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Fitbit, Inc.
- July 12, 2016
( 184,100)
Top Developer
The free Fitbit app is designed to work with Fitbit activity trackers and smart scales.
- Wirelessly SYNC your Fitbit Surge, Charge HR, Charge, Flex, One or Zip tracker with your Android device.
Learn more about Fitbit activity trackers and smart scales at www.fitbit.com
- Wirelessly SYNC your Fitbit Surge, Charge HR, Charge, Flex, One or Zip tracker with your Android device.
- Track your DAILY GOALS and progress over time for STEPS, DISTANCE, CALORIES BURNED and more
- Use GPS to track your RUNS, WALKS & HIKES
- LOG ACTIVITIES like YOGA and WORKOUTS to keep you on track
- LOG FOOD to see how many CALORIES you consume each day, and manage your WEIGHT over time
- COMPETE with FRIENDS with daily or weekly CHALLENGES and compare stats
- ALL DAY HEART RATE TRACKING with Fitbit Surge and Charge HR
Learn more about Fitbit activity trackers and smart scales at www.fitbit.com
See list of supported devices at www.fitbit.com/devices
Tracking my sleep patterns Lately my sleep records haven't been charting even though I 'Begin sleep' and tap I'm awake' every morning. I make the effort to do this am/pm but it's sad to see that it doesn't track consistently but only every other day.
Don't have any problems. Sometimes it does freeze for a while, same as all other apps, but restart always helps.
Love It! I find it extremely motivational, I didn't even want to take it off to charge. The information on the face of the watch is a bit odd to maneuver through but overall self explanatory. The app is sort of the same way. There's tons of information but just takes a while to figure it out. I wish the food-intake portion on the app was a little more user friendly and a bit more exciting. Overall a great product that I'd recommend to my closest friends.
S7 edge compatibility Great app and great product when it comes to the design and all of its functions are amazing. The downfall is its not fully compatible with the latest phone out which is the S7 edge! How do you come out with a new product not making it work with 100% compatibility with the latest phone out? When will this be fixed? I'll give 5 stars if i could fully us 100% compatibility such as answering or rejecting calls from the blaze, also reading the notification such as text, and have full music control!!
I bought a fitbit charge in December and the strap became faulty resulting in it separating from the face. I contacted Fitbit by email and their service was first class. They gave me a reference number, asked me to photograph the fitbit showing the fault alongside the reference number and email them this. Within ten days from initial contact with them I had a new replacement fitbit Alta (as they had no fitbit charge available) delivered to my door. They kept in contact with me giving regular updates on what was happening - excellent service. Well done to the customer service team.
Good but needs improvement It's hard to put on and tapping to change screens takes sometimes more then one tap. Not instant. Gets sweaty underneath during workout. Would recommend but know its not easy to put on. Overall I do like it and find it very helpful to reach my goals. It's great motivation. The style of it is cool and contemporary.
What's New
*Bug fixes and performance improvements
Check out details on sync support for Android:
Lifestyle companion to track your fitness, weight, diet, food & sleep.
Create the body of your dreams with a professional fitness trainer FitProSport!


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Wet Nose Therapy Dog T-Shirt
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Warm snuggles, little puppy kisses, and all the devotion in the world -- for free! How can any human therapist measure up to all that?
- 100% cotton
- Reads "My Therapist Has a Wet Nose"
- Machine wash warm with like colors; tumble dry medium
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- Shirt imported. Country of origin will vary by size and color
Garment Measurements:
Sizing in inches/centimeters:
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For bulk pricing on these and other items, please contact our Customer Service department at firstname.lastname@example.com.
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This is my favorite t-shirt. I love the slogan and it starts a lot of conversations. It represents how I feel about my dog. As for the material it self I love the weight of the fabric. Washes very well. Nice fit. Liked so much I have 3 brown ones and now have 3 navy.
- October 2, 2015
I bought a brown one for myself and had the same color shipped to my brother in another state. I was concerned that it would be too small for him, but I went by the sizing suggestion, and he said it fits great. He loved the shirt as much as I did. Always glad to shop through Greater Good, as it helps so many charities.
- August 30, 2015
Love this T-shirt! I spent more than usually do, but goes to worthwhile cause.
- June 19, 2015
I got this one for my husband who is seeing a therapist! He gets more comfort and sympathy from our dogs than his therapist. Unfortunately the dogs can't refill his prescriptions!
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Really cute and sizing is perfect...I ordered the brown and it is really pretty....
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Beavers Bend Luxury Cabin Rentals, located minutes from scenic Broken Bow Lake and Beavers Bend State Park, offers ... read more...
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A-OK Motel in Broken Bow offers rooms with king, queen, and double beds. In-room refrigerators and microwaves are ... read more...
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Frisco ISD Board Unanimously Approves Attendance Zone ChangesIt's official, some students in the Frisco Independent School District now know they will be attending new schools next year. Monday night, Frisco ISD trustees finalized new attendance boundaries.
Frisco ISD Board Could Finally Decide On New Attendance ZonesIt's a battle over school boundaries! At a meeting happening tonight Frisco Independent School District officials could finally decide what's next for thousands of students.
Major Milestone For New Dallas Cowboys Headquarters In FriscoA major milestone has been reached in Frisco. Construction on the new Dallas Cowboys headquarters has reached the halfway point.
Frisco High School Students Walk Out In Support Of PrincipalDespite being asked, over school loud speaker, by Frisco Independent School District Superintendent Dr. Jeremy Lyon not to, hundreds of students from Frisco High School left their classrooms and took to the streets Tuesday.
New Frisco School Bus Goes Up In Flames, With Students OnboardIt could have been disastrous... a rolling school bus, with two special ed students onboard, catches fire. But quick actions by the driver and a school monitor helped everyone escape unhurt.
Some Collin County Parents Stunned By School Redistricting PlansThere are few issues in Frisco that will get people more upset than redrawn school boundaries. A new proposal, dropped in front of parents Monday evening, is sending shock-waves through some homes.
Many Parents Learned Thursday Why A Frisco Principal QuitParents knew Corbell Elementary School principal David Wehmeyer suddenly resigned, but were surprised to find out why. According to the Frisco ISD, Wehmeyer quit after police alleged he stole some student medication.
Frisco School Board Approves $800 Million Bond ProposalThere has been a major decision in the Frisco Independent School District. Monday night the school board voted to approve a referendum for voters to decide on a nearly $800 million bond proposal.
Some Frisco Parents Upset Over New School BoundariesOne subject that will bring the emotions of many a parent to a boil is -- school re-zoning. Changing a child's school district is not what most parents want, but it was a topic on the table in Frisco Monday night.
Collin County School District Closes Some Schools To New StudentsThe Frisco ISD is bursting at the seams. It's growing so quickly, that for the first time, the school district closed off some elementary schools to new students -- weeks before classes even began.
Fast-Growing Frisco ISD Prepares For Thousands Of New StudentsAs one of the fastest growing districts in the nation, hundreds of new teachers – and thousands of new students - are expected in Frisco for the 2013-2014 school year.
Frisco Population Growth Due In Part To School SystemFamilies from across the state and nation are moving to the city of Frisco. While they all have their various reasons for packing up and heading out, many go to the city for one main reason: the schools.


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those programs relating to agriculture; aquaculture, including mariculture
Please help us! 3 Ways You Can Help
Vote Smart Just The Facts
"Experts nearly all recommend Vote Smart."
"Vote Smart is a bright light in an often desultory civic culture."
- Bill Moyers
"We think so highly of Vote Smart that we are distributing their materials to all of our affiliates."
Legislative Committees
Hawaii Senate Committee
Room 229
T Th Sa 2:45pm
Web Address: http://www.capitol.hawaii.gov/committeepage.aspx?comm=AGL
- There are no members currently assigned to this committee.


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Digital Charities is a group of people working together to share web knowledge and help each other deliver compelling web campaigns and content for our supporters. New members and ideas for sessions are always welcome.
We meet on the first Wednesday of the month, usually at the offices of one of our members, in London. We also have an online Slack team, and we're on Twitter and Facebook too, obviously.
Below are notes of past meetings and advanced warning of future ones.


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Join Date: Mar 2008
Location: Lake in the Hills
Posts: 608
Gamertag: H3rtZ DoNuT
Tips and Strategies
Sup guys, I've made a video that shows how to beat "Brawl for it All" mode easy and fast.
Part 1
Part 2
Tips and Strategies
Using those tips i was able to beat the game on Truculent and Impossible without much trouble.
If you guys have any questions or need help with this game feel free to ask.
Tips and strategies from Rex Pugna, lead AI/Gameplay engineer for FaceBreaker.
To play Truculent you should have a good understanding of all the mechanics of the game. I recommend reading the manual, there is a lot of good and entertaining information in there. Also, In general, this instructions assume that you have a fairly fast character that has a strong breaker like Ice.
Kekoa - Punch Kekoa until he dodges your punch, then dodge his punch back and continue. You can beat Kekoa with a FaceBreaker this way.
Steve - When Steve disappears to teleport immediately use your breaker button (Y or triangle) and hit him while he is invisible. Steve will be stunned for a moment allowing you to punch him once or twice. He also tends to change the rhythm from fast jabbing to hooks. When he is throwing hooks don't chase his punches, wait for him high or low as he will most likely continue to switch high and low.
Ice - Ice is a tough fight but he is quite predictable. He has a tendency to punch low so the best thing to do is defend low with dodge and wait for an opening to throw Ice in the corner. The use two hooks and a BoneBreaker followed by a HayBreaker.
Sparrow - She is a tough to fight toe-to-toe because she uses a very smart strategy (two jabs followed by a parry), however she is easy to beat once you realize she will dash-attack you constantly. Punch her with a hook and run away so she try to dash-attack you and defend here attack with dodge or parry. If you dodge her punch and hit her you'll have a few seconds so you can connect one more punch followed by a BoneBreaker and a HayBreaker. Run away after connecting the last HayBreaker and repeat the process. Be patient, she sometimes tries to trick you with the dash-attack by not releasing the punch right away.
Voodoo - Don't get hit his halitosis attack. Dash back to avoid being hit. Voodoo always throws his bad breath at you when you miss with a HayBreaker. You can normally escape if you dash back in time and have enough space behind you. If you get poisoned by it try walking back if you have space or mash buttons as fast as you can. Corner Voodoo with throws and in the corner use the same strategy Sparrow uses namely punch- punch-parry. After you punch him twice stop and charge a hook and Voodoo will likely punch into your charge and you will dodge him giving you the opening for a hook followed by a BoneBreaker also followed by a HayBreaker (if your character is fast). Learn this strategy well as it will be very effective against other users and AI characters.
Romeo - If you beat him on Fierce, you should be able to use the same strategy in Truculent and Impossible. You can chain 10 parry-parry-punch exchanges to get the combo meter to FaceBreaker level and finish it off with parry- parry-punch-FaceBreaker. BTW, this is a good fight to win the Untouchable achievement/accomplishment.
Spin - He is basically the same strategy as Sparrow but he is way harder. You will likely be hit with a SkyBreaker or FaceBreaker if you try to fight him toe-to-toe because he is so fast. With Spin you have to wait for him to dash- attack you then you dodge his punch (patience, he is even more tricky than Sparrow) and follow up with a hook a BoneBreaker and a HayBreaker. Wash-rinse-repeat. BTW, he will always try to dash attack you after you hit him with a hook or a breaker if you are far away enough.
Brick - Any time there is separation between you and Brick he will try his Stomp attack. Anytime you hit him with a dodge or parry hook that gives you separation dash attack him immediately and you will likely hit him in the middle of the Stomp giving you a chance to punch him again and do a BoneBreaker followed by a HayBreaker. If Brick's back is on the ropes you should run away to the opposite corner and dash attack him when he is about to get close enough.
Kiriko - Same strategy as Romeo but with dodges. Keep the L-stick pointed at Kiriko all the time to close distance. Once she is on the ropes you will be able to pull a FaceBreaker on her after a dodge punch.
Molotov - With Molotov you have to control space correctly and know the range of his HayBreaker. Charge a hook and walk back and around the ring (use all the ring). Hit molotov when he misses his HayBreaker and follow that with a punch a BoneBreaker and a HayBreaker. This should give you just enough separation so he will try his HayBreaker again. If you continue charging a hook and walk back and around the ring he will walk forward to try to catch you and he will try his HayBreaker at the edge of his range giving you the chance again to repeat the process. If you are cornered, don't panic, hold high (or low) and wait for him to punch you. After you dodge his punch and hit him with the hook use the throw button (B or circle) to reverse the positions and start walking back again. If you do this right you can win The Best D is a Good O achievement/accomplishment on this fight.
Socks - Socks is relatively easy to beat in the corner, especially if you use the punch-punch-dodge or punch-punch-parry strategy. He is really hard to fight in the middle of the ring so the easiest way to corner Socks is to dash back into your corner and use throw (B or circle) to reverse positions when he is getting close to you.
Tiki - If you beat Socks Tiki should be an easy fight. the only disconcerting thing about Tiki is that he changes rhythm from jabs to hooks. When he is jabbing try to dodge his jabs. When he is hitting you with hooks wait for him by holding down high or low and he will eventually switch and you will be able to dodge him. BTW, there are two ways to get away from Tiki when he is jumping on the turnbuckles: a) dash back to the opposite corner and he will fall down from the air in front of you and b) use the throw button (B or triangle) as he is approaching to hit you and you'll catch Tiki right from the air and do a body slam on the mat and it will hurt him a lot.
BFR - The Friendly Robot has three stages:
BFR Stage 1 - On the first stage he has a Stomp special attack just like Brick but you should not get separated from him so he will not get to use the Stomp. On this first stage he uses hooks continuously and stays with the same punch (high or low) most of the time. When you are in front of the robot, take a guess (high or low) and prepare to parry his punch. If you miss, the next punch will be the same punch 99% of the time so you should switch and try to parry his next punch. After you hit him with the parry hook follow that with a breaker and dash in to take another guess and repeat the cycle. I recommend using parry instead of dodge because the damage from the parry punches (you know they do more damage right) will offset the damage you take from guessing wrong.
BFR Stage 2 & 3 - You should not try to fight. Get separation by any means possible (dodge, parry, throw, HayBreaker, etc) dash wall the way back into your corner and wait for him to shoot a rocket. You can dodge the rockets by holding the high or low punch buttons (when he crouches down use low). He will shoot a couple of rockets and then come at you. Be patient, if you stay in the corner and you are fighting diagonally across the ring he will come close to you and shoot another rocket instead of hitting you. If you successfully dodge this rocket he will end up flying all the way back to his corner and continue shooting rockets from there. Repeat this process and you should beat him easily. You will loose if you try to fight toe-to-toe so if/when he hits you get separation again by dodge or parry. Don't panic and you should be OK.
Join Date: Dec 2007
Location: Central, NJ - USA
Posts: 154
Gamertag: RenegadeANIM4L
Thanks for the tips they're working like a charm.
Join Date: Feb 2008
Location: Ukiah,CA
Posts: 83
Gamertag: jakekh1983
thanks for the help. once i learned each fighters weakness it was pretty easy.
Join Date: Jan 2008
Location: Lincolnshire, UK
Posts: 45
Gamertag: Uncl Ad
Thanks bud...very benificial..
Join Date: Jan 2008
Location: Vault 13
Posts: 748
Never mind I did it.
Last edited by DreadfulCorpse; 10-18-2008 at 01:38 AM.
Join Date: Jan 2008
Posts: 285
Gamertag: KemikTheViking
Totally awesome tips many thanks
I've been trying to beat BFR for hours and didnt realise it was just as simple as dodging his lasers I was trying to beat the crap out of him and loosing everytime. Used your tip and beat him first time round
Many thanks!
Join Date: Nov 2008
Location: Mexico
Posts: 273
Gamertag: Alaska Has Eyes
I´ll try,maybe i can finally hit 1K
Join Date: Feb 2009
Posts: 14
Gamertag: ReBoRN I xX I
I cant do the reversal thing when fighting socks he just kicks my ass over and over and over... PS Im using VooDoo
Join Date: Jan 2009
Location: Heist-op-den-Berg, Belgium
Posts: 937
Gamertag: dennoman
I just finished getting 1k on this game. Thank you so much to the author, it was absolutely VITAL to helping me figure everything out.
An essential point of reference, don't know why this hasn't been stickied. I just finished my guide to Impossible difficulty, maybe we could combine the two into one big sticky. That would definitely be the penultimate guide for anyone that's struggling with this game.
Achievement Hall of Fame
Banned User
Join Date: May 2009
Posts: 865
Gamertag: NoLifeDGenerate
This game is insane. I did find one thing that kind of helped me with the robot stage 2-3 though. If he's coming at you and you know it's to beat you senseless: dash forward, hit him once or twice, then throw him into the corner and get back as fast as you can. Hopefully, he'll go back to shooting rockets. This seems a little safer then just letting him come to you and trying to dodge/parry.
Join Date: May 2007
Location: Lexington, KY USA
Posts: 354
Gamertag: park diesel
Just wanted to thank the OP, I never would have finished this game without this thread. I know this game isn't popular, but this thread should be stickied as it is very much needed for gamers. For those just starting out, I know it seems really hard, I thought I'd be sitting at 425 GS forever, but if you follow the advice and get familiar with the game 1000 GS is very possible.
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Marketing + Graphic Design Internship. As a Marketing & Graphic Design Intern you have a creative eye and skill set to create compelling imagery that enhances...
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Create implementation ready designs. We are looking for a UI/UX Design Intern to help with the process for creating screens for user-flows....
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Directs seasonal staff or civil engineering interns as assigned. Experience with AutoCAD Civil-3D software pertaining to municipal infrastructure design....


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IEEE membership options for an individual and IEEE Xplore subscriptions for an organization offer the most affordable access to essential journal articles, conference papers, standards,
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The main requirement of multimedia stream transfer is to guarantee the quality of service contracted between the user and the network. We are particularly interested by the synchronization constraints between different data streams flowing through a high-speed network. Our approach aims to regulate the streams progress reducing the gap observed between these streams and assuming that intermediate nodes in the network are capable of performing appropriate procedures that can be pre-defined by the user and the network manager. This intelligence allows the network nodes to manage the data streams, unlike passive carriers, by accelerating data units arriving after critical delays. We particularly address the case of active-ATM technologies and define for this purpose the active-ATM architecture that is needed for multimedia flow synchronization.
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Argument Between Two Sioux Falls Brothers Turned Violent, One Stabbed
A Sioux Falls man was arrested following a fight Monday morning with his brother that resulted in knife wounds
A Sioux Falls man was arrested following a fight Monday morning with his brother that resulted in knife wounds
Sioux Falls Police are investigating three car burglaries in the city.
Sioux Falls Parks and Recreation will host free summer fun for the entire family at the Kids' Nite in the Park event Tuesday June 28, 2016 at McKennan Park
Caddyshack, Zombieland, and the Big Lebowski are a few of the movies ready to debut in Rooftop Cinema in downtown Sioux Falls.
Uber is getting closer to being green-lighted to hit the streets of Sioux Falls.
Sioux Falls police are searching for a missing man according to Sergeant Kevin Henkel.
Police Spokesman Sam Clemens says 18-year-old Conner Hanson faces a number of charges after he reportedly broke into an apartment on Saturday
Nationally Ranked University of Sioux Falls Football Season Tickets Available
A woman who was leaving work on Friday night in Sioux Falls was startled to find a man sitting in her car.
A car wash mishap resulted in body work for a car, and construction work for a Sioux Falls car wash.


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Jets 3 Islanders 2
UNIONDALE, N.Y. Mark Stuart, Andrew Ladd and Devin Setoguchi scored second-period goals and Al Montoya made 28 saves for Winnipeg.
The Jets won their second straight to start a six-game trip. Winnipeg defeated New Jersey on Monday.
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text:
Pr�sentation de l'�diteur
MATLAB has become the standard software tool for solving scientific and engineering problems due to its powerful built-in functions and its ability to program. Assuming no knowledge of programming, this book guides the reader through both programming and built-in functions to easily exploit MATLAB's extensive capabilities for tackling engineering problems.
The book starts with programming concepts, such as variables, assignments, and selection statements, moves on to loops, and then solves problems using both the programming concept and the power of MATLAB. In-depth coverage is given to input/output, a topic fundamental to many engineering applications.
The third edition of MATLAB: A Practical Introduction to Programming and Problem Solving has been updated to reflect the functionality of the current version of MATLAB. It features new and revised end-of-chapter exercises, stronger coverage of loops and vectorizing, and more engineering applications to help the reader learn this software tool in context.
- Presents programming concepts and MATLAB built-in functions side-by-side
- Systematic, step-by-step approach, building on concepts throughout the book, facilitating easier learning
- Sections on common pitfalls and programming guidelines direct students towards best practice
Biographie de l'auteur
PhD Boston University
Department of Mechanical Engineering at Boston University, and Associate Chair for the Manufacturing Engineering undergraduate program within the department. She has been the course coordinator for the Engineering Computation courses at Boston University for over twenty years, and has taught a variety of programming courses using many different languages and software packages.


text:
Friday, 27 May 2016
Griff Gristle: Here Be Monsters Pinup
I was delighted to be asked to contribute a pinup to the fabulous sounding (and looking) Griff Gristle: Here Be Monsters comic which at the time of writing this is nearly finished its succesful Kickstarter campaign (and completely smashed it!).
The book is from the writing team of Rob Jones and Michael Sambrook, with art by the talented Rory Donald and published by Madius Comics. I met up with the guys at Thought Bubble last year and they're not only very talented and creative guys but they ooze enthusiasm for comics, both their own and those published by others. So with that in mind I was only too happy to help out.
Above is my contribution, where I join other talented sea dogs such as Vince Hunt, Ian Laurie, Neil Ford and Matt Rooke in all doing pinups for the book.
The bar was well and truly raised by those guys, I hope I got somewhere close to them.
Posted by Andy Bloor at 09:45 No comments:
Thursday, 19 May 2016
Birmingham Comic Festival thoughts...
It's been a while, about time I posted my thoughts on the recent Birmingham Con.
Getting there and setting up was easy, organisation was good as was the venue, sadly the 2 hour 'early bird' idea wasn't and it took a while before anyone bought anything - I don't think it was just us, speaking to others there it seemed very slow!
Luckily it did pick up and we sold some books, not as many as I'd liked. I had periods of being pretty busy then others where it was very quiet. Had lots of nice feedback on the books and my work but all in all the con was a bit of a damb squib for me, and a pretty long day too! I was quite glad to pack up and go home truth be told.
It was nice to meet up with super artist (and all round nice guy) Chris Imber and to catch up with Jason Cobley (making his first con appearance for years with his wonderful 'Amnesia Agents' book) but looking back on it, it wasn't the event I had hoped, and although great to see Mums and Dads bring their kids along (most dressed as superheroes) it does seem that maybe indie comics were not what they were expecting.
For those that did well, I'm pleased for you. If I continue with the self publishing into next year (which I am deciding about) then this is one con I will leave off the list.
Posted by Andy Bloor at 09:39 No comments:
Wednesday, 20 April 2016
Next con: Birmingham Comics Festival!
Welcome back to my sadly neglected blog!
Busy as ever behind the scenes, with some news I hope to share soon about my upcoming projects.
In the meantime, this Saturday I will be exhibiting at the Birmingham Comics Festival. First time I will have done this con as I was on holiday last year when it was on.
I will be selling my usual wares, copies of Midnight Man (now onto the 3rd printing!) plus the limited sketchpacks (which include the book, badge, A5 mini-print and an original sketched card), copies of Andy-Thology, prints and artwork.
My good friend and comics/film enthusiast John will be with me to help lend a hand on the day, which is always good!
I've booked more cons for later in the year as well, so far I'm doing:
Cardiff Independent Comic Con, Cardiff - Saturday June 25th
ICE, Birmingham, Saturday 10th September
Nottingham Comic Con, Nottingham - Saturday 29th October
Sadly I didn't get a table for Thought Bubble, although I've kindly been offered to table share with a comic professional (super nice bloke) but I'm still hoping I get something on the reserve list. Hopefully will have news on that later in the year.
A few other cons I'm looking at too, Bristol is a firm possibility, just looking into it.
Mo (Ali, co-creator of Midnight Man) and I have had some more great feedback on the book too from some unexpected people. Hopefully we can share more on that soon too. That book just continues to find more fans, which we couldn't be happier about.
Posted by Andy Bloor at 02:53 No comments:
Tuesday, 9 February 2016
Pipedream Comics reviews!
Had a couple of great reviews courtesy of the guys at Pipedream Comics.
The review for Midnight Man is here.
The review for Andy-Thology is here.
Alex from Pipedream came over to my table last weekend at the True Believers con, had a good chat with him and it was great to hear how much he liked the books. Thanks so much for the kind words Alex!
True Believers itself was a fun day. Sales were ok, about on a par with last year really. It's such a great venue and carries with a good atmosphere with parents taking their kids (most dressed up as superheroes along with the many cosplayers) that you can't help but not enjoy the day.
The less said about the drive home in truly horrendous rain and windy conditions the better!!
Posted by Andy Bloor at 08:04 No comments:
Friday, 5 February 2016
OK True Believers!! let's go...
Looking forward to heading back to Cheltenham Racecourse tomorrow for the True Believers comic festival. Had a good time last year and the buzz around this years con has been building and building so I'm hopeful it's going to be a good day.
Be nice to meet up with friends again and get the comic creating buzz back which I've lost of late.
I'll be selling copies of Midnight Man (just took receipt of the third!! printing of the book yesterday again great service by Rich Hardiman of Comic Printing UK - can't recommend this guy enough) Andy-Thology, both Wolfmen books, prints and original art.
Again I'll have special 'limited edition' sketchpacks of Midnight Man available, which, along with the regular comic contain a mini A5 print, badge and an original sketched card. All for just £6!!
Posted by Andy Bloor at 03:38 No comments:
Friday, 15 January 2016
Midnight Man gets an 'honourable mention' from Curiosity of a Social Misfit
Back to business, and nice to notice that Patrick Scattergood over at the Curiosity of a Social Misfit site gave Midnight Man an 'honourable mention' in the best graphic novels of 2015 category for his end of year awards.
Considering the amount of books published, I consider that an award it itself, so thanks very much Patrick!
You can check the post out here!
Nice to see my friends over at Accent UK get a few mentions and a runner up spot for Stephenson's Robot #1 (which features my design work) as best comic book! Well done guys.
Posted by Andy Bloor at 09:21 No comments:
Monday, 11 January 2016
RIP Simon Bloor
I wanted to pay my own tribute here to my dear brother Simon, who sadly passed away November 15th 2015, aged just 45.
My thoughts go to his wife, Elizabeth and his three children, Alex, Benjamin and Joseph.
It was thanks to Simon that I got into drawing, as he was much better than me growing up and I wanted to draw like him. He got me into comics too and he too was very fond of sci-fi, especially Star Trek.
Not a day goes by where I don't think of him, and I wanted to write these words just to say that I miss him terribly. RIP my dear brother.
Posted by Andy Bloor at 02:46 No comments:
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text:
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text:
"Culture Pilots" awarded as "Success Story" by the European CommissionDie Anfänge der "Culture Pilots", reichen zurück ins Linzer Kulturhauptstadtjahr 2009. Für "Linz09" entwickelte das BFI Oberösterreich ein interkulturelles Bildungs- und Integrationsprojekt, das Migrantinnen zu Kulturlotsinnen ausbildete. Diese boten in weiterer Folge... read more
Digital revolution – an opportunity for allWhile the ENTELIS Seminar 2015, which took place in Dublin, Tom Bieling, Design Research Lab (Berlin, University of Arts) and Sabine Siemsen (Hagen, FernUniversität) both invited by EVBB, gave a lecture. The designer Tom Bieling presented in a very entertaining way... read more
Close to the Decision Makers – The EVBB in BrusselsOn January the 4th the European Association of Institutes for Vocational Training has set a new milestone and established its new office in Brussels. Located in the European Quarter the EVBB will continue to fly its flags for the support of vocational training and... read more
Vocational Education and Training for Europe – The EVBB
The European Association of Institutes for Vocational Training (EVBB) is a European umbrella association whose members are state associations, associations, coordinating institutes of education and educational providers at a national, regional and local level.
The objective work done by the EVBB is the qualitative improvement of vocational education and training in European countries and an increase in the efforts being carried out in education at a European level.


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Kevin Clapp is halfway to the finish! He crossed the halfway point at 01:52:14, about 15 minutes ago. His pace is 8:33 and his predicted finish is 3:44:28 -- which means he actually sped up from miles six to 13.
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Looks like a great start.
Next questions:
10ft for 11 ft hammock
double or single layer
woopies or buckles
Type: Posts; User: Sky kid
Looks like a great start.
Next questions:
10ft for 11 ft hammock
double or single layer
woopies or buckles
If it happens, expect to see more stealth camping with cuben fiber cloaking devices installed. :)
Thanks.. Storyteller and I have hit about 7 of the Percy Priest islands since our first Bear island hang with ya'll . Bear is a good one for bigger groups, but for smaller groups, Luau island is...
Rock Island, home of Jackson Kayaks. I love that place, great falls, and an awesome place to pitch a hammock and relax in front of. I can't wait to see what the finished product looks like.
was there a weight difference between the nylon you have, and the argon? curious.
This looks really nice and a great price. If this is still here tomorrow, I might have to jump on this one. Gotta ask the wife.
A walk in the woods shuttle service. Good guys.
Very cool.
I'm guessing it has everything to do with the weight.
m90, momentum 90.Momentum 90™T
Momentum 90™ T is an ultralite downproof taffeta finished with a high quality DWR. Great feeling...
Lol, every time I see the word Dutch oven on here, I immediately think it's a new titanium dutchware type of oven. That could be a tad pricey. ;) The peach cobbler sounds amazing.
Everyone seemed extremely nice, and neat to see familiar faces,, and meet newbies. Got to see some really neat homemade gear, true innovators. We made it through...
Hated to leave early. You guys were great to hang with last night!
Wrong side of the river. It's in Lebanon. ;). Sounds like a fun time. Weather should be great!
That looks super nice!
So, I just found out from Adam, that this is the same as m90, and is the 15 denier fabric. He mentioned that they will change the name again soon.
Just curious.. I'd hat to order and find out that it meant something else worth shipping back to them. CS? Climashield? no idea here... :confused:
If anyone has a pic, that would be awesome, as...
Would you consider a trade?
Transaction complete. Please remove.
$200 now? sweet deal!! This won't last long..
Sold pending funds to Bigmuddy..PM me your email
I have an Enlightened Equipment Revelation X 10 degree quilt for sale. 6 feet long. It's the Wide model.
I've only used it about 4 times. Excellent condition. No defects, odors, or stains....
This looks like a great deal. I'm going to have to check with the wife.. ;)
Lived in WA for 7 years. Miss the mountains, don't miss the rain.
Did you ever use this is the hammock, or just on the ground? I'm curious how it did in the hammock. Also, how long is the Regular?
Wow, with that r rating, I could go to ground in winter, no problem. Looks like the new style, also. Sweet mat
I think its gotta be an understanding going into it, that the cottage vendors will make great stuff, and most likely here in the USA. I've had to wait on orders, but I knew what I was getting into,...
Cool vid. What was that song at the end. Good stuff..
Dang. Nice pickup.
funds received, sold to John4. Thanks to ErickSaint for his help moving the sale along.. ;)
curious how much this one weighs...
sold pending funds to John4
Is right at 19 ounces. It's the original neo air, 2011 version. I've been offered 70, but really think it's worth it at 75 plus 5.. Maybe I'll try it over on wb if this doesn't sell soon.
I'll give it one last try
Bumper time
Bumpity bump
Hey guys, i'm selling my Thermarest Neo Air Mattress SOLD!! TO John4. It was $170 brand new, and I don't need it anymore. Great in the hammock or on the ground. This is so light...
I always take mine. Use them if I need them. Had some fellow ground dwellers complain, but I think they were just jealous... :)
She took your dog? Cold blooded....
Nice pics by all.
Great idea on the trek pole water grabber....
Great video, as usual. I love the parachute idea. Wonder how much it weighs.. ;)
In, grandtrunk has a WBBB? lol.. nice..
glad you are on the board STT. Plenty of fun to be had out there in them thar woods..
Good stuff.
Word! Can't wait for it...
Seems to me that I've always encountered at least one shady person, at the shelters on the AT. I've felt safer in the hammock, than on a slab with 15 other people I don't know. I'd rather stealth...
Looks like a nice setup! Enjoy the ride.
I second the long hunter loop. Some good water sources, and is as close as you'll get to being out there.
Looks like a great place to have a group hang. Let's do it!
Here is mine.


text:
Love Hurts
- Height: 20m
- Bolts: 5
- Ascents: 162
- Description:
- Vertical Face Wet in rain Afternoon sun NW
- Ethic: inherited from Kangaroo Point
Climbing at 'KP' has a long, rich history and the cliffs are used by a huge number of climbers of very varied styles and abilities. For these reasons ethics here are complicated. However here are some undisputed points:
- Avoid lowering off directly through the anchors. All lowering off and top roping should be through quickdraws attached to the anchors. If you are last then rethread and rap off to avoid wearing the anchors.
- Don't ever chip holds.
- Don't paint on any new initials.
- Retrobolting is acceptable only after consensus is reached with a large cross section of the 'KP' climbing community. New bolts that don't meet this criteria are chopped, often within a few days.
- Tags:
Walk off
Rap off
<5 min
Route History:
First Free Ascent: Andrew Barry & Paul Hoskins, 1984
Located in Left Main Wall approx:
Long/Lat: 153.034342,-27.478173
Some content has been provided under license from: © Australian Climbing Association Queensland (Creative Commons, Attribution, Share-Alike 2.5 AU)
Route Grade Citations
|Community registered grade
|ACA Route Register
|Kangaroo Point Climbing Guide
|Kangaroo Point
Route quality
Overall quality score: 61%
Lead Styles
Comments keywords
pumped challenging crux difficult tough hard super fun cool nice good sweet satisfying wicked awesome scary fall rest interesting fingers awkward roof layback thrutchy easy mantle undercling lip crack
Learn about creating circuits.
Activity Configure
Check out what is happening on Love Hurts (note there is an unresolved system issue where ascents logged before 2010 are not displayed in the stream below). If you just want to see beta then click on view ascents with beta.


text:
Hello Oliver, and also hello Shami,
I have not read through the text of this post, but react from a more over-view perspective.
I carry out a significant amount of Anti-Fraud work, as a Civilian, and I deal with a number of raging psychopaths, as so wonderfully pin-pointed in the 20 items in the Hare Psychopathy Checklist.
My targets abuse and wrap around their little fingers the laws to do with protecting the rights of the individual.
At this point in time I have two fraudulent Harassment Notices against me filed on the central Police record of such notices, that can be accessed from any Police Station Front Desk, and three bogus Complaints about me filed at my Institute of Chartered Accountants in England and Wales; although in both environments SOME people are, at long last, waking up to where the smoke-generating fires actually are burning; and it's not my pants are not on fire, but those of my targets… three Police arrests should soon take place, within the Thames Valley Force area…but it often resembles trying to run in setting tarmac…
For me the relevant laws bring themselves into gross disrepute, since they offer so many opportunities for criminals and worse, and for their totally bent Solicitors, simply to take the piss out of EVERYBODY, until and unless the likes of Theresa May cuts through all of the crap and gets the very obviously desirable result achieved. Exit stage airport – terminally…. YES!
I'm glad that I'm on the same side as said Mrs May, who really knows how to read The Riot Act, con moto; as to the Police Federation recently.
I note for balance that I know of as many bent Chartered Accountants, for some of whom the writing is on their comfortable and affluent walls, as I do bent Solicitors.
There seem to be far fewer bent or psychopathic members of the Bar, who then go on to be QCs and Judges – save for the likes of the Barrister whose assault on a witness in a sex abuse trial led to the personal re-disintegration and suicide of that witness, who had suffered mightily from the two accused in sexual assaults and abuse in childhood – who were then convicted and sentenced.
SURELY some wise people recognise that a particular line was crossed on that day, and in that Criminal Court, by that Barrister, and NOT by anybody preparing the case.
A witness's natural rights were so severely assaulted as to lead to her suicide, and to her husband's and children's' bereavement.
That was, for me, a terrible, terrible injustice; mistakes are bound to be made, since we are fallible human beings – but that seems to have been less of an innocent mistake, and more of the product of an unworthy attempt to discredit a witness, so as to try to protect a pair of utter scumbags and super-perverts.
I am, fortunately for me, a far, far more resilient witness, and I fend off attempts to intimidate me, or to bring me discredit. I am becoming very adept in using such attempts to discredit their originators, and far more effectively…… I return their gifts with bells, whistles, and bombs attached…metaphorical and legal, of course..
Yours, George Gretton


text:
Tattoo Submission: Kim J (Germany) - 31st October 2012
This is one of my five tattoos. A tiny heart – Love it a lot.
This is one of my five tattoos. A tiny heart – Love it a lot.
Anja (Zug) "first tattoo"
Samantha (Newark, Delaware) "Done By Tj Delany at Agaru Tattoo in Delaware"
Johanna (Uppsala, Sverige)
These are mine and my boyfriend's unintentionally matching tattoos, both on our wrists. Mine is the small one on the right, his is the left. He's in the Navy and is based in the Middle East for a year. They keep us anchored to each other whilst we're living over three thousand miles away from one another.
"To live would be an awfully big adventure"
I got it as a beginning to my gap year in between high school and college. It is a reminder to never let my depression get the best of me, and also to take on any adventure that comes my way.
Stéven F (Paris) ""Lorsqu'on est heureux à Paris, on peut être heureux n'importe où. Surtout à Paris" L'amouràtrois"
Caroline (Denmark) "En trekant som er et tegn for båndet mellem mig og min to søstre." www.anewsituation.blogspot.com
Astrid (Copenhagen) "I got my tattoo in Berlin this summer 2012, It is my sisters name "Alfa Liv" and i chose the name for her, and it means "the first life" And she is what is most important for me and she always comes first in my life."
I thought I'd do this post even though it isn't tattoo related because quite simply I love blogs. I love blogging and I like reading blogs, I follow hundreds. I follow all of the big ones that every...


text:
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Tech Thursday: 15% off 1+ Yards of Sport Lycra, Performance Knit & Performance Pique Fabrics Through 11:59 P.M. EDT July 28th.
A successful architect & commercial interior designer; these days I am loving my transition to product, graphic and surface design! Please contact me via my site for licensing info and join my growing list of licensees "izing" their products!
© 2008-2015 Spoonflower Inc


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Florida Georgia Line with Dallas Smith and Chris Lane: Tuesday, April 15 at Rexall Place. Tickets: $39 to $59 plus service charges. On sale: Friday, Dec. 13 at Ticketmaster.
Florida Georgia Line bring the Good Times — and warmer weather? — to Edmonton
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Best Fictional Mothers
The Top TenXW
1Molly Weasley from Harry Potter
She loves all her children dearly, & even welcomes Harry & Hermione into her own home as her own children (Harry especially, as he is an orphan, & his own uncle & aunt treat him terribly).V1 Comment
2Hinawa from Mother 3
It's spelled Hinawa, people. :/ Regardless, I love this character. She's funny, fair, and must I say very brave to sacrifice herself to save her kids: Lucas and Claus.
3Akiko Minase from Kanon
4Ms. Brisby from The Secret of Nimh
5Trisha from Full Metal Alchemist
6Bambi's Mom From Bambi
7Rosalina from Super Mario Galaxy
8The Delarosa from Homestuck
9Mortisha from The Addams Family
10Kanta from Lucky Star
The Contenders
11Donna from The Cleveland Show
12Kala from Tarzan
13Marge Simpson from The Simpsons
14Mrs. Jumbo from Dumbo
15Belle-mere From One Peace
16Claire from The Cosby Show
17Ursa from Avatar: The Last Airbender
18Kya from Avatar: The Last Airbender
She sacrificed herself for her daughter, although she didn't know she was going to die.
19Lily Potter from Harry Potter
20Narcissa Malfoy from Harry Potter
BAdd New Item
Most Iconic Songs by Male Artists/BandsTop Ten Saddest Musician Deaths of the 20th Century Best Video Games of 2015 Best Songs of 2015 Top 10 Online Flower Ordering Websites Best Car Insurance Companies Best Fitness Center Chains and Gym Franchises Best Home Workout Programs Best Alternatives to YouTube Best Web Hosting Companies
Related ListsTop Ten Scariest Fictional Monsters Top Ten Greatest Fictional Characters Top Ten Interesting Similarities Between Fictional Characters And/or People Top Ten Most Inspiring Fictional Characters Most Powerful Fictional Characters
List StatsUpdated 29 Jul 2016
32 listings
2 years, 321 days old
2 years, 321 days old
Top Remixes
1. Molly Weasley from Harry Potter
2. Ms. Brisby from The Secret of Nimh
3. Hinawa from Mother 3
2. Ms. Brisby from The Secret of Nimh
3. Hinawa from Mother 3
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Kendrick Daye is lounging in his Midtown studio, surrounded by work: The carpet is covered in bright drips of paint and scraps of paper left over from the collages and paintings he creates under the name GreatEclectic. His computer screen glows with an inbox full of messages about his graphic design work and Art Nouveau magazine, the slick urban art and culture publication he started in 2007. The neckline of his shirt has been chopped open and exposes a sprawling chest tattoo that spells "Crazy" in an ornate script. He runs his ring-covered fingers over a wispy moustache, past the beauty-mark piercing under his bottom lip. "I'm more into style than fashion," he says. "I don't really care about fashion. Honestly, there's a pile of clothes in my closet and I just pick whatever I see first."
Daye's sense of style informs everything he does. The Basquiat-and-Warhol-cribbed spirit that runs throughout his celebrity-themed solo show, Want to See a Sad Boy Smile? Pay Him, opening April 14, is just as visible in the pages of Art Nouveau. When he blogs something like "Kanye West Has Every Right To Be An Asshole" for the magazine, those subjects usually show up in his visual work. From painting to publishing to music to graphic design, Daye sees little use in drawing distinctions. "Art is everything. It's the way you dress, what music you listen to, where you live, what you eat. Your entire life is art — that's your biggest masterpiece, you know?"
That principle is certainly visible in Daye's apartment, where the couch has been hastily reupholstered with bright red fabric, one wall painted day-glo green, and every available surface covered in some sort of project. Everything around him bears his mark. "It's usually extremely loud in here," he says, explaining that he listens to long mixes — a group of songs about money for example — as way of entering into the mood he needs to create.
Once he's there, though, Daye says his work doesn't take long, "The process is quick no matter what I'm doing — if it's design, if it's making pieces for show — it's always a quick process. I just get it done and get it out of the way," he says.
That immediacy and play between mediums have come to define Daye's work as a promoter. Not long after moving from Miami to Atlanta to attend Morehouse College in 2005, Daye started putting on events and parties that brought together musical performances, fashion shows, live painting and art exhibits, such as 2010's Super Pop! and FUNKshion. Through the events and the magazine, Daye's found himself around more than a few local artists he admires — musician and painter Corinne Stevie Francilus, interdisciplinary photographer Nikita Gale, self-styled art star and painter Fahamu Pecou among them — but has neither embraced, nor been embraced by, the local gallery scene.
Daye says that finding a gallery in 2009 for his two-person show with Francilus, Extraordinary Machine, was a frustrating process. "No one was trying to show our work anywhere," he recalls. Since then, he's resolved to produce his exhibitions without any outside help. "It's better to do everything on your own," he says. "I hate having someone over my shoulder telling me what to do."
Operating with such a strong DIY ethic has its challenges, though. "If the venue doesn't have sound, you have to get sound. The venues, none of them have lights, so you have to get lights," he says. "I never enjoy any event, but I refuse to let someone else do it. I have to do everything because I feel like it won't get done."
Daye says the added expense and effort is worth knowing that he hasn't compromised his vision. "When people think of art, they think of the snooty museum type of art, but urban art and street art came out of the fact that [those artists] weren't included in the museum and what contemporary art is supposed be," he says.
That iconoclastic vision alludes to Daye's childhood memories of his parents. His father was a talented portrait artist. "He was really good at drawing. There were portraits all over my grandmother's house of random people, of cartoon characters, all types of stuff," he says. But Daye mostly credits his creativity to his mother, an event planner who knew how to bring people together on a slim budget without sacrificing the style necessary for orchestrating a wedding or making the centerpiece for a dining table. "I consider her more of an artist than my father," says Daye. "He had that technical skill but she was more creative. What she can pull off with nothing — to me, that's what being creative is."


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John M.
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nice pl;ace for me to have lunch
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Outside Ballyhoo Grill, a sign made to look like a colorful speedboat beckons to passersby, hinting at the smorgasbord of fresh seafood to be found within. A tropical theme permeates the space, with live music setting a relaxing mood and nautical decor—such as a surfboard, an alligator head, and a stuffed and mounted kraken—adorning the walls. Guests dine on surf 'n' turf plates, fish tacos, pulled pork, and burgers as they share friendly conversation and clink cocktail glasses and mugs of draft beer.


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Sachin + Babi
Bevan Sleeveless Top
$ 99 Gilt
$ 250
Well-heeled fashionistas around the globe love Sachin + Babi, the exquisite line by husband and wife designers Sachin and Babi Ahluwalia. Pretty meets edgy in this collection, featuring silk tops with fringe accents, leather skirts in eye-popping colors, and textured, belted trenches.
Flattering, feminine picks for work to weekend
Here today, gone tomorrow: stylish apparel and accessories not to be missed
Contemporary clothing for the all-encompassing woman, by the husband-and-wife duo
Make an entrance they won't forget


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Posted by ESPN.com's Heather Dinich
CLEMSON, S.C. -- As Maryland headed into its locker room trailing 17-6 at the half, freshman defensive tackle A.J. Francis stopped at the 50-yard line, knelt down and said a quick prayer.
At this point, prayer might be the Terps' best option.
Maryland is making Clemson look like, well, like the No. 9 team in the country. You remember, how everyone thought the Tigers would look heading into that Alabama game? Right.
The Tigers look hungry. They look tough. They are playing physical, despite what defensive coordinator Vic Koenning said earlier this week (or maybe because of it).
Maryland looks disoriented. It is.
Defensive coordinator Chris Cosh is going to have a hard time explaining this one. James Davis and C.J. Spiller are having their way with his defense and will both have well over 100 rushing yards before the game is over. Davis already has 100. They're throwing it just to amuse themselves, because there's certainly no need to at this point.
Clemson punter Jimmy Maners must be so bored. They've only needed him once. The Tigers have 14 first downs to Maryland's three. They have 260 yards of total offense to the Terps' 93. They've controlled the clock.
This is not a game where turnovers are important. Heck, Clemson handed it to Maryland twice and the Terps couldn't get in the end zone. They couldn't ask for more -- one pass even bounced off a receiver's back and into the hands of Ronnie Tyler for a lucky first down.
Maryland's offensive line is overrated and underperforming. The Terps have come within inches of the end zone and couldn't score a touchdown.
Francis has the right idea. It's going to take a miracle for Maryland to win this one if it comes out and plays the same way in the second half.


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Hey I have an 04 acura tl, I just bought it a week ago and its making a howling sound when I start drivin but it doesn't kick in until about 20-30km and gets louder as I go faster. If I take my foot off the gas it stops its only when I'm giving gas, what can this problem be? Wheel bearing perhaps?
Tuesday, September 4th, 2012 AT 4:41 PM


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none of the bars and screws lined up right on the bed. the canopy is very weak my daughter threw a ball up in the air and it landed on it and it riped and was a ball from a crane machine. i had it for three days and returned it to the store for a different one.


text: The Institute for Business in Society convenes events and programs of broad impact to address critical issues related to business and society. Upcoming events and initiatives include: June, 2016 CREATING GROWTH AND RESILIENCE IN BUSINESS Helping local, post start-up companies scale and grow A day of problem-solving, practical toolkits and networking for organizations looking to take their business to the next level June 23, 2016 UVA Darden School of BusinessSo you've survived the startup phase of your business. How do you now scale and grow? At this conference, faculty from the UVA Darden School of Business and industry experts will assist small and medium-sized Virginia business owners in exploring the scaling phase of entrepreneurship. Participants will learn from peer companies, panelists, Darden Professor Ed Hess, author of Grow to Greatness: Smart Growth for Entrepreneurial Businesses, as well as keynote speaker John Bassett, author of Making It in America: A 12-Point Plan for Growing Your Business and Keeping Jobs at Home. Hosted by the Institute for Business in Society, this conference will include panel discussions, networking sessions and a hands-on workshop to provide support and tools to entrepreneurial business leaders. We will address growth across various business functions, such as: Accounting Financing HR and Payroll International Trade Marketing and Sales Attendees will participate in a hands-on workshop with Professor Ed Hess and receive a complimentary copy of his book, Grow to Greatness: Smart Growth for Entrepreneurial Businesses, which addresses the top ten business growth challenges and how to overcome them. For an overview of the agenda, please click here. Registration While there is no charge to attend this conference, registration is required and by invitation-only. For more information, please contact us at email@example.com.


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Aminoacylase 3 (AA3) mediates deacetylation of N-acetyl aromatic amino acids and mercapturic acids. Deacetylation of mercapturic acids of exo- and endobiotics are likely involved in their toxicity. AA3 is predominantly expressed in kidney, and to a lesser extent in liver, brain, and blood. AA3 has been recently reported to interact with the hepatitis C virus core protein (HCVCP) in the yeast two-hybrid system. Here we demonstrate that AA3 directly binds to HCVCP (Kd~10 μM) that may by implicated in HCV pathogenesis. AA3 also revealed a weak endopeptidase activity towards the N-terminus of HCVCP.
Hepatitis C virus (HCV) affects ~3% of the world population [1]. HCV infection is associated with liver steatosis, cirrhosis, hepatocellular carcinoma, cryoglobulinemia, insulin resistance and diabetes 2 mellitus. The HCV core protein (HCVCP) plays an important role in the HCV assembly. It is formed from a single polyprotein of about 3000 amino acids by host signal peptidase to yield p23, a 191 a.a. protein attached to the endoplasmatic reticulum (ER) membrane that is subsequently cleaved in the C-terminus generating a 177(179) aa. mature soluble protein p21 [2]. After the release from the ER membrane and trafficking to the cytoplasm, HCVCP associates with lipid droplets that is an important step in the HCV maturation [2,3]. The hydrophobic domain 2 (118–170 a.a.) is involved in the association of HCVCP with lipids, whereas the soluble basic N-terminal domain 1 (1–117 a.a.) interacts with the viral RNA that is responsible for the nucleocapsid formation [2]. A recent study [4] demonstrated that of basic residues located in two clusters (cluster 1, aa. 6–23; cluster 2, aa. 39–62) within the N-terminal 62 amino acids of HCVCP, only four basic amino acids residues of cluster 2 (Arg50, Lys51, Arg59, and Arg62) are essential for the production of infectious viral particles. Nevertheless mutation of these residues did not interfere with HCVCP subcellular localization, HCVCP–RNA interaction, or HCVCP oligomerization [4].
The N-terminal domain also interacts with a number of host proteins including transcription factors, receptors, and protein kinases [2,3,5–7]. The interaction of the N-terminal domain with the proteasome activator PA28γ in the nucleus is involved in the degradation of HCVCP mediated by 20S proteasome [8,9].
Recently a new binding partner of HCVCP in the host cell was indentified using the yeast two-hybrid system, namely aminoacylase 3 (AA3) [10]. AA3 is expressed in the kidney, liver, brain, blood [11,12], and mediates the deacetylation of N-acetyl L-amino acids with aromatic side chains as well as the deacetylation of mercapturic acids (S-conjugates of N-acetyl cysteine) derived from the glutathione (GSH) dependent detoxification of exo- and endobiotics [13]. The deacetylation of N-acetylaromatic amino acids formed from protein catabolism is a salvage mechanism given that the majority of eukaryotic proteins is Nα-terminally acetylated [14]. The AA3 mediated deacetylation of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, the excretable product of the GSH-dependent detoxification of trichloroethylene, in renal proximal tubules [15] is responsible for acute renal failure induced by trichloroethylene [12,13]. The AA3 mediated deacetylation of a mercapturic acid generated during the GSH-dependent detoxification of a toxic aldehyde 4-hydroxynonenal is neurotoxic [16]. Inhibition of AA3 completely protects neuronal cells from 4-hydroxynonenal toxicity [16].
AA3 is expressed in liver and blood; therefore the AA3–HCVCP interaction may potentially play a role in the HCV pathogenesis. Given the potential false-positive signals in the yeast two-hybrid system, we performed this study to determine whether AA3 and HCVCP proteins interact.
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) except Complete protease inhibitors cocktail that was purchased from Roche (Indianapolis, IN, USA). The following peptides were synthesized: STLPKPQRKT (HCVCP-short) and STLPKPQRKTKRNTIRRPQDVKFPGG (HCVCP-long) corresponding respectively to aa. 2–11 and aa. 2–27 in HCVCP, SSLPGSREPL corresponding to aa. 2–11 in mouse AA3, and STENVEGKSPN corresponding to aa. 2–12 in the human sodium bicarbonate cotransporter NBCe1-A (SCL4A4). The N-terminally acetylated (Ac) versions of all peptides were also synthesized. The N-terminal acetylation of these proteins (after the cleavage of a methionine) was either predicted [17] or was demonstrated experimentally for the HCVCP expressed in insect cells [18]. The purity of all peptides was 95.7–98.1% as was confirmed by HPLC and mass-spectrometry (MS).
Mouse AA3, with high structural and sequencing homology (with human AA3 [11,12,16], was used in this study. Mouse AA3 was expressed in E. coli as an N-terminally Strep(II)-tagged protein using the pRSET vector (Invitrogen, Carlsbad, CA, USA). Sequences of all constructs were confirmed by bi-directional sequencing using an ABI 310 sequencer (Perkin Elmer, Foster City, CA, USA). E. coli was grown to the optical density of 0.6, and then 1 mM isopropyl β-D-thiogalactopyranoside was added. Three hours later, bacterial cells were precipitated by centrifugation at 6000 g for 20 min and resuspended in PBS. The procedure was repeated twice, and the cells were disrupted in a BugBuster™ HT Protein Extraction Reagent (Novagen, Madison, WI, USA) containing Complete protease cocktail (Roche). Mouse AA3 was purified using affinity chromatography on Streptactin Sepharose (GE HealthCare, Piscataway, NJ, USA) in the presence of Roche complete protease inhibitor cocktail. The enzyme was eluted from a Streptactin Sepharose column with 3 mM desthiobiotin in 50 mM Tris-HCl, pH 7.5, 0.2 M NaCl, and desalted on a PD MiniTrap G-25 column (GE Healthcare). Purified AA3 was >99% purity judged by SDS-PAGE.
The HCVCP cDNA coding aa. 1–177 was recloned from the pcDNA 3.1 vector containing HCVCP genotype 1 provided by Warren Schmidt (University of Iowa) to the pRSET vector (Invitrogen, Carlsbad, CA, USA) as a C-terminally 6xHis tagged protein and expressed in E. coli. The expressed HCVCP was solubilized in 9 M urea in the presence of Roche protease inhibitors cocktail, and purified using metal affinity chromatography on a Ni IMAC FF column (GE HealthCare). Protein was eluted with 200 mM imidazole in 50 mM Tris-HCl, pH 7.5, 0.2 M NaCl, 0.03 % dodecyl β-D-maltoside, and desalted on PD MiniTrap G-25 column.
Equal volumes of 0.1 mM solutions of mouse AA3 and HCVCP in 0.1 M Tris-HCl, pH 7.5, were mixed. The solution was absorbed on electron microscopic grids immediately after mixing. Grids were washed with milliQ H2O and negatively stained with 1% uranyl acetate. Micrographs were recorded on a 4k×4k CCD camera at 30000–200000× magnification in a FEI Tecnai F20 electron microscope operated at 200 kV.
The affinity of HCVCP and peptides to mouse AA3 was measured using the surface plasmon resonance method in a Biacore T100 (GE HealthCare). AA3 was immobilized on a CM5 Biacore chip using matrix activation by N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1:1 mixture). 50 mM Na-phosphate buffer, pH 7.5, was used as running buffer. Mouse AA3 (10 μg/ml in running buffer) was captured on flow cell 2 at a density of 300 response units at a flow rate 5 μl/min. HCVCP and peptides were injected over flow cells 1 and 2 at a flow rate 30 μl/min. The complex was allowed to associate and dissociate for 150 s and 420 s, respectively. The surface was regenerated with 50 μl of 1 M NaCl (for peptides) or 50 mM NaOH (for HCVCP) in running buffer. Various concentrations of peptides (1 μM – 10 mM) and HCVCP (0.01 μM – 1 mM) were injected over both flow cells, and the response curve on the flow cell 1 was subtracted from the flow cell 2. The data were fitted by the Kinetics/Affinity analysis with the Biacore T100 evaluation software, version 1.1.
The reaction assay contained 3 μM AA3, 1 mM peptide or 0.1 mM HCVCP in 50 mM Tris-HCl, pH 7.5, and 1 mM CoCl2. Cobalt (II) was added to the assay because it has been shown to significantly activate AA3 [12]. After incubation for 18 h at 37°C, an aliquot was diluted in milliQ H2O to a final peptide concentration 50 μM and injected (100 μl/injection) onto a reverse phase HPLC column (polymeric resin, 150 × 1 mm, 5 Å, Phenomenex, Torrance, CA, USA) equilibrated in water/formic acid (100/0.1, v/v) and eluted (50 μl/min) with an increasing concentration of acetonitrile (time/% acetonitrile: 0/0, 60/100). The effluent from the column was passed in series through a fixed wavelength UV detector (215 nM) and then an Ionspray™ source interfaced with a triple quadrupole mass spectrometer (PE Sciex API III+) that was scanning from m/z 300–2000 (orifice 65 volts, 6 secs/scan). Accession of spectra and deconvolution of the multiply charged ion clusters into true molecular weight spectra was performed using the instrument supplied sofware (MacSpec™ version 3.3, PE Sciex). Cleavage sites in the peptides were determined using the EXPASY FindPept tool (http://www.expasy.org/tools/findpept.html).
The deacetylating activity of AA3 towards the Nα-acetylated peptides was determined in an assay, which in a total volume 2 ml contained 1 mM peptide, 3 μM AA3 in 50 mM Tris-HCl, pH 7.5, with or without 0.1 mM CoCl2. The reaction was started by adding AA3. Aliquots of 500 μl each were taken after 0, 1, 2 and 3 h incubation at 37°C, and analyzed using an Acetate detection kit (R-Biopharm, Darmstadt, Germany) as recommended by the manufacturer. The boiled for 2 min wt-AA3 was used in the control experiments.
AA3 did not deacetylate any of the N-terminally acetylated peptides used in this study, namely Ac-HCVCP-short, Ac-HCVCP-long, Ac-AA3 and Ac-NBCe1-A. This is in agreement with the preferential deacetylation by AA3 of N-acetyl aromatic not aliphatic amino acids. In addition, no corresponding deacetylated peptides were detected in the assay using a LC-MS technique (data not shown).
Precipitation was observed immediately after mixing of AA3 with HCVCP in support of the direct interaction of these proteins. Individual proteins did not demonstrate any aggregation (Fig. 1, left). HCVCP particles of 2–3 nm are seen that probably represent different projection of HCVCP monomers and/or dimers. In agreement with a previous study [19], AA3 is mainly represented by dimers. A heterogeneous population of round particles of different size (20–60 nm) assembled in significantly larger (>1500 nm) irregular branched structures was seen under electron microscope in the suspension formed after mixing AA3 with HCPCP (Fig. 1, right).
To quantitate the interaction of HCVCP with AA3 we used the surface plasmon resonance method. The Kd value for interaction of HCVCP with AA3 was 10.1±4.3 μM (Table 1). A moderately high affinity of HCVCP to AA3 suggests that they may physically interact in the organs/tissues where they are co-expressed, for example in the liver and blood.
To locate the HCVCP region responsible for this interaction and given that interactions of the core protein with different protein partners are mostly mediated by its extreme N-terminal part [2,4], we synthesized short (STLPKPQRKT) and long (STLPKPQRKTKRNTIRRPQDVKFPGG) HCVCP peptides corresponding to aa. 2–11 (HCVCP-short) and 2–27 (HCVCP-long).
N-acetylated HCVCP-short peptide (Ac-HCVCP-short) had the Kd value of 7.3 mM, whereas the non-acetylated short peptide bound to AA3 with a higher affinity (Kd=1.6 mM). Both Ac-HCVCP-long and HCVCP-long peptides demonstrated higher affinities to AA3 (170±13 and 142±11 respectively) than the corresponding short peptides but lesser affinities than the full-length HCVCP (Table 1). Again N-acetylation decreased the affinity of long peptide. The data suggested that the N-terminal acetylation impaired the binding of a peptide to AA3 likely via decreasing of the pI value of the acetylated peptide. Indeed the N-acetylation of HCVCP-short decreases its predicted pI value from 11.17 to 10.06, and HCPCP-long from 12.02 to 11.58. To further confirm this hypothesis, we synthesized two peptides with the pI values below 6, namely SSLPGSREPL corresponding to aa. 2–11 in mouse AA3 (calculated pI=5.72), and STENVEGKSPN corresponding to aa. 2–12 in the human electrogenic sodium bicarbonate cotransporter NBCe1-A (calculated pI=4.53). Both non-acetylated and N-acetylated peptides were used. Again as with HCVCP peptides, acetylated peptides of AA3 and NBCe1-A were used because of the predicted acetylation of the second residue (Ser2) after the cleavage of a methionine [17]. Both acetylated and non acetylated AA3 and NBCe1-A peptides demonstrated poor binding to the enzyme (Kd>10 mM). Figure 2 illustrates the dependence of the affinity of peptides to AA3 from their pI values. The Kd value is significantly decreased at the peptide pI>10.
Although the Kd values for peptide–AA3 interactions were quite similar to the Km values of AA3 substrates [12,13], and the binding of the peptides to the active site of AA3 could not be excluded, the presence of several exposed negatively charged areas in the mouse AA3 atomic structure [20] suggests that multiple sites on the surface of AA3 may be involved in the interaction with HCVCP.
The binding of AA3 to HCVCP peptides suggested that AA3 may potentially deacetylate them although the substrate specificity of AA3, which deacetylates N-acetyl aromatic amino acids and mercapturic acids (N-acetyl-S-conjugates of L-cysteine) with bulky side chains [13], did not support this suggestion. In agreement with the substrate specificity, AA3 did not generate acetate during even 24 h incubation with N-acetylated HCVCP and other peptides. Instead, a peptide hydrolysis was detected using LC/MS (Fig. 3A–D). Three peptides (STLPKP, STLPKPQ and STLPKPQR) were detected after incubation of both N-acetylated and non-acetylated short HCVCP peptide (STLPKPQRKT). No C-terminal peptides were detected. The results suggested that AA3 has endopeptidase activity towards the HCVCP peptides. This is in agreement with the structural similarity of the active site of AA3 and carboxypeptidase A [20]. Surprisingly, both Ac-HCVCP-long and HCVCP-long were not hydrolyzed by AA3. Given the higher affinity of HCVCP-long than HCVCP-short to AA3, the larger size of HCVCP-long may prevent its productive orientation towards the enzyme peptidase site. Both the N-acetylated and non-acetylated AA3 peptide (SSLPGSREPL) were hydrolyzed between Gly5 and Ser6 and both moieties (SSLPG and SREPL) were present (Fig. 3E–H). Both acetylated and non-acetylated NBCe1-A peptides were not hydrolyzed (data not shown). The endopeptidase activity of AA3 was Co2+-dependent; it was not detected without this metal ion. The endopeptidase activity of AA3 was likely attributed to the enzyme active center since the inactive H21A mutant of AA3 did not hydrolyze the peptides (data not shown). The endopeptidase activity of AA3 was not due to protease contaminants since standard protease substrates insulin B chain and casein were not hydrolyzed (data not shown). The Co2+-dependent endopeptidase activity of AA3 was inhibited by ~50% in the presence of 1 mg/ml Pefabloc SC suggesting that a serine residue(s) may be involved in mediating this activity. It should be mentioned that a serine residue is not present in the hypothetical active site of mouse AA3 [20] although 20 serine residues are present in AA3 monomer [11,12].
Our study for the first time demonstrates that HCVCP directly interacts with AA3. This interaction is mediated by the highly basic extreme N-terminus of HCVCP. The interaction between AA3 and HCVCP may play an important role in HCV pathogenesis. Binding of AA3 to the HCV capsid may form a specific shield protecting the virus from the recognition by the host immune system. From this standpoint it is important to determine whether AA3 is present on the surface of the HCV particles in vivo.
Our study provides the first evidence for the presence of endopeptidase activity in a mammalian aminoacylase. Dipeptidase and carboxypeptidase activity of several microbial aminoacylases have been reported [21–24]. Cleavage of the neighbor amino acids in the C-terminus of HCVCP-short suggests that AA3 also possesses carboxypeptidase activity. This is in a good agreement with the similarity of the crystal structure and catalytic mechanism of AA3 and carboxypeptidase A [20]. AA3 does not demonstrate preferences for amino acids in the position P1 and P1' of the substrate since the enzyme cleaves the peptide bonds between both charged and uncharged amino acids. The cleavage occurs only in the C-terminal and central part of peptides not in their N-terminus. The net charge of the N-terminal part of peptides seems to be important for their cleavage by AA3 with the preference to a positive charge. The N-terminus of the best hydrolyzed peptide, HCVCP, has a positive charge, the N-terminus of a significantly less hydrolyzed AA3 peptide is near neutral, while the non-hydrolyzed NBCe1-A peptide has a negative charge in the N-terminal half. The absence of the hydrolytic activity of AA3 towards the higher affinity HCVCP-long peptide and whole HCVCP argues for the existence of additional binding site(s) distinct from the enzyme active site. In agreement with this hypothesis, if the hydrolysis occurs, the dissociation of products should be significantly decelerated. According to the N–end rule, the N-terminal amino acids with a bulky or charged side chain are considered to be destabilizing and target proteins to the site of intracellular ubiquitin–dependent proteolysis [25]. The ubiquitin–mediated degradation of the HCVCP was demonstrated previously [9,26,27]. Cleavage of the N-terminus of HCVCP mediated by AA3 may expose destabilizing amino acids and affect the turnover rate of HCVCP in the cell.
The authors thank Dr. Warren Schmidt (University of Iowa) for providing the pcDNA3.1(+) plasmid with the HCVCV genotype 1b, and Dr. Young S. Hahn (University of Virginia Health Sciences Center) for providing the pGEX:Core192 plasmid with the HCVCV genotype 1b, fused with GST. This work was supported in part by the NIH grant ES012935 and the NIH Training Grant T32-DK-07789. The sponsors were not involved in the study design, collection, analysis and interpretation of data, in the writing of the manuscript and in the decision to submit the manuscript for publication.
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