd bet it's not Stoops.
Jared L (Eugene)
If Hue Jackson gets head coaching offers, what do the Raiders do?- Who's more valuable to them, Cable or Jackson?
Bill Williamson (2:30 PM)
Interesting question. The offense is better this year. I'm not sure what would happen there if Jackson gets interest, espcially if it's from Denver. I know Al Davis loves Jackson ... and we know he doesn't love Denver.
Bill Williamson (2:30 PM)
Interesting question. The offense is better this year. I'm not sure what would happen there if Jackson gets interest, espcially if it's from Denver. I know Al Davis loves Jackson ... and we know he doesn't love Denver.
65-TossPowerTrap (Leawood, KS)
Do you think the Chiefs may have used too much McCluster too fast in his first game back from missing 5 weeks last week. He was used as a slot receiver, kick returner and running back, and though he had a 60+ yard reverse called back on penalty and had some nice receptions, he also fumbled as a back and nearly got re injured. With Jones and Charles, do you think McCluster should be limited to receiver and kick returner?
Bill Williamson (2:31 PM)
No, I think he is a guy you unleash.
Nathan Cossey (Grandview, MO)
IF Cassel makes the game Sunday how do you think the Chiefs will do?
Bill Williamson (2:32 PM)
Depends on how effective he is.
Richard (Encinitas, CA)
Which game are you going to this week and why?
Bill Williamson (2:32 PM)
KC-SD .... Do Ineed to say why? This is a biggie.
Kyle (Fresno, CA)
Hey Bill, the Raiders have had trouble in their two previous early start game against Tennessee and Pittsburg. Do you think this week will be different against JAX?
Bill Williamson (2:36 PM)
Well, they've trying to address it by practicing early. That's all they can do.
Brodie (Kansas City, MO)
Bill, will you show some love after I put up 2 TD and over 200 yds passing this weekend against the Chargers and take a commanding 3 game lead over them?
Bill Williamson (2:39 PM)
You got it.
Perry (Rochester, NY)
Bill...realistically... do you believe the Bolts will ever win a championship as long as Norv's our coach?? He seems to be an offensive guru, but not really a true "leader of men"...
Bill Williamson (2:40 PM)
I'm not ready to close the door on Turner. This is a good team and it will be a good team next year regardless what happens in the next four weeks.
Marty (Salem, OR)
How do you like the Raiders chances this weekend in J-Ville? and do you think they are capable of running the table?
Bill Williamson (2:43 PM)
Other than the Denver game, they have some tough games. But if the Raiders show up and play hard, they can win all four games. But they have to show up.
jesse (las vegas)
bill, if the chiefs lose this week and the raiders win , what do you think the chances are that it comes down to week 17 winner takes all
Bill Williamson (2:45 PM)
There would still be some things that would have to happen -- Week 16 would be huge for Oakland at home against the Colts. It's still a long shot, but boy, would that be a fun ending to the season or what?
ken (charlotte)
i think it is a travesty that raiders will not make the playoffs but will more than likely sweep the division, what do you think?
Bill Williamson (2:47 PM)
A travesty?It's all about wining the most games. Do better against the rest of the AFC if you want to make the playoffs.A travesty?
D. Bowe (WR Heaven)
Will I catch a ball let alone a TD this weekend?
Bill Williamson (2:48 PM)
I'd think so.
Justin (Kansas City)
In your opinion, how has Glenn Dorsey progressed since being drafted?
Bill Williamson (2:50 PM)
He's come along well this season. I'm not sure he'll be the constant game changer he wa sin college, but he's becoming a plus-player.
Josh (Michigan)
How long before the NFL outlaws tackling?
Bill Williamson (2:52 PM)
Maybe next week.
josh (KC)
Croyle has never had the talent around him as he does now in KC. The last gane he started he was efficient...which is what the Offense is about in KC.....With that bveing said do you see the Game Plan changing all that much if Cassel can not play. Also Croyle as twice there arm as Cassel if only he can take a hit KC should be fine...your thoughts?
Bill Williamson (2:53 PM)
I think the key for Croyle will be rust and health. Can he make it through the game? You don't want Tyler Palko out there.
Bruce (Oakland)
Do you see the Raiders 7-9 when the smoke clears? They have several big ones left. Can you point to the game you would consider the big one for the Raiders?
Bill Williamson (2:54 PM)
This is the big one and if they win it, then the next won it the big one. The Raiders have to keep winning.
Tod (Arizona)
Why wouldn't Gruden be a great fit in Denver? He gets to play against Al twice a year, I think he likes Tebow. If he could bring the Glory years back to Denver he'd be almost as big a hero as Elway!
Bill Williamson (2:54 PM)
Ryan (NJ)
BW, have you heard if Denver has any interest in making sure they keep the 3-4 for next year?
Bill Williamson (2:55 PM)
It all depends on the new coach.
Albert (Otay, CA )
Do you think the Chargers make playoffs?
Bill Williamson (2:57 PM)
They put themselves in a bad, bad spot last week. But if they beat the Chiefs and the Raiders lose at Jax, they be back in business. deal is changing every week. That's why it's so fun for everyone.
Jason (San Diego, CA)
Bill, MVP of each AFC West team to date?
Bill Williamson (2:57 PM)
KC: CasselOak: McFaddenSD: RiversDen: Orton
Daylon (Wyoming)
It Studesville runs the table and the team responds well to him you see any chance of him being the new HC?
Bill Williamson (2:58 PM)
There's a chance, but you you really see Denver going 4-0?
Kevin (San Diego)
Ron Rivera as Head Coach of San Diego? As a Bears fan, I think it will be a great move.
Bill Williamson (3:00 PM)
I could see Rivera getting a gig somehwere next monht. Wouldn't be surprised if he gets an interview in Denver.
Bill Williamson (3:00 PM)
Great hour folks, enjoy the big Week 14 action in the AFC West. Talk to you next week.
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text:
Reviews and ratings of local lawyers.
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Learn when to hire and how to choose the right attorney.
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1 total
July 26, 2010 9:17pm
Miranda: To Speak or Not to Speak. That is the Question. If you are being questioned by the police about a potential crime, should you speak to the police? Contrary to some lawyers advice, the answ
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text:
Google Publisher Tag (GPT) is an ad tagging library with which you can dynamically build ad requests. GPT takes key details from you such as ad unit name, ad size, and custom targeting, builds the request, and displays the ad on web pages or mobile apps.
websites using the $ symbol on their website - meaning it may accept payment in this currency used in Israel.
Google Analytics is a service offered by Google that generates detailed statistics about a website's traffic and traffic sources and measures conversions and sales. Google Analytics can track visitors from all referrers, including search engines and social networks, direct visits and referring sites. It also displays advertisin...
Google+ platform websites integration using the Google+ platform tag.
AddThis, the new brand name for Clearspring Technologies, is the leader in socially connecting publishers, services and advertisers to audiences on the open web. Reaching more than 1.3 billion unique users monthly, AddThis' social tools and analytics are used on more than 14 million unique domains to distribute and track digital...
This site uses the viewport meta tag which means the content may be optimized for mobile content.
The Apache HTTP Server is an open-source HTTP server for modern operating systems including UNIX, Microsoft Windows, Mac OS/X and Netware. The goal of this project is to provide a secure, efficient and extensible server that provides HTTP services observing the current HTTP standards. Apache has been the most popular web server ...
PHP is a widely-used general-purpose scripting language that is especially suited for Web development and can be embedded into HTML.
Google Apps is a cloud-based productivity suite that helps you and your team connect and get work done from anywhere on any device. It's simple to setup, use and manage, allowing you to work smarter and focus on what really matters.
Mandrill is a scalable and affordable email infrastructure service, with all the marketing-friendly analytics tools you've come to expect
The DOCTYPE is a required preamble for HTML5 websites.
Allows a website to define how a page is rendered in Internet Explorer 8, allowing a website to decide to use IE7 style rendering over IE8 rendering.
Websites using https protocol.
Google's hosted library for web fonts. Allows websites to choose and use fonts from a free, wide variety of fonts.
Meta descriptions are HTML attributes that provide concise explanations of the contents of web pages. Meta descriptions are commonly used on search engine result pages (SERPs) to display preview snippets for a given page.
Sites who has this tag are verfied by google to use WebMaster tools
The http-equiv attribute provides an HTTP header for the information/value of the content attribute. The http-equiv attribute can be used to simulate an HTTP response header.
A canonical link element is an HTML element that helps webmasters prevent duplicate content issues by specifying the "canonical", or "preferred".
UTF-8 (8-bit UCS/Unicode Transformation Format) is a variable-length character encoding for Unicode. It is the preferred encoding for web pages.
A scheme.org entity that represents an organization such as a school, NGO, corporation, club, etc.
A scheme.org entity that represents a person (alive, dead, undead, or fictional).


text:
another 682b joke
by livergirl
Battle of the apostles (and anyone mentioned in the Bible):
1 David (James)
4 Stephen (Steven Gerrard)
6 John (Terry)
15 Matthew (Upson)
16 James (Milner)
9 another Stephen (Stefan Kießling)
10 Luke (Lukas Podolski)
13 Thomas (Müller)
15 Peter (Piotr Trochowski)
16 Philipp (Lahm)
and don't forget (im)Manuel (Neuer) at the base.
Germany won because they base themselves in immanuel 😛 . if Aaron (Lennon) the other Peter (Crouch) played, maybe England will stand a better chance 😉


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|Home | About | Journals | Submit | Contact Us | Français
DOCK8 and MyD88 have been implicated in serologic memory. Here we report antibody responses were impaired and CD27+ memory B cells were severely reduced in DOCK8-deficient patients. Toll-like receptor 9 (TLR9)- but not CD40-driven B cell proliferation and immunoglobulin production were severely reduced in DOCK8-deficient B cells. In contrast, TLR9-driven expression of AICDA, CD23 and CD86, and activation of NF-κB, p38 and Rac1 were intact. DOCK8 associated constitutively with MyD88 and the tyrosine kinase Pyk2 in normal B cells. Following TLR9 ligation, DOCK8 became tyrosine phosphorylated by Pyk2, bound the Src family kinase Lyn and linked TLR9 to a Src-Syk-STAT3 cascade essential for TLR9-driven B cell proliferation and differentiation. Thus, DOCK8 functions as an adaptor in a TLR9-MyD88 signaling pathway in B cells.
Maintenance of serologic memory requires maturation of naïve B cells into memory B cells and long-lived plasma cells1. This involves interactions between ligand-receptor pairs that include CD40 ligand–CD40, BAFF or APRIL with TACI or BCMA, and TLR ligands–TLRs. TLR9 ligation on naïve B cells promotes proliferation, immunoglobulin secretion and maturation into memory B cells2–4, and synergizes with CD40 and TACI in causing B cell activation5, 6. The TLR9 ligand CpG is an adjuvant for antibody responses in mice7, 8. Defective B cell responses to TLR9 ligation has been observed in patients with common variable immune deficiency characterized by poor antibody production and decreased generation of memory B cells and plasma cells9.
Following ligand binding, TLR9 associates with and signals via the adaptor protein MyD88 (ref. 10). Resting B cells express several TLRs including TLR4 in mice and TLR6, 7, 9 and 10 in mice and humans. MyD88 is important for antibody responses to T dependent (TD) antigens administered with lipopolysaccharide (LPS) or CpG as adjuvants11–13, although exceptions have been noted14. MyD88 does not appear to play a role in the antibody response to TD antigens administered with alum as adjuvant, or certain bacterial antigens15–17, but is important for the antibody response to viruses12, 18, 19.
The dedicator of cytokinesis-8 (DOCK8) is one of eleven members of the DOCK180 superfamily20. DOCK proteins have characteristic DOCK homology region-1 (DHR-1) and DHR-2 domains. The DHR-1 domain is important for DOCK protein targeting to membranes, through its binding of phosphatidylinositol (3,4,5)-triphosphate (PtdIns(3,4,5)P3)21. The DHR2 domain binds to Rac-Rho family GTPases, and can function as an exchange factor for these GTPases21. The biological functions of DOCK8 include regulation of cell migration, morphology, adhesion and growth20. A mouse genetic screen for mutations that disrupt the persistence of the antibody response identified loss-of-function mutations in Dock8 (ref. 23). DOCK8 mutant mice exhibit T cell lymphopenia, and their B cells fail to develop into marginal zone (MZ) B cells and to persist in germinal centers (GCs) and undergo affinity maturation. Reconstitution experiments indicate that DOCK8 expression in B cells is important for the normal persistence of GCs22, suggesting that DOCK8 expression in B cells plays a key role in serologic memory.
Recently, mutations of DOCK8 have been shown to account for a combined immunodeficiency in humans characterized by increased susceptibility to viral skin infections, severe allergy, elevated serum IgE, eosinophilia, T cell lymphopenia and impaired antibody responses23, 24. We analyzed the response of B cells from DOCK8-deficient patients to the TLR9 ligand CpG. DOCK8 was found to mediate a novel MyD88 signaling pathway, which is essential for TLR9-driven B cell proliferation and immunoglobulin production.
Ten patients aged 3.5–15 years with homozygous mutations in DOCK8 were studied (Supplementary Table 1). None had detectable DOCK8 protein in lysates of peripheral blood mononuclear cells (PBMCs) or Epstein-Barr virus (EBV) transformed B cells (data not shown). All had typical clinical characteristics of DOCK8 deficiency (Supplementary Table 2). Five patients, from whom serum was available prior to initiation of immunoglobulin replacement therapy, showed defective IgG antibody response to tetanus toxoid (TT), hepatitis B vaccine (Hep. B), TT-conjugated Haemophilus influenzae type B vaccine (HiB) and conjugated pneumococcal polyvalent vaccine (PV) (Table 1). The IgM TT antibody response was significantly decreased in these patients (Supplementary Fig. 1). Two of these patients, aged 8 and 15 years mounted a brisk early antibody response 8 weeks after a booster dose of TT, which fell below the protective level twelve and fifteen months later (Fig. 1a). This response is in contrast to >99% of normal children, in whom protective antibody titers persist five years after TT booster vaccination 25, 26.
Flow cytometry analysis of PBMCs revealed that the percentage of CD3+ T cells was significantly decreased in the patients compared to age-matched healthy controls, as previously reported23, 24, while the percentage of CD19+ B cells was normal or increased (Fig. 1b). There was a severe deficiency in the percentage of circulating CD19+CD27+ memory B cells in all patients examined, with CD19+CD27− naïve B cells accounting for virtually all (>95%) their B cells (Fig. 1c,d). The percentage of circulating IgD+CD27+ MZ-like B cells was decreased in the patients compared to controls (Supplementary Fig. 2), consistent with the findings in DOCK8 mutant mice22. These results indicate that DOCK8 is important for the generation of memory B cells and serologic memory in humans.
The TLR9 ligand CpG ODN 2006 (thereafter referred to as CpG) acts selectively on human B cells27, and has no detectable effects on non-B cells28. PBMCs from DOCK8-deficient patients were severely deficient in their capacity to proliferate and to secrete IgM and IgG in response to CpG stimulation, compared to PBMCs from age-matched normal subjects, which included shipping controls (Fig. 2a). In contrast, DOCK8-deficient PBMCs proliferated and secreted IgM normally following stimulation with anti-CD40 plus interleukin 21 (IL-21), and secreted about half the amount of IgG as normal PBMCs (Fig. 2b). PBMCs from the patients proliferated and secreted IgE in response to anti-CD40 plus IL-4 to an extent comparable to normal PBMCs (Fig. 2c).
The severely impaired response of DOCK8-deficient PBMCs to CpG could not be simply explained by the lack of memory B cells. Highly purified naïve B cells from normal subjects (>95% CD27−, Supplementary Fig. 3a), proliferated to CpG to an extent comparable to total B cells isolated from the same subjects (Fig. 2d). Consistent with previous reports2–4, the amounts of IgM and IgG secreted by CpG-stimulated naïve B cells were reduced to respectively ~one-half and ~one-third of those secreted by CpG-stimulated total B cells (Fig. 2d). In contrast, purified B cells from DOCK8-deficient patients, which were virtually all naive, completely failed to proliferate and to secrete IgM and IgG in response to CpG (Fig. 2d). Proliferation and secretion of IgM and IgG in response to anti-CD40 plus IL-21 was comparable in normal naïve B cells and DOCK8-deficient purified B cells (Supplementary Fig. 3b). The decreased IgG secretion of DOCK8-deficient PBMCs to anti-CD40 plus IL-21 was most likely secondary to lack of memory B cells, because purified normal naïve B cells exhibited a comparable decrease in IgG secretion in response to anti-CD40 plus IL-21 relative to total B cells (Supplementary Fig. 3b). The failure of B cells from DOCK8-deficient patients to respond to CpG was not due to increased apoptosis and cell death, as determined by staining with Annexin V and propidium iodide (Supplementary Fig. 4). These results indicate that B cells from DOCK8-deficient patients have an intrinsic and selective defect in their ability to respond to CpG.
CpG stimulation of B cells induces the expression of AICDA, which encodes activation-induced cytosine deaminase (AID) that is critical for isotype switching, and upregulates the expression of the low affinity IgE receptor CD23 and the co-stimulatory molecule CD86 (refs. 4,29). CpG stimulation induced comparable AICDA mRNA expression in PBMCs and B cells from DOCK8-deficient patients and controls (Fig. 3a), and comparable increases in the percentage of CD19+ B cells that express CD23 and CD86 (Fig. 3b,c). The mean fluorescence intensity (MFI) of these antigens on B cells was comparable between DOCK8-deficient patients and controls (Fig. 3b,c). NF-κB and the MAP kinase p38 are activated by CpG stimulation and play an important role in the induction of AICDA, CD23 and CD86 expression30–32. CpG stimulation caused comparable IκBα and p38 phosphorylation in EBV-B cells and PBMCs from DOCK8-deficient patients and controls (Fig. 3d and data not shown). CpG-driven IL-6 secretion by EBV-B cells was not significantly different between patients and controls (Fig. 3e). CpG stimulation causes plasmacytoid dendritic cells (pDCs) to secrete interferon-α (IFN-α). This induction requires MyD88-dependent activation of IRF7 (ref. 33). Stimulation of PBMCs with the CpG type A ODN 2216, which activates TLR9 in human pDCs, resulted in comparable secretion of IFN-α in patients and controls (Fig. 3f)
The DHR2 domain of DOCK proteins can activate Rac and/or Cdc42 (ref. 21) and CpG activates Rac1 in DCs34. To examine Rac1 activation, a fusion protein of glutathione-S-transferase (GST) and the GTPase binding domain of p21-activated kinase (GST-PAK) was used to precipitate GTP-bound Rac1 and Cdc42 from cell lysates, followed by immunoblotting with antibodies to Rac1 and Cdc42. Stimulation with CpG resulted in comparable Rac1 activation in EBV-B cells from normal and DOCK8-deficient subjects (Fig. 3g), but caused no detectable Cdc42 activation (data not shown). Thus, CpG activates Rac1 in B cells, but DOCK8 is not essential for this activation.
STAT3 is important for B cell proliferation and differentiation in response to anti-CD40 plus IL-21 (ref. 35,36). Stimulation of normal PBMCs with CpG induced STAT3 phosphorylation on residue Y705 (Fig. 4a). In contrast, CpG-driven, but not IL-21-driven, STAT3 phosphorylation was defective in DOCK8-deficient PBMCs (Fig. 4a). CpG-driven, but not IFN-α induced, STAT3 phosphorylation was defective in purified DOCK8-deficient B cells (Fig. 4b), indicating that the defect is B cell autonomous. IL-6 and IL-21 caused comparable STAT3 phosphorylation in purified B cells from patients and controls (Fig. 4c). Densitometry scanning analysis demonstrated that CpG-driven STAT3 phosphorylation in B cells from DOCK8-deficient patients was 18±7% of control (n = 3 each, P < 0.01), whereas IL-21-driven STAT3 phosphorylation was comparable (Fig. 4d). The defect in CpG-driven STAT3 phosphorylation in DOCK8-deficient B cells was not secondary to lack of memory B cells, because CpG caused comparable STAT3 phosphorylation in purified naïve B cells and unfractionated B cells from normal controls (Fig. 4e). These results demonstrate that DOCK8 deficiency selectively impairs CpG-driven STAT3 phosphorylation in B cells.
The role of STAT3 in CpG-driven B cell proliferation and differentiation was investigated by examining PBMCs from six patients with autosomal dominant hyper-IgE syndrome (AD-HIES), resulting from different dominant-negative mutations in STAT3 (Supplementary Table 3). PBMCs from these patients had significantly impaired proliferation and IgG secretion following stimulation with CpG (Fig. 4f). There was no significant difference between PBMCs from AD-HIES patients and controls in their ability to proliferate and secrete IgE in response to anti-CD40 plus IL-4 (Fig. 4g). As previously reported35, PBMCs from AD-HIES patients failed to proliferate and secrete IgG in response to anti-CD40 plus IL-21 (Supplementary Fig. 5).
Because IL-6 caused STAT3 phosphorylation in DOCK8-deficient B cells, we examined whether exogenous IL-6 corrects their defective response to CpG. Addition of rIL-6 to DOCK8-deficient PBMCs failed to correct their defective proliferation and IgG secretion in response to CpG (Fig. 4h, and data not shown). These results indicate that in addition to STAT3 phosphorylation, other signals that are important for CpG-driven B cell proliferation and differentiation are impaired in DOCK8-deficient B cells.
TLR9 protein expression was comparable in B cells from patients and controls (Supplementary Fig. 6a). Correct subcellular localization of TLR9 is essential for its function37. Lack of DOCK8 might result in TLR9 mislocalization, which could interfere with the ability of TLR9 to activate STAT3. EBV-B cells from normal subjects and DOCK8-deficient patients were stimulated for 90 min with CpG, permeabilized and simultaneously stained for TLR9 and either endoplasmic reticulum (ER Tracker®), the early endosomal marker EEA-1 or the late endosomal-lysosomal markers LAMP-1 (also known as CD107a). Fluorescent microscopy revealed that the subcellular localization of TLR9 was comparable in normal and DOCK8-deficient EBV-B cells (Supplementary Fig. 6b). Thus DOCK8 has no detectable role in TLR9 trafficking in B cells.
The tyrosine kinase Syk activates STAT3 in B lymphoma cells38, 39. Stimulation of normal PBMCs with CpG caused Syk phosphorylation on residue Y352, the target of Src kinases (Fig. 5a). In contrast, it caused negligible Syk phosphorylation in DOCK8-deficient PBMCs (Fig. 5a). The defect in Syk phosphorylation was specific to CpG because B cell receptor (BCR) crosslinking with anti-IgM caused comparable Syk phosphorylation in patients and controls (Fig. 5b). Densitometry scanning analysis demonstrated that CpG-driven Syk phosphorylation in PBMCs from DOCK8-deficient patients was 16±8% of controls, (n = 3 each, P < 0.001), while BCR-driven Syk phosphorylation was not significantly different (Fig. 5c).
The pentapeptide Syk inhibitior SYKINH-61 was used to examine whether Syk activation is important for CpG-driven STAT3 phosphorylation. SYKINH-61 inhibited CpG-driven STAT3 phosphorylation in normal PBMCs (85±5% inhibition, n = 3), but had no effect on IL-21-driven STAT3 phosphorylation (Fig. 5d), or CpG-driven phosphorylation of p38 (data not shown). Moreover, SYKINH-61 inhibited CpG-driven proliferation and IgG production in normal PBMCs in a dose-dependent manner, but had no effect on CD40–IL-21-driven proliferation and IgG production (Fig. 5e,f).
The role of Syk in CpG-driven STAT3 phosphorylation was verified using RNA silencing. The mouse B cell line CH12 faithfully reproduced the response of primary human B cells to CpG stimulation by exhibiting Pyk2, Syk and STAT3 phosphorylation (Supplementary Fig. 7a). shRNA knockdown (KD) of Syk, but not introduction of non-silencing (NS) shRNA, strongly reduced Syk protein expression and strongly inhibited CpG-driven STAT3 phosphorylation in CH12 cells (Supplementary Fig. 7b–d). These results place Syk upstream of STAT3 activation by CpG in B cells.
CpG may bind to sensors other than TLR9 (ref. 34). To determine if DOCK8 dependent CpG-driven phosphorylation of Syk and STAT3 proceeds through the engagement of TLR9 and its adaptor protein MyD88, we compared the response of purified splenic B cells (>95% B220+ cells) from Tlr9−/− and Myd88−/− mice and their genetically matched wild-type controls. CpG stimulation resulted in the phosphorylation of Syk and STAT3 in B cells from wild-type mice (Fig. 5g,h), as in normal human B cells. In contrast, CpG stimulation caused minimal Syk and STAT3 phosphorylation in B cells from Tlr9−/− and Myd88−/− mice. Furthermore, CpG failed to cause proliferation and IgG secretion by B cells from Tlr9−/− and Myd88−/− mice (Supplementary Fig. 8). Together, these results indicate that CpG engages TLR9–MyD88 to cause DOCK8-dependent phosphorylation of Syk and STAT3, which is essential for B cell proliferation and differentiation.
Stimulation of normal PBMCs with CpG resulted in tyrosine phosphorylation of proteins with molecular weights that correspond to those of Src family kinases (54–56 kDa), their target Syk (72 kDa), and Pyk2 (110 kDa), a tyrosine kinase that functionally interacts with Src kinases (Fig. 6a). In contrast, it caused minimal protein tyrosine phosphorylation in PBMCs from DOCK8-deficient patients. This defect was selective to CpG stimulation, because BCR crosslinking caused comparable protein tyrosine phosphorylation in PBMCs from DOCK8-deficient patients and controls (data not shown), consistent with the normal activation of B cells from DOCK8 mutant mice following BCR crosslinking22.
Pyk2 is auto-phosphorylated on tyrosine residue Y402 (ref. 40). Activated Src family kinases (thereafter referred to as Src) are autophosphorylated on Y residues that correspond to Y416 in Src40, and are recognized by anti-pY416Src. Immunoblotting lysates of PBMCs with phosphospecific mAbs to pY402Pyk2 and pY416Src demonstrated that stimulation with CpG caused Pyk2 and Src phophorylation in normal PBMCs (Fig. 6b). In contrast, CpG stimulation caused minimal phosphorylation of Pyk2 and Src in DOCK8-deficient PBMCs (Fig. 6b), demonstrating that CpG-driven tyrosine phosphorylation of Pyk2 and Src is DOCK8 dependent. Pyk2 and Src were phosphorylated after CpG stimulation of B cells from wild-type, but not Myd88−/− mice (Fig. 6c), indicating that TLR9-driven phosphorylation of Pyk2 and Src, like that of Syk and STAT3, is dependent on MyD88.
We used selective inhibitors of Pyk2 and Src to examine the roles of these kinases in CpG-driven activation of B cells. The Pyk2 inhibitor Tyrphostin A9 strongly inhibited CpG-driven tyrosine phosphorylation of Pyk2, Src, Syk and STAT3 (Fig. 6d-g). In three experiments the inhibition was 84±3% for Pyk2, 81±6% for Src, 95±4% for Syk and 97±2% for STAT3. Tyrphostin A9 had no detectable effect on IL-6 driven STAT3 phosphorylation, or CpG-driven p38 phosphorylation (data not shown). Tyrphostin A9 strongly inhibited CpG-driven IgG secretion by 90±2% (n = 3, P < 0.01), but not CD40–IL-21 driven IgG secretion (Fig. 6h). Similar results were obtained with the structurally different Pyk2 inhibitor PF562271 (data not shown). The role of Pyk2 in CpG-driven STAT3 phosphorylation was verified using shRNA knockdown in CH12 B cells (Supplementary Fig. 7b–d).
The Src inhibitor PP2 had a modest effect on CpG-driven Pyk2 phosphorylation (Fig. 6d), but more strongly inhibited CpG-driven phosphorylation of Src, Syk and STAT3 (Fig. 6e–g). In three experiments, inhibition by PP2 was 28±13% for Pyk2, 80±8% for Src, 71±9%, for Syk and 63±11% for STAT3. The modest inhibition of Pyk2 phosphorylation is consistent with the ability of Src to activate Pyk2 (ref. 40), and suggests feedback amplification of Pyk2 activation by Src. The less than complete inhibition of Syk and STAT3 phosphorylation could be due to the ~20% residual Src activity. Alternatively, a Src-independent link between Pyk2 and Syk–STAT3 might exist. PP2 inhibited CpG-driven IgG secretion by 70±12% (n = 3, P < 0.05), but had no effect on CD40–IL-21 driven IgG secretion (Fig. 6h). Similar results were obtained with the structurally different Src kinase inhibitor SU6656 (data not shown). Taken together the above results suggest that TLR9 ligation activates a Pyk2-Src-Syk cascade that results in STAT3 phosphorylation.
Our results place DOCK8 and Pyk2 downstream of MyD88. To examine whether DOCK8 associates with MyD88, 293T cells were co-transfected with vectors encoding Myc-tagged DOCK8 and HA-tagged MyD88, or HA-tagged myocardin-related transcription factor-A (MRTF-A) as a control. DOCK8 was found to co-precipitate with MyD88, but not with MRTF-A (Fig. 7a). To examine whether DOCK8 associates with MyD88 in B cells, DOCK8 immunoprecipitates from normal EBV-B cells were probed for MyD88. DOCK8 associated weakly with MyD88 in unstimulated cells, but this association increased following CpG stimulation (Fig. 7b).
Pyk2 was reported to interact with MyD88 in macrophages41. This interaction was mapped to the proline-rich C-terminal region of Pyk2 and the death domain of MyD88. Probing of MyD88 immunoprecipitates from normal EBV-B cells with mAb to Pyk2 revealed that Pyk2 associates with MyD88 (Fig. 7c). This association was not altered detectably by CpG stimulation and was independent of DOCK8, as Pyk2 co-precipitated with MyD88 from lysates of EBV-B cells from DOCK8-deficient patients (Fig. 7c). Pyk2 was readily detected in DOCK8 immunoprecipitates from normal EBV-B cells (Fig. 7d). This association increased following CpG stimulation. The constitutive association of DOCK8 and Pyk2 was independent of MyD88 and was observed in EBV-B cells from a MyD88-deficient patient that express no detectable MyD88 protein (Fig. 7d). However, CpG stimulation did not increase the association of DOCK8 and Pyk2 in the absence of MyD88. These results suggest that DOCK8 forms a complex with MyD88 and Pyk2.
The YEEVK motif (amino acids 628–632)in the DHR1 domain of DOCK8 also exists in cofilin-1 where it is a target of Pyk2 phosphorylation42. This motif is located within a sequence in DOCK8 (HNKSPDFYEEVKIKL, amino acids 621–635), which is a potential binding site for the SH2 domain of Src. Probing DOCK8 immunoprecipitates from EBV-B cells with mAbs to phosphotyrosine and Src revealed that CpG stimulation caused tyrosine phosphorylation of DOCK8 and its association with Src (Fig. 7e). Both events were abrogated by the Pyk2 inhibitor Tyrphostin A9. The Src family member Lyn is expressed preferentially in B cells43. Probing Lyn immunoprecipitates with a pY416Src mAb demonstrated that CpG stimulation causes tyrosine phosphorylation of Lyn in normal B cells (Fig. 7f). Furthermore, CpG stimulation caused robust association of Lyn with DOCK8, which was abrogated by addition of the Pyk2 inhibitor Tyrphostin A9 (Fig. 7g). These results indicate that TLR9 ligation in B cells causes Pyk2-dependent DOCK8 phosphorylation and recruitment of Src and/or Lyn.
We present evidence that DOCK8 functions as an adaptor that links TLR9 via MyD88 to a Pyk2-Src-Syk-STAT3 signaling cascade, and we demonstrate that this pathway is essential for TLR9-driven B cell proliferation and immunoglobulin production.
DOCK8-deficient patients had impaired ability to sustain a protective antibody response, similar to DOCK8 mutant mice, and lacked circulating CD27+ memory B cells. DOCK8-deficient B cells failed to proliferate and secrete IgM and IgG in response to CpG. This defect is cell-autonomous, specific to CpG and not accounted for by the lack of memory B cells. Because of constraints in obtaining sufficient amounts of blood from children for B cell purification, our experiments used PBMCs, which were stimulated with the B cell selective TLR9 ligand CpG ODN2006, and/or EBV-B cells. Whenever possible, purified B cells from at least two patients were also used.
CpG stimulation normally upregulated the expression of AICDA, CD23 and CD86 in DOCK8-deficient B cells and resulted in normal activation of NF-κB and p38, and normal IRF7-dependent secretion of IFN-α in DOCK8-deficient PBMCs, indicating that these events occur independently of DOCK8. Unexpectedly, given the reported GEF activity of DOCK proteins for small GTPases21, CpG activated Rac1 independently of DOCK8. CpG activation of Rac1 in pDCs uses a non-TLR9 sensor and DOCK2 (ref. 34). A similar pathway could be operative in B cells.
A central finding in this study is that CpG causes STAT3 phosphorylation in B cells in a DOCK8-dependent manner. Impaired STAT3 phosphorylation in DOCK8-deficient B cells was specific for CpG stimulation, and was not secondary to the lack of memory B cells. The critical role of STAT3 in CpG-driven B cell proliferation and differentiation was demonstrated by the observation that these responses were impaired in patients with dominant-negative mutations in STAT3. The failure of IL-6, which caused STAT3 phosphorylation in DOCK8-deficient B cells, to correct their defective response to CpG indicates that other DOCK8-dependent signals are also required for CpG-driven B cell proliferation and IgG secretion. These may include signals delivered by Pyk2, Src and Syk which trigger the activation of phospholipase C-γ, phosphatidylinositol-3-OH kinase and B cell linker protein (BLNK)44–46.
Syk was shown to play a critical role in CpG-driven STAT3-dependent B cell proliferation and differentiation. CpG stimulation of B cells caused Syk phosphorylation, which was severely impaired in DOCK8-deficient B cells, placing Syk downstream of DOCK8. The Syk selective inhibitor SYKINH-61 blocked CpG-driven STAT3 phosphorylation, placing Syk upstream of STAT3, and inhibited CpG-driven B cell proliferation and IgG secretion. CpG stimulation of B cells caused DOCK8-dependent phosphorylation of Pyk2 and Src. DOCK8 was demonstrated to link TLR9-MyD88 to a Pyk2-Src-Syk-STAT3 cascade in B cells, which was shown to be essential for TLR9-driven B cell activation. The observation that CpG-driven phosphorylation of Pyk2, Src, Syk and STAT3, and B cell proliferation and differentiation were dependent on TLR9 and MyD88, indicate that CpG engagement of TLR9-MyD88 results in DOCK8-dependent B cell activation. CpG-A DNA has been reported to induce tyrosine phosphorylation of proteins, including Syk, in monocytes and macrophages independently of TLR9 and MyD88 (ref. 47). Differences in the ODNs and target cells used may account for the difference in the requirement for TLR9 and MyD88.
DOCK8 was found to exist in a complex with MyD88 and Pyk2. MyD88 was not essential for DOCK8-Pyk2 association, and DOCK8 was not essential for MyD88-Pyk2 association, although it was essential for Pyk2 phosphorylation following TLR9 ligation. Following CpG stimulation, DOCK8 became more strongly associated with MyD88 and Pyk2, underwent tyrosine phosphorylation and associated with Src and/or Lyn. We propose the following model of DOCK8 dependent TLR9 signaling. Ligation of TLR9 by CpG causes recruitment and stabilization of a preexisting MyD88-Pyk2-DOCK8 complex, which results in autophosphorylation and activation of Pyk2. Pyk2 then phosphorylates DOCK8 causing it to recruit Src kinases, including Lyn, via their SH2 domain, releasing them from auto-inhibition. Src then activates Syk, which drives STAT3 activation. The proposed pathway is likely a simplification. Src and Syk can phosphorylate Pyk2 (ref. 40), and Src and Pyk2 can synergize to cause STAT3 phosphorylation48, 49. Future experiments will determine whether the DOCK8-dependent TLR9-MyD88 signaling pathway we have identified is used by other receptors that signal via MyD88 in B cells and other cells. Preliminary data indicates that this pathway is activated by TLR4 ligation in PBMCs (data not shown).
DOCK8 deficiency results in impaired immunological synapse in B cells22 and may impair the ability of T cells and DCs to drive antibody production by B cells. Mice with selective DOCK8 deficiency in B cells, and mice in which the interaction between MyD88 and DOCK8 is disrupted will help define the contribution of DOCK8-dependent MyD88 signaling in B cells to the impaired serologic memory in DOCK8 deficiency. Given that TLR9 ligands are vaccine adjuvants and the role of TLR9 in autoantibody responses to self DNA50, the TLR9 signaling pathway we have described may be important for developing better vaccines and understanding and treating autoantibody-mediated diseases.
Ten patients with established DOCK8 mutations and six with STAT3 mutations were studied. Informed consent was provided by adult donors or by the children's parents or guardian. Protocols used in the human studies have been approved by the Committee on Clinical Investigations at Children's Hospital, Boston, MA, USA.
Human PBMCs were isolated from heparinized blood obtained from patients and normal donors using Ficoll-Hypaque (GE Healthcare). Naïve and total B cells were purified from PBMCs by negative selection using the B cell isolation Kits II (Miltenyi Biotec) according to the manufacturer's recommendation and contained >96% CD19+ cells. Cells were suspended in RPMI-1640 containing 10% heat-inactivated FCS (Hyclone), 2 mM L-glutamine, 50 μg/ml streptomycin and 100 U/ml penicillin (medium). Epstein-Barr virus (EBV) transformed cell lines on patients and controls were established as described51.
PBMCs (1 × 106 and 1.5 × 106 cells/ml for proliferation and immunoglobulin production respectively) or B cells (1.5 × 106 cells/ml) were cultured with medium, CpG ODN 2006 or 2216 (0.1 μM, Invivogen), or anti-CD40 (5 μg/ml, clone 626.1) in the presence of either recombinant (r) human interleukin 21 (IL-21, (50 ng/ml, Miltenyi Biotech), or rIL-4 (5 ng/ml, R&D Systems). The following kinase inhibitors were used: Syk inhibitor (SYKINH-61, a kind gift of F. Uckun, University of Southern California, Los Angeles); Pyk2 inhibitor (Tyrphostin A9, AG 17), and Src inhibitor (PP2) from EMD biosciences. Proliferation was assayed 4 days later by assaying for 3H-thymidine incorporation. IgM, IgG and IgE production was measured in 14-day culture supernatants by ELISA as previously described52. CpG-mediated upregulation of CD23 and CD86 expression was analyzed by flow cytometry after 48 h. IL-6 and IFN-α in culture supernatants were measured after 24 h using ELISA kits from Invitrogen. Apoptosis was evaluated on day 3 after stimulation. IgM specific tetanus toxoid (TT) antibody in sera was assayed using ELISA kit from IBL International.
Anti-human monoclonal antibodies to the following were used for staining: CD3 (HIT3a), CD19 (HIB19), CD27 (M-T271), CD23 (M-L233), CD86 (2331(FUN-1)) and IgD (IA6-2) (BD Biosciences); CD123 (AC145) and BDCA-4 (AD5-17F6) (Miltenyi) with the appropriate isotype controls. Annexin V–FITC was purchased from Biovision Inc. Data collected with a FACSCalibur cytometer (BD Biosciences) were analyzed with CellQuest software (BD Biosciences) or FlowJo software (TreeStar).
RNA was extracted from cultured B cells on day 5 using TRIzol (Invitrogen) and was reverse transcribed by Supercript II RT (Invitrogen) according to manufacturer's instructions. Q-PCR for AICDA was performed using primers and an ABI Prism 7300 sequence detector from Applied Biosystems. Relative RNA expression normalized to GAPDH RNA abundance was calculated by the relative standard curve method as outlined in the manufacturer's technical bulletin (Applied Biosystems).
PBMCs, B cells or EBV-transformed B cell lines (1 × 106 cells/condition) were unstimulated or stimulated with either CpG (2.5 μM), IL-21 (10 ng/ml), IFN-α (1000 U/ml), Biosource), rIL-6 (2 ng/ml, R&D systems) or anti-human IgM (10 μg/ml, Jackson Immuno Research Labs). Total cell lysates were then separated by SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes. The following antibodies were used for immunoblot analysis: anti-DOCK8 (#HPA003218, Sigma-Aldrich); anti-STAT3 (79D7), anti-BLNK (#3587), anti-Pyk2 (5E2), anti-Lyn (C13F9) and antibodies to phosphorylated p38 (D3F9), IκBα (ser32/36, 5A5), STAT3 (Tyr705, D3A7), Syk (Tyr352, #2701), Pyk2 (Tyr402, #3291), and Src family (Tyr416, #2101) (Cell Signaling); anti-IκBα (sc-847), anti-IKKγ (Fl-419), and anti-HA (Santa Cruz Biotechnology); anti-Syk (4D10.1), anti-Src (CT, NL19), anti-Myc (9E10), and anti-phosphotyrosine (4G10) (Millipore). The densitometric analysis of the scanned bands was evaluated using the NIH Image program J 1.440 software.
A wild-type human DOCK8 construct was generated with a Myc tag by PCR amplification of pCR4-TOPO human DOCK8 cDNA (Open Biosystems) using standard cloning techniques. 293T cells were transfected with plasmids encoding Myc-tagged DOCK8, HA-tagged MyD88, and HA-tagged MRTF-A (R. Treisman, Cancer Research, UK), using Lipofectamine LTX (Invitrogen). After 16 h, cells were lysed with buffer containing 0.75% NP-40. Immunoprecipitation was performed using a monoclonal anti-HA (HA-7) agarose conjugate (Sigma-Aldrich), were separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with antibodies against Myc, MyD88 and HA. For immunoprecipitation (IP) experiments in EBVs, IP and MyD88 blotting was done with anti-MyD88 (H00004615-PW2 pair, Novus).
RAC1 activation was assessed by pull-down from lysates with a fusion protein of gluthatione-S-transferase and the PBD domain of PAK1 (Pierce Laboratories) as previously described53. Immunoblot of precipitated samples and aliquots of the cell extracts for loading controls was then performed with RAC1 antibody (Pierce Laboratories).
EBV transformed B cells stimulated with CpG for 90 min, were spun onto poly-D-lysine-coated coverslips (50 μg/ml, Sigma). Localization of TLR9 to endoplasmic reticulum was confirmed using ER Tracker® Red marker and Alexa 488-conjugated (green) anti-TLR9 (26C593.2, Imgenex Corp). Cells were loaded with ER Tracker® Red (Invitrogen) per vendor's protocol, fixed in 4% paraformaldehyde, permeabilized with 0.04% saponin in 1% BSA54 and incubated with anti-TLR9. Coverslips were mounted on slides using Prolong Gold with DAPI antifade mounting medium (Invitrogen). Cells on coverslips were fixed in 4% paraformaldehyde, permeabilized with 0.25% Triton X-100, blocked with 10% FCS in PBS, incubated with Early endosome (EEA1, 1G11) and late endosomes/lysosomes (LAMP1, H4A3) monoclonal antibodies (Abcam) for 1 h at 25°C, washed and stained with Alexa 555-conjugated (red) anti-mouse IgG (Invitrogen). Counterstaining with TLR9 antibody and slide mounting was performed as described above. All images were acquired using a Nikon Eclipse Ti inverted microscope, and were processed using NIS elements AR3.0 software and/or Adobe Photoshop.
pGIPZ lentiviral vectors encoding green fluorescent protein (GFP) as well as shRNAs specific for murine Pyk2, Syk and a non-silencing (NS, scrambled) plasmid were purchased from Open Biosystems. CH12 cells were transfected by electroporation with 0.5 μg/ml of each plasmid and transduced cells were selected using puromycin. GFP-positive cells were subcloned and tested for Pyk2 and Syk protein expression by immunoblotting.
TLR9- and Myd88-deficient and the corresponding C57BL/6 and BALB/c wild-type control mice were used at 8–12 weeks of age according to the guidelines of the Animal Care Committee of Children's Hospital, Harvard Medical School, Boston, MA, USA.
B cells were purified from splenic cell suspensions by negative selection using CD43-conjugated magnetic beads from Miltenyi. Purified B cells (>95% B220+) were suspended in complete medium containing 50 μM β-mercaptoethanol and were cultured (106 cells/ml) with medium or CpG ODN 1826 (3 μg/ml, Invivogen). Proliferation was assayed day 4 by measuring 3H-thymidine incorporation. Day 6 IgG was measured in culture supernatants by ELISA. For immunoblot studies CpG ODN 1826 (2.5 μM) and mouse rIL-21 (10 ng/ml, R&D Systems) were used.
Student t-test was used for statistical analysis.
We thank K. Eurich for technical assistance, A. Rambhatla and A. Chen (University of California, Los Angeles) for their help in DNA sequencing, the members of the Geha laboratory for useful discussions, J. Kagan and H. Oettgen for commenting on the manuscript, and the patients and their families for donating blood. SYKINH-61 was a kind gift of F. Uckun (University of Southern California, Los Andeles). The MyD88-deficient EBV-B cell line was a kind gift of J.-L. Casanova (The Rockefeller University), A. Puel and C. Picard (Hopital Necker-Enfants Malades, France). This work was supported by USPHS grants P01AI076210 (RSG), T32AI007512 (RSG), R01AI083503 (RSG), R21AI087627 (TAC) and K08AI076625 (DM), a grant from the Dubai Harvard Foundation for Medical Research (RSG and LN), grant PASMP3-127678/1 from the Swiss National Science Foundation (MR) and grants from the Clinical Immunology Society (EJ) and the Manton Foundation (EJ and LN).
AUTHOR CONTRIBUTIONSH.H.J., D.R.M., E.J, M.J.M., N.R., A.B., I.R., H.B., and M.R. performed the experiments. L.S., S.B, R.W., G.D., M.D., W.A., I.B., S.B., N.K., H.O., A.P., M.K., G.L. and I.R., provided patient blood samples. K.F. and D.G. provided mice. J.M. and L.N. provided advice. S.K, R.C. and T.C. performed DNA sequencing. H.H.J. and R.S.G. wrote the manuscript.
The authors declare no competing financial interests.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Daytona Bicycle Center
Xlab's Mini Bag is a small seat bag perfect for carrying the essentials. Can fit a tube, tire levers, inflator, patch kit, and two 16-gram CO2 cartridges. Small enough to tuck under your saddle or inside your Super Wing or Carbon Wing rear carrier. It's made from tough Cordura, is easily attached with the hook-and-loop straps, and it works with round and aero seatposts.
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Fairbanks – Nov. 7, 2016 – 4:15 p.m. -Alaska's state and federal wildfire suppression agencies are deploying personnel to the Lower 48 to help battle wildfires in the drought-stricken southeastern U.S. In the last two weeks, more than a dozen personnel from state and federal agencies in Alaska have been sent to Alabama, Georgia, Kentucky, […]Continue reading
Moose Creek Fire north of Palmer is 100 percent contained
Friday, Oct. 21 – 4 p.m. – The Moose Creek Fire north of Palmer along the Glenn Highway is now 100 percent contained, according to the Alaska Division of Forestry. Alaska Division of Forestry firefighters toured the fire perimeter on Friday morning and declared the 303-acre fire fully contained, according to Incident Commander Phil Blydenburgh. […]Continue reading
Rain and snow help push containment up to 83 percent on Moose Creek Fire north of Palmer
Friday, Oct. 21 – 10:25 a.m. – With a little help from Mother Nature, firefighters on Thursday continued to gain ground on a wildfire smoldering along the Glenn Highway north of Palmer. The 303-acre Moose Creek Fire is now 83 percent contained, according to a perimeter map released by the Alaska Division of Forestry on […]Continue reading
Moose Creek Fire north of Palmer now 70 percent contained
Thursday, Oct. 20 – 10:40 a.m. – Containment on the Moose Creek Fire north of Palmer is now at 70 percent and fire managers are optimistic full containment on the late-season wildfire will be achieved before the end of the weekend. "If (the weather) stays the way it is right now we're hoping for full […]Continue reading
Winds offer no respite for firefighters battling wildfires in Matanuska Valley
Monday, Oct. 17 – 7:15 p.m. – Windy weather made for another busy day for wildland firefighters in the Matanuska Valley. In addition to continuing their battle with a stubborn, late-season wildfire north of Palmer, Alaska Division of Forestry firefighters had to contend with three different wildfires caused by high winds blowing trees onto power lines. […]Continue reading
Wildfire north of Palmer makes run toward Glenn Highway; high winds, limited resources pose serious challenge
For the third straight day, high winds and limited resources are posing a serious challenge to firefighters contending with a late-season wildfire along the Glenn Highway north of Palmer. The 300-acre Moose Creek Fire started early Saturday morning as the result of an escaped debris burn and firefighters have been battling the wind-driven blaze around […]Continue reading
Wildfire near Sutton is 25 percent contained; firefighters continue effort in harsh conditions
Sunday, Oct. 16 – 4:10 p.m. – A wildfire in the Matanuska Valley near Sutton is now 25 percent contained and firefighters continue to battle the blaze in harsh conditions. The Moose Creek Fire is now estimated at 300 acres, an increase of 84 acres from Saturday evening. The increase in acreage is due to better […]Continue reading
Smokejumpers respond to fire near Koyuk
Friday, Sept. 2, 2016 – BLM Alaska Fire Service Smokejumpers are responding to a fire burning in tundra about two miles east of Koyuk. The fire was reported by villagers at approximately 1:30 p.m. today. Agency personnel flew over the fire and reported the fire as about 10 acres in size, 15 percent active and burning with […]Continue reading
Hunters advised to be careful with fire during hunting season
With hunting season underway, the Alaska Division of Forestry and BLM Alaska Fire Service ask hunters to use caution with anything that could cause a wildfire. While abundant rainfall, shorter daylight hours and cooler temperatures have decreased fire danger across most of the state, all it will take is a few hot, dry days to […]Continue reading
Mid Salcha Fire now 90 percent contained
July 23, 11:25 a.m. – Containment on the Mid Salcha Fire increased on Friday while the estimated number of acres burned decreased. Due to better mapping, the burned acreage total for the wildfire near the Salcha River south of Fairbanks is now estimated at 1,049 acres, down from the previous estimate of 1,164 acres, according […]Continue reading


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Englewood students feed the hungry using green thumbs
ENGLEWOOD - Middle school students at Dwight-Englewood School are growing fresh food for clients at a local food pantry to help fight hunger.
Nine sixth-grade students planted vegetable seedlings – including squash, beans, tomatoes, herbs and cabbage – at the Third Street Community Garden on May 28 as part of the school's service learning project. One of the site's eight 10-by-12-foot garden beds was designated specifically to grow food for the Center for Food Action, an Englewood-based food pantry.
The Third Street Community Garden was built in 2012 in collaboration with the city and local community groups such as Northern NJ Business Volunteer Council (BVC) and the Bergen County Habitat for Humanity.
Jennifer Johnson, communications director at Center for Food Action, said fresh food was something the center rarely receives. The center was considering creating its own community garden before learning about the Third Street garden from Council President Lynne Algrant.
When people eat solely from prepackaged foods, there is a greater risk they may develop high blood pressure and diabetes, said Johnson.
"We're giving our clients the opportunity to have healthier diets," said Johnson.
The sixth-grade service project focused this year on hunger in New Jersey, said Tasha Urbanowski, Dwight-Englewood's sixth-grade dean who teaches the school's garden class.
She said the project helped students get involved in the local community and help out their neighbors.
"The idea is for kids to recognize there is a large world around them," said Urbanowski and for them "to see their place in it and be a part of it."
The collaboration between Dwight-Englewood and the Center for Food Action began in January, when Johnson discussed the food pantry with the students. Since then, eight groups throughout the entire grade learned how to prepare a garden by making fertilizer, testing soil, composting and planting seeds in the school's greenhouse.
"It's something for a good cause," said Rudolph LeDuc, a sixth-grade student. "It doesn't hurt us, it actually helps us… you feel really good."
Five volunteers, such as Jen Anderson from AJR Landscaping and Alan Kostelnik, a Center for Food Action volunteer, will care for the crops in the garden that the children planted on a daily basis. He believed clients will enjoy seeing the vegetables in their monthly bag of food, which usually includes canned foods and pastas.
"It's fun to see the expression on their faces when they see fresh food," said Kostelnik.
Johnson and Kostelnik both expressed a desire to have clients eventually work on the gardens and learn how to grow fresh food.
Email: firstname.lastname@example.com


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- Repair your camping mattress with relative ease using this universal repair kit.
- Make fast, permanent repairs to your self-inflating mattress in the field.
- Use the repair kit anywhere to repair tears and rips quickly and permanently.


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Sony Walkin Interview For Freshers/Exp As Data Analyst
Sony Walkin Interview For Freshers/Exp Candidates For Data Analyst Positions in Delhi Locations.Interested and Eligible Candidates can attend For Walkin Interviews on Given Dates. Further Details Like Total no of Vacancies, Education Qualification,Experience Details, Location of Job and Detailed Description of Job is Given Below.
Sony Walkin Interview For Freshers/Exp
Company Name : Sony India Pvt.Ltd
Designation : Data Analyst
No Of Positions : Multiple Jobs
Qualification : Any Graduates
Experience : 0 – 2 years
Location : Delhi
Salary : As Per Industry
Job Description :
- Candidate should have good Excel Knowledge and efficient in Data Entry.
- Candidate should have SAP Knowledge.
- Sony Walkin Interview For Freshers/Exp As Data Analyst
Company Profile :
We are a Japanese multinational conglomerate corporation headquartered in Tokyo, Japan. Our diversified business includes consumer and professional electronics, gaming, entertainment and financial services. We are one of the leading manufacturers of electronic products for the consumer and professional markets.Sony Corporation is the electronics business unit and the parent company of the Sony Group, which is engaged in business through its four operating segments – electronics (including video games, network services and medical business), motion pictures, music and financial services.
At Sony, our mission is to be a company that inspires and fulfills your curiosity. Our unlimited passion for technology, content and services, and relentless pursuit of innovation, drives us to deliver ground-breaking new excitement and entertainment in ways that only Sony can. These make Sony one of the most comprehensive entertainment companies in the world.
Apply Mode : Walkin
Latest Walkins :
Walkin Date : 17th Feb 2017
Walkin Time : 10:00 AM
Interview Venue Details :
Sony India Pvt.Ltd,
A-31,Mohan Co-operative Industrial Estate,
Mathura Road,New Delhi-110044.


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While being tested during the F1 Grand Prix.
by Lauren Hogan, 19 April 2017
A busy intersection has had new technology installed to recognize and display the faces of jaywalkers.
by Bailey Hu, 18 April 2017
It looks like the newly-formed environmental police force is finally cracking down on air polluters.
by Justine Lopez, 17 March 2017
Fifteen police dogs in the capital have been equipped with VR cameras to help ensure public security.
by Justine Lopez, 13 March 2017
When police arrived, they found her belting out ethnic songs incoherently.
by Jocelyn Richards, 24 February 2017
The "Chaoyang Masses" app is named after Beijing's well-known volunteer crime fighters called the Chaoyang qunzhong (Chaoyang masses).
by Justine Lopez, 15 February 2017
Seventy-seven people were arrested in connection with organized prostitution at three exclusive nightclubs in the capital.
by Justine Lopez, 08 February 2017
Monkey King was driving.
by Bridget O'Donnell, 08 February 2017
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Explore the UK's leading showcase of fine food and drink
Grannys Secret
Handmade, cold pressed juices from fresh squeezed fruits. 100% Fruit; Nothing added and nothing taken away. Blackcurrants are the richest in Vitamin C - more than Oranges and kiwi..It is a good source of antioxidants.
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Available in 200ml bottles & 700 ml bottles. Shelf life 18 months....
This juice is great as a mixer with tonic water, as it creates 'pink foam' when pored in the glass. Ideal with some ice and a slice of lemon as a long drink....
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Colin Anderson via Getty Images
LONDON — A new sex app has been developed that allows strangers to control a woman's vibrator remotely.
"Miss On the Go" app has been unveiled by a Canadian entrepreneur, who wanted to extend the possibilities of online dating, the Daily Star reported.
The package includes the vibrator which lasts up to 2.5 hours, ten times longer than the average man. And it could be controlled remotely from anywhere in the world.
They can also see how many people the user has controlled before, as well as rate their performance.
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Definition of laquear
: coffer 4a
Love words? You must — there are over 200,000 words in our free online dictionary, but you are looking for one that's only in the Merriam-Webster Unabridged Dictionary.
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Origin and Etymology of laquear
Latin; akin to Latin lacus lake, basin — more at lake
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Dr. Dimitris Schinianakis received his diploma in Electrical and Computer Engineering from the Electrical & Computer Engineering Department, University of Patras, Greece, in 2005 and his Ph.D. from the same department in 2013.
His current research interests include 5G/IoT security architectures, cryptography, post-quantum and homomorphic cryptography, and computer arithmetic.
Since 2008, in parallel with his Ph.D. studies, he is employed in Nokia and in 2014 he was assigned as a Product Line Manager in Nokia/Munich with focus on Microwave Radio technology. In 2015 he joined NOKIA Bell Labs as a Security Research Specialist, focusing on IoT and 5G security strategies.
He is the recipient of a paper award from IEEE in 2006, and an award for a tutorial presented in ICECS 2009. He has authored more than 15 conference and journal papers and he has presented tutorials in international conferences on "Cryptographic Hardware Design". He was a special session speaker on ISCAS 2014 in Melbourne on "Alternative Number Representation Systems". He is also a co-author of the books "Secure System Design and Trustable Computing" and "Embedded Systems Design with Special Arithmetic and Number Systems", both by Springer-Verlag. He regularly reviews for conferences like IEEE ISCAS, ICECS, etc. and for journals like IEEE Transactions on Circuits and Systems I, IEEE Transactions on VLSI, and the Elsevier Journal of Systems and Software.
He is a Member of the IEEE and a member of the Technical Chamber of Greece.
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Transitions sometimes take me forever to mull just the right starting place. "Forever" might equal three days or more. For a productive writer who likes writing, that mulling stage seems like a dead end. I think some people might consider that to be Writers Block, but I don't think that way.
I think positive, that this mulling stage is only a nesting time for when just the right idea, or the right scene makes for a good transition in the story line. Frequently, transitions are used to jump the scene ahead to save space. Other transitions can be done with change of POV or with mood.
The next thing is Who Owns the Scene, in whose head are we? That is most important in every scene. Who owns the scene, or is it narration?
To balance this, consider what the scene is and who it most affects? That is who owns the scene. Sometimes just stopping and weighing who owns the scene makes for an easy transition, that is a change of POV.
Take the scene, consider who is most affected by it, which character feels most deeply about it, add a powerful first jump line and you're generally off and running with a transition.


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396 Hannifin Drive, Blacklick, Ohio
$167,9003 Beds1+ BathsFor Sale Under Contract
$167,9003 Beds1+ BathsFor Sale Under Contract
396 Hannifin Drive, Blacklick, Ohio 43004 - THIS WONDERFUL HOME LOCATED IN CRAWFORD FARMS HAS JUST BEEN PROFESSIONALLY PAINTED AND IS IN TURN KEY CONDITION. OFFERING CATHEDRAL CEILINGS IN THE GREAT ROOM, FULLY APPLIANCED EAT-IN KITCHEN WITH CERAMIC TILE FLOORING. 3 BEDROOMS AND 1 FULL BATH UP. THE LARGE FAMILY ROOM IS LOCATED IN THE LOWER LEVEL WITH THE HALF BATH/LAUNDRY ROOM. THE GROUND DECK IS LOW MAINTENANCE AND THERE IS A NEWER 6FT PRIVACY FENCE. 2 CAR ATTACHED GARAGE. THE HOME BACKS UP TO CRAWFORD PARK! NO BACK DOOR NEIGHBORS!
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text:
Reports: 'Happy Days' star Erin Moran dead at 56
San Antonio teen wins big on reality TV show
Spurs' Kawhi Leonard's incredible night not enough as...
Dewayne Dedmon is out for Game 4 against Grizzlies
How much money do you need to make to buy a home in San Antonio?
Sex with 12-year-old student gets teacher 10 years in prison
Beyonce, pregnant with twins, is being fat-shamed
Photos: Fiesta's Alamo Heights Night Friday was not to be missed
Mexico says 2 top drug traffickers killed near US border
Armed robbers hit multiple locations on the North Side...
Monthly Archive for December, 2011
Garrett fires up Cowboys with speech at team hotel
NEWARK, N.J. – Jason Garrett apparently closed out 2011 with one heck of a speech on the eve of the Cowboys' win-or-go-home game with the New York Giants. "Jason Garrett just gave the greatest speech …
McBriar's season over
NEWARK, N.J. – The Cowboys will be without two-time Pro Bowl punter Mat McBriar tonight after they placed him on season-ending injured reserve Saturday. McBriar has been plagued by a left foot injury …
NFL Fearless Forecasters: Jan. 1
http://blog.mysanantonio.com/nfl/files/2011/12/NFL-FORECASTERS-WEEK-17.jpg"> …
Jerry says it one more time: Garrett's job is safe
For the umpteenth time in the past month or so, Jerry Jones has issued Jason Garrett a strong vote of confidence. Asked about speculation from some in the media that Garrett's job could depend on the …
Giants' Jacobs reveals what fuels his hatred for Cowboys
Brandon Jacobs says Cowboys fans "get on my nerves" and are even worse than Eagles fans. The outspoken New York Giants running back loves to talk trash about Dallas and he was on a roll Thursday, …
Warner suggests bum hand, cold weather will combine to give Romo problems Sunday night
Still nursing a swollen right hand, Cowboys quarterback Tony Romo had limited participation in practice Thursday for the second straight day, according to the team's injury report. "The swelling still …
Is Spencer really having 'a very good year?'
IRVING – Outside linebacker Anthony Spencer is second on the Cowboys in sacks (6.0) and pressures (18). But over the last three games, he has zero sacks and only one pressure. Those facts were pointed …
Witten: Romo as "good as he always is"
IRVING – The Cowboys listed quarterback Tony Romo as being limited in Wednesday's practice because of his bruised right hand, but tight end Jason Witten painted a different picture. Tony did good," …
Romo throws passes in practice
IRVING – Tony Romo was at practice Wednesday, throwing passes during the portion of the practice open to the media. Romo wore a glove on his bruised right hand while the players stretched. When he …
Ware, Ratliff land on NFC Pro Bowl roster
IRVING – The Cowboys landed only two players on the NFC Pro Bowl roster. Outside linebacker DeMarcus Ware and nose tackle Jay Ratliff made it as starters. It's the sixth selection for Ware and the …


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Do the 5:2 Fasting Diet
Edited by Leomar Umpad, Eng
I would like to thank Tracy Kiss for the awesome video on 5:2 Fasting Diet. I organized it into a wiki for anyone with a hearing impairment, or for those who just prefer to read the steps. Hope you enjoy it. Hi, my name is Tracy Kiss. I'm 26 and I am a mother of two children. I have been a vegetarian since I was 5 years old - that's 21 years now. I basically want to try the 5:2 Diet as a way to lose 40 pounds.
- 1Here is a diary of everything I ate for 30 whole days. Throughout the 30 days, you pause twice a week to culture your daily intake of calories. On the days when you don't fast, which is 5 days in a week, you can eat whatever that you want. As you can see I had takeaways; I had birthday parties; I had cake, ice cream, pizzas, and spring rolls in a cheap shop - everything and anything.I'm really greedy with my food as you can see.Advertisement
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- 14Advertisement
Video: Do the 5:2 Fasting Diet
Referencing this Article
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Do the 5:2 Fasting Diet. (2016). In VisiHow. Retrieved Apr 27, 2017, from http://visihow.com/Do_the_5:2_Fasting_Diet
MLA (Modern Language Association) "Do the 5:2 Fasting Diet." VisiHow, visihow.com/Do_the_5:2_Fasting_Diet Accessed 27 Apr 2017.
Chicago / Turabian VisiHow.com. "Do the 5:2 Fasting Diet." Accessed Apr 27, 2017. http://visihow.com/Do_the_5:2_Fasting_Diet.
If you have problems with any of the steps in this article, please ask a question for more help, or post in the comments section below.
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text: We've now got loads of JCSC essays and other guff. Can I have some opinion on whether it is worth publishing these on the downloads or stick to more general odds and sods. (next to go on are a variety of forms from DGD&D's CD. ATFAs, blank CRs and more general forms would be useful too if anyone's got them).


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Archive for the 'Health Tips' Category
The Benefits of Using Coconut Oil in Your Hair!
The benefits of using coconut oil in your hair!
- Rub in your hair can shield it from damaged cause by heat styling and UV rays.
- Antibacterial and anti fungal compounds also help stop dandruff and hair loss.
- Rub a dab of the oil through your hair saturating the roots wait 20 minutes then shampoo as usual. (You won't need to apply a hair conditioner).
- Rub a dab in your hands then through hair to help cut frizz.
- Rub a dab on your split ends to de-frizz them.
- Rub some in your hair and leave in longer (2 hours) for a more intense hair conditioning.
- Rubbing a small amount in your scalp daily can stimulate hair growth.
How to use Coconut Oil step by step instructions:
Celebrate 10/31 – Halloween
Today is Oct. 31
- Halloween
- Frankenstein Day
- Books For Treats Day
- National Caramel Apple Day
- National Knock-Knock Jokes Day
- National UNICEF Day
- Scare a Friend Day
- National Magic Day
Halloween Safety Tips
Frankenstein Download (free)
Caramel Apple Recipes
Halloween Safety TIps
Trick and Treat Safety Tips
CPSC Gov Halloween Safety Tips
CPSC Gov Halloween Tips
Reader's Digest Tips
Ultimate Halloween Safety Guide – Discovery Health


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Edwin Chan 1 Developing a search strategy 台大新竹分院家醫科 陳宗伯 / 陳孟侃
Edwin Chan Evidence-Based Medicine (EBM) Resources 2
Edwin Chan Systematic Reviews and Meta- Analyses The Cochrane Database of Systematic Reviews The Database of Abstracts of Reviews of Effects (DARE) 3
Edwin Chan Critically-Appraised Topics (Evidence Syntheses) Clinical Evidence DynaMed National Guideline Clearinghouse 4
Edwin Chan Critically-Appraised Individual Articles (Article Synopses) The ACP Journal Club Evidence Updates Dartmouth EBM Database 5
Edwin Chan Unfiltered Resources [Finding an original study] Pubmed Medline 6
Edwin Chan Background Information/Expert Opinion UpToDate eMedicine National Guideline Clearinghouse 7
Edwin Chan 8 Outline Strategic approach to locating studies Designing a search strategy Tips to search PubMed Documenting a search
Edwin Chan 9 Contexts Clinical care Clinical education Clinical research
Edwin Chan 10 Finding as much as possible and being strategic Use multiple sources and start with the highest yield Electronic databases -Cochrane Review Group register -Cochrane Central Register of Controlled Trials(CENTRAL) -MEDLINE -EMBASE -Other databases-depending on your topic Reference lists of trials and reviews Handsearching journals and conference proceedings Personal communication
Edwin Chan 11 Designing a search strategy Formulate a focused question(PICO) Identify search terms from the elements of the focused question Combine the search terms Search the database
Edwin Chan 12 Focused question Translate the clinical problem into a focused question Dose preoperative education improve outcomes in patients undergoing lower limb joint replacement?
Edwin Chan 13 Extract the PICO terms from the focused question Does preoperative education improve outcomes in patients undergoing lower limb joint replacement? Patients or problemHip or knee replacement Intervention(s)Preoperative education,information,instruction ControlRoutine or standard care OutcomePain,QOL,mobility,LOS designRCT,systematic reviews
Edwin Chan 14 Note Not all PICOD terms are equally useful for searching Typically start with P & I C is useful if a specific comparator is named e.g. placebo or active treatment D is useful for limiting search to gold-standard designs Do not have to use all PICOD elements,sometimes just 2 will suffice Searching is an iterative process (trial and error ) Don ' t expect to get it right the first time
Edwin Chan 15 Searching the database -2methods Using Free-text terms directly search PubMed bibliographic database using the searchable PICO terms just identified Using MeSH terms MeSH thesaurus is a standardized dictionary of terms first search the MeSH thesaurus for MeSH terms then search the bibliographic database using the selected MeSH terms
Edwin Chan 16 http://www.ncbi.nlm.nih.gov/pubmed
Edwin Chan 17 Outline your Strategy Which of the PICOS elements will I initially use? Shall I do both MeSH &/or free-text searches? Shall limit the search
Edwin Chan 18 lnitial search strategy Start with a MeSH search of the P term Followed by a free – text search of the I term Combine the 2 searches and check the yield Patients or problemHip or knee replacement[MesH] Intervention(s)Preoperative educationm,information,instruction[Fr ee-text] ControlRoutine or standard care OutcomePain,QOL,mobility,LOS designRCT,systematic reviews
Edwin Chan Searching with MeSH terms 19
Edwin Chan 20 Search for the MeSH term/s corresponding to the P term/s
Edwin Chan 21 Result of MeSH thesaurus search …
Edwin Chan 22 Combine MeSH term/s with OR
Edwin Chan 23 Search PubMed using combined MeSH terms …
Edwin Chan 24 Search PubMed using MeSH terms as Major Topics only …
Edwin Chan 25 MeSH Search Results Scroll down to See syntax
Edwin Chan 26 Search syntax in Search details box …
Edwin Chan Searching with free-text terms 27
Edwin Chan 28 Free-text searching for the I term "preoperative education" OR "preoperative instruction" OR "preoperative information"
Edwin Chan 29 Free-text Search Results
Edwin Chan 30 Check search history by going to Advanced search view …
Edwin Chan 31 Combine P and I searches using AND(Advanced search)
Edwin Chan 32 Preview of search results (#of hits)
Edwin Chan 33 Select additional search limits if necessary
Edwin Chan 34 Search with limits
Edwin Chan 35 Result of limit search, click on Display Settings arrow
Edwin Chan 36 Tips:In order to minimise bias don ' t limit search on Language Source(which journal, database etc) Publication status (full article, conference abstract, unpublished etc) Publication date
Edwin Chan 37 Searching with Clinical Queries
Edwin Chan 38 Search for specific study aims
Edwin Chan 39 Select Abstract format from Display Settings … To save references in format exportable to RevMan, choose the MEDLINE display
Edwin Chan 40 Save or e-mail search results using the Send to menu
Edwin Chan 41 Print from the web browser
Edwin Chan 42 Documenting your search important for you, so you coa remember what you ' ve done important for users of you review document What you searched (databases etc) When you search ed (year) How you searched (syntax)
Edwin Chan 43 Managing you search history
Edwin Chan 44 Saving the search syntax in …
Edwin Chan 45 You ' re My NCBI account
Edwin Chan 46 Click on search syntax name to rerun search
Edwin Chan 47 Saving the search syntax in you ' re my NCBI account(alt)
Edwin Chan 48 Name the search syntax & save
Edwin Chan 49 Specify any e-mail update options in the Settings dialog box
Edwin Chan 50 Return to the list of your Saved Searches
Edwin Chan 51 Manage searches from Saved Search list-search, delete,edit
Edwin Chan 52 Search strategy summary 3.Publication types e.g.RCTs 1.Patient/Problem (MeSH headings OR text words) 2.Intervention (MeSH headings OR text words) AND
Edwin Chan 53 What if … Too many references is your topic too broad? is your question focused enough ? have you used enough PICO elements? have you mistakenly used OR instead of AND to combine across PICO elements try using limits try using NOT to exclude specific articles
Edwin Chan 54 What if … Too few references is your topic too focused? try using MeSH searches too capture all synonyms have you used too many PICO elements? have you mistakenly used AND instead of OR to combine within PICO elements don ' t use NOT have you left the limits function on by mistake? try checking known references for relevant key words are you searching the right database? is the terminology just too unstandardized & inconsistently used in this field?
Edwin Chan 55 Additional points No right time to stop searching;be guided by returns and resources Ideas for search terms from citatins you know of;try them and check returns Use a referencing database to manage citations Can import references into RevMan 5- display searches in MEDLINE format & save
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