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H. C. Andersen is, according to many, the world's greatest fairytale narrator, and the "The Princess and the Pea" is one of his most popular renditions. This performance was premiered in 2005 and was intended as a tribute to the poet in the occasion of the 200th anniversary of his birth. ... ..
Theater Performance: If we carefully read the adventure, we discover that history has a sort of villain. For example, the queen does everything she can to prevent the prince from getting married. The queen's sovereignty becomes even clearer in the performance than in the adventure because we have made a frame story. Here we meet the puppet theater player Adam Abel von Contrafei who travels from place to place to perform ... THE PRINCESS ON THE PEA! what the puppeteer, however, does not know when his theater performance begins, is that the queen has stolen the king's crown, thereby gaining the right to decide everything and everyone. Including the puppeteer! And now she forces him to build a mountain of mattresses and duvets, as well as placing a princess on top of it all. - Dread and horror! "Fortunately, Von Contrafei, with the help of the audience, rips the crown from the queen's head. And so the king gets his crown, and the prince is allowed to marry the princess. - Hip hooray!
The play is performed in Norwegian.
Saturday 27th of April. 1.00pm. Cinema vestibule. Price: 100NOK / 120NOK (group of 4+)
Adresse: Moerveien 1 1430 Ås Problemer med å kjøpe eBilletter? Send en mail til firstname.lastname@example.com


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Can I Add or Change the List of Scrolling Donors?
Currently, only the top ten donors will be displayed. If there are fewer than ten donors, all your current donors will show up in the scrolling list. Otherwise, you will see only the top 10 donors.
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subset: commoncrawl


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A gorgeous warm fuchsia pink (bubblegum pink) acai berry bead strand necklace. Approx 70cm drop – total length approx. 140cm.
This is the price for one strand. The strand is long enough to be doubled over or also used as a statement bracelet, doubled over many times.
Made in Brazil.


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0 Answers
Can my home insurance raise their price for owning a bully breed if it's a service animal?
Asked by: insurance 592 views Home Insurance
I am getting a pit bull as a service dog; she will be official. My question is this: in the state of Colorado, can the insurance company raise the price of our home insurance for having a 'bully breed', if she is a service dog? Or is that an infringement upon my rights as a disabled person?
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My children love this question. "What time is it Mammy?" is a regular utterance in our house. Why they need to know the time, I'm not sure. They are only 5 and 7 after all, and during the school holidays we're all more relaxed about bedtimes and mealtimes, so it's even less important. With no school day to start or appointments to get to, we can chill out about the time. And yet they still like to know.
I encourage them to have a go themselves. My 5 year old can tell me the o'clock, and is starting to get to grips with half past. The 7 year old has got the hang of quarter past and quarter to, but when they asked me the time at twenty to eight the other evening, the discussion that followed got rather complicated. How is seven-forty the same as twenty to eight? Why, when the long hand is pointing to the eight, does that make forty minutes? Or twenty minutes to something?
Part of the problem is, of course, the fact that we have different ways of saying the same thing. So half past three is the same as three-thirty; ten forty-five is a quarter to eleven; twelve o'clock is midday (or midnight if your children can stay up that late). The five year old happened to ask me, at midday today if it was morning or afternoon. She likes to make sure she hasn't missed a meal, so this is a common question; if it's afternoon she should have had her lunch. We were in the car at midday and so had to hurry home to eat! But was it morning or afternoon? Well, neither, at the exact moment she asked me. And that started another discussion about time.
With preschoolers, parents can help teach time by talking about times of the day, and the order in which we do things. "It's breakfast time now, then we'll get dressed and after that it's time to go to the toddler group." Think back over the day with your two or three year old and talk about what they've done, and the order that things happened in. This gets them understanding that time is sequential, and that there are certain times for certain activities. Make a clock with a paper plate, some hands cut out of cards and a split pin so you can move the hands around, and let them play around, looking at the numbers and beginning to show them the time on the hour. This will be great preparation for starting school and learning to tell the time. Talking about the past as well is as important as times of the day. When you were a baby; when I was a little girl; in the olden days (which is very subjective, as was brought home to me when we were watching Back to the Future a few weeks ago, a film guaranteed to cause confusion around time concepts, and my 7 year old asked me if it was made in the olden days? Yes, when I was your age!)
Show your child different types of clocks and watches. My watch has no numbers on the face, but my children still like to look at it and see if they can work out the time. Our alarm clock in the bedroom is digital and they know that this is a different type of clock, although understanding how the digital display relates to an analogue clock face is tricky. Making a sundial is a great, and very simple activity. Next time you spend a sunny day on the beach, put a stick in the sand and mark where the shadow falls, then keep coming back to it during the day and marking the hours, talking about the way the sun moves across the sky and the shadow moves. "What's the time Mr Wolf?" is a game I remember from school. Play this with your children; as well as being great fun, they start to learn the language of telling the time. There's loads of online games to help kids with time telling too. One of my favourites is on the bbc bitesize page but there are lots more out there.
Once children have been at school for some time and can confidently count in ones, they will begin to learn to count in twos, tens, and then, importantly, in fives. This step is vital for advanced time telling later on, as when you can count in fives up to sixty, you can count round the clock and get to 10 past, 25 past or 55 minutes past. And then if you can subtract you can make the connection between knowing that there are sixty minutes in an hour, so 55 minutes past is 5 to the next hour. We don't do this calculation of course, every time we look at a clock. Experience and practice teaches us that when the long hand is pointing to the 11 on a clock face, it's five to the hour.
Learning to tell the time is difficult; children won't grasp it overnight, and it takes years to progress from playing with a home made paper plate clock to being able to confidently state that the time is twenty to eight from looking at a watch with no numbers. But we can help our kids by talking to them about time, playing games with them, and helping them to use clocks and watches themselves.


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The Denver Handyman You Can Actually TrustIf you need work around the house, hire him you must! LET'S FINISH YOUR TO-DO LIST TODAY!
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When you choose Best Denver Handyman Services, the answer to all of these is the same – a resounding "Yes!"
Of course, our sterling reputation for punctuality, honesty, and quality work means you don't even have to ask them in the first place. In fact, we are so confident in your ultimate satisfaction that we guarantee our services!
We can offer this guarantee because we understand that a key element in your satisfaction is making sure you know exactly what to expect – and, to that end, we start your project by walking through it together.
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Kevin was great to work with. I would trust him with any project in my home. His communication was prompt, he returned my inquiry the following day, and we had a plan to work on my project the same week. He arrived on time and treated my home (and my pets) like his own. He kept me posted on the work, questions he had on the end result. There wasn't a time that I was hesitant or concerned. He wrapped up the project within the time frame he quoted and the price matched the quote and was very reasonable. I will definitely want to work with him again and would highly recommend him. Thank you again for the quick help and amazing craftsmanship.Christine K.
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We supply all materials for the work we perform. This enables us to offer our 100% satisfaction guarantee. (We cannot guarantee materials we weren't able to carefully select ourselves.)
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Major projects are quoted and scheduled based on the scope and requirements presented. Any work performed that is not part of any quoted project will be billed at the rates listed here on our website, unless other arrangements have been made.
All work is done with safety in mind. If you have requests that do not fit within safe working conditions, we will let you know what it will take for us to maintain everyone's safety.
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English Korean Dictionary
Click on a word to display the translation.
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The Solution:
Venue Manager Peter Johnston consulted with Aussie Broadband to find a solution to their ongoing issues.
Aussie Broadband's team recommended a Licensed Microwave Corporate Broadband Solution which provided a Corporate Grade symmetrical service with SLA and 1:1 contention ratio which would solve all ongoing issues.
Aussie Broadband's team also recommended & then installed a Hosted PABX Phone System. This included new Handsets & a Dedicated Voice Service (delivered by Licensed Microwave as well). The Dedicated Voice Service provides professional grade voice quality & call dropouts and quality issues have now been solved. The Hosted PABX has also provided Auto Attendant, In Dial Numbers, Internal Extensions & Personalised Voice Mail now provide Sale & District Greyhound Racing Club with a professional solution.
The Results:
Peter is pleased with the outcome since the Licensed Microwave & Hosted PABX solutions have been implemented, it has solved his constant & ongoing issues. The benefits are now being felt for not only internal staff but also patrons & external clients alike.
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Our team of talented and friendly staff would love to help your business. Simply tell us the services you are currently on and what you're looking for and we will help ensure your services and setup are the best for your business.request a consultation


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Last night Owen and I have a night to ourselves without T. He went to a "meeting" as Owen called it. Really he was at a research thingie. :) Needless to say, we were on the go all night. It is literally a battle to get Owen to go down and last night wasn't anything different.
It started off with a bath that last for about 45 minutes. He was squeaky clean! Ha! After that he got into his new jammies that Nana gave him. He said they were "monster cookie" pajamas. Not cookie monster, "monster cookie". They were so cute on! Once we were dressed, had apple juice, and blankie, we headed upstairs.
I decided to call my dad because it had been a few days since we have talked. I didn't get much in before my dad wanted to talk to Owen. He loves talking to his PawPaw. I don't know what they talk about, but Owen always has a smile on his face.
It was getting close to bedtime and I noticed he was getting tired. He told me he wanted to go in the "big bed" SHOCKING! We went down there at about 8 & I was SO tired! I was dozing off and he would lay his head on me and say "wake up mommy" Oops! I was tied. After about 45 minutes of trying to convince him to RELAX, I took him to his bed. I just don't sleep well while he is in our bed.
He finally relaxed and went to sleep about 9'ish. Tony got home about 10:30 and I was still up working on my PowerPoint for class. I finally decided to give it up and head to bed. Little did I know I would be woken up at 11 by Owen crying.
As soon as I hear him I pop right out of bed. I went in there and this is how our conversation went:
Owen-Mommy I am crying
Owen-I am said
Me-Why are you sad?
Owen-I want to go with you in the big bed!
Owen-Please! It will be fun! Please get my blankie & pillow and take me with you.
Me-Ok. I will take you.
Owen-We can snuggle and watch cartoons
I gave in and took him with me. I am such a sucker and he wasn't kidding when he wanted to snuggle. I didn't realize that snuggling meant laying right on top of me. I work up at 1 and took his little butt back in his bed. I rested well for the rest of the night.
Off to relax and make some coffee for tomorrow.
Wednesday, September 23, 2009
Pajamas & PawPaw
Last night Owen and I have a night to ourselves without T. He went to a "meeting" as Owen called it. Really he was at a research thingie. :) Needless to say, we were on the go all night. It is literally a battle to get Owen to go down and last night wasn't anything different.
Posted by Owen's Mommy at 9/23/2009 07:08:00 PM 1 comments
Sunday, September 20, 2009
Sun, Sand, & Sanity, Oh My!
It has been many years in the making...a {VACATION}!!!! It will be 5 years in May since we have had a true vacation (7 days) on the beach, but that's all about to end! We have already booked our condo in FL for my Fall break! I am so excited, but I think I am more excited for Owen to see the ocean! I had really hoped to be thin, but that's a joke. HaHa! I am going to make the best of my week and really try to get some sanity back.
The weekends go by so fast. Its like I am so happy it's Friday and then I wake up and it's Sunday. :( I started off my weekend by seeing Wicked with two of my wonderful friends, April & Cori. The play was AMAZING! We ate dinner at Maggiano's and then off to see the play. I was tired from a long week, but I seemed to managed. I would highly recommend the play. I have waited 3 years to see it & FINALLY I got to. Well worth the wait & money.
Owen has started a new thing at bedtime. When it's time to hit the sacks, he starts saying "I want to lay in the big bed(our bed)" and it is really hard to deny him! So, like a sucker of a parent I allow him to lay with me before bed. Most times it's only for about 10 minutes, but other nights he falls asleep. This was taken yesterday.
Yesterday we did our normal dinner..."Las Thomas" as Owen calls it. Nothing better than taco, beans, rice, salsa, and guacamole.
Today was the first home Titans game and they really stunk it up! Before Tony left for the game O had to put his jersey on & I HAD to get a picture! He looks like such a big boy in his KVB jersey! We lost the game, but I gained some really cute pics!
After Tony got home from the game, he put his hat on O's head...he wore ir for an hour. Even on our way to HHGregg.
While I was trying to make my crack, AKA Starbucks, Owen insisted on playing with the flour. Once again, a sucker of a mother gave in! He loves scooping the flour an dumping it out. What did it hurt? NOTHING!
Well, I am off to bed! It has been raining here for about a week and I am feeling a little suicidal! I couldn't live in Seattle! I need to see the sun!!! Please GOD, let the sun shine this week. I am going a little crazy. OK, so I am crazy, but it is getting worse!
Posted by Owen's Mommy at 9/20/2009 09:00:00 PM 1 comments
Saturday, September 12, 2009
Milkshakes, hula hoops, & music...
It has been {FOREVER} since I have posted a blog, but there was a good reason...my camera was "sick". Something was wrong with the zoom & my year warranty expired the day before I turned it in. I knew it was going to take 6 weeks & I waited till the last possible minute to give it up. Basically there is going to be 6 weeks of Owen's life that doesn't have photos.
Monday was a sad day...my washer went out. I currently have my Nanny's old ones & I swear they are like 30 years old. I knew this day was coming, but I wasn't prepared for it. We spent all week looking for a new pair and finally decided on these Samsung pair. We don't have them, but they are scheduled to be here on Monday. I am looking forward to having some quiet ones. When we are washing clothes with the old ones, Owen says "shaky shaky". They are SO loud.
Every weekend we must go to the car show at Chick-Fil-A for three reasons.
1. To dance to the music
2. Milkshake
3. Hula Hoop
Owen has such a blast & everyone loves watching him dance. All the cars there are really old & the average age is about 60. We fit in...HaHa!All-in-all it's a great time.
I have been busy with work...it is kicking my butt this year. Starting off great, but a lot more work that my other years.
Headed to bed...will most more soon.
Enjoy the pics!
This pic Owen has figured out he can get out of the outfit by putting his hands in it.
Posted by Owen's Mommy at 9/12/2009 09:53:00 PM 1 comments


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This Toiletry Bag will allow you to keep all your essential toiletries together in the one convenient location. It is compact yet spacious. It is made from a waterproof nylon and polyester material to protect the items inside. It has zippers enabling you to close the compartments and when open has a hook allowing you to hang the bag up if you need to.
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weight: .15kg so not to heavy inside your luggage
Colours available: black, navy, grey, pink (Note: the toiletry bag does not come with the toiletries inside it)
Shape: box shape
Full refund provided if item not as described. Purchaser to pay return postage.


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Drake Shares Behind Barz Freestyle (Audio + Video)
Drake drops some new rhymes following the release of his double album "Scorpion." After dropping his highly-anticipated double album Scorpion last Friday, Drake has shared a new "Behind Barz" freestyle with Link Up TV. The nearly 3-minute clip features some brand new rhymes from the Toronto rapper and comes in advance of an OVO Sound Radio episode which promises an "exclusive post-Scorpion mix."
Though he names no names, Drake hints at secrets he's keeping to himself for the time being: "We came around and you showed us the most / I know so much shit that I cannot expose / I keep it inside and I laugh on my own."
Drake's Scorpion is the first album to hit 1 billion total streams in a week. It was certified platinum the day of its release due to the immense singles sales it received from "God's Plan" and "Nice For What." It is certain that the album will debut at No. 1 on the Billboard 200, though it will only be announced Sunday, along with the official sales tally for the week.
The new episode of OVO Sound Radio airs Saturday night at 6PM EST. Will new Drake music be premiered? Tune in to find out.


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Apple Wins the Minds of High-Tech Professionals
Scientists, engineers, software engineers and programmers, and other high-tech workers in the U.S. think that Apple is the "most technologically advanced" company, based on a nationwide online survey of 1,582 high-tech professionals. Google comes in second and Microsoft third. The survey was conducted by the Technology Advisory Board® (www.technologyadvisoryboard.com), an online panel of technology professionals who are paid to take part in technology surveys. The top-10 "most technologically advanced" companies are shown below.
Apple is reaping the brand-image benefits of its invasion and conquest of the
smartphone market. "Most technologically advanced" is largely a hidden
brand-image dimension and, therefore, especially valuable in silently promoting
the acceptance of all Apple products. Apple is winning the war for the minds of
the technology world.
Complete Results
The survey was conducted during the first quarter of 2013 and included high-tech
professionals in all 50 U.S. states, plus Washington, D.C. The sample came from
the Technology Advisory Board®, an online panel of scientists, engineers, and
other high-tech professionals. Participants were shown a list of 64 major high-tech
companies and asked to choose the "most technologically advanced" companies (up
to 5 choices).
The Technology Advisory Board® is owned and operated by Decision Analyst, a global research and analytical consulting firm headquartered in the Dallas-Fort Worth area.
Margin of Error
The data was collected in January and February of 2013. The margin of error is less than 2 percentage points, plus or minus, at a 99% confidence level. For any questions about this survey please contact firstname.lastname@example.com.
About American Consumer Opinion®
American Consumer Opinion® (www.acop.com) is a worldwide network of people who get paid for sharing their opinions and ideas by answering online surveys. Members' participation helps companies make decisions on new products, advertising campaigns, package designs, and marketing strategy. American Consumer Opinion® is owned and operated by Decision Analyst, a global marketing research and analytical consulting firm headquartered in the Dallas-Fort Worth area in the United States.


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|23 Nov 2015
Windows 8.1 64 bit
BSOD when playing csgo
Cousin getting a BSOD when playing CSGO.
|My System Specs
|24 Nov 2015
Windows 7 Home Premium 64-Bit SP1
Hello jjoner the BSOD experts want to see you follow this tutorial.
Blue Screen of Death (BSOD) Posting Instructions
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I've just recently installed a fresh new versions of windows 7 on my new 256gb samsung ssd pro. I've installed all necessary windows 7 drivers from windows update. About 30min into a video game (csgo) I get a BSOD :( I only get this BSOD while playing the game. This had never happened prior to...
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A committee of local motorcycle enthusiasts and former Windber Dog Run organizers, known as Heaven's Dog Pound, will host a party June 1 at the Windber Recreation Park ballroom.
The party is to honor members of the committee who have died and raise money for the charity. Member Jodi Gahagen said that three of the original eight members are still alive.
"We decided we wanted to do something special in memory of the members who passed away," Gahagen said. Her sister, Kathy Gahagen-Lucas, was also a member, but she was killed by a drunk driver on July 31, 2009.
Brett Lucas, Gahagen-Lucas' son, said that the party is a perfect way to honor his mother and the other deceased members.
"I just wanted to carry on a tradition they had for so many years and to honor them for all their hard work and dedication throughout the years," Lucas said.
"My mom would be proud of our efforts."
Back in August 1989, the Dog Run began as a group of eight friends on a motorcycle run. In that time, the group raised more than $600,000 for the community.
More than 20 years later, the Dog Run has become synonymous with Windber and charity. Heaven's Dog Pound is attempting to continue that tradition by donating scholarships to Windber Area School District in memory of their deceased members.
From last year's party, the group donated basketball jerseys, homecoming fireworks and a marble park bench to Windber.
This year's event, from 5 to 11:30 p.m., is to include live bands, 50/50's and a basket raffle. More information is available by calling 814-243-1015 or 814-467-6503


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In the great state of Kansas and, more specifically, in the city of De Soto, Funds4Claims is the best choice as your lawsuit funding source for all of your litigation finance needs. Funds4Claims offers quick and easy, low cost settlement loans to De Soto and all of its surrounding areas. Funds4Claims is a lawsuit funding company that offers low-cost lawsuit loans to residents of De Soto and surrounding cities. With Funds4Claims it is easy for residents of De Soto to qualify for lawsuit funding, we do not require personal or business credit. The only requirement for a lawsuit cash advance is that you have a valid lawsuit. Our lawsuit financing can help you pay for the cost of litigation or for any other expenses you need money for, we do not carry restrictions on how you use your lawsuit money. Funds4Claims is a highly respected lawsuit funding company that cares about their clients. We strive to provide financial stability for people living in De Soto going through the extensive court processes associated with a lawsuit. In addition, Funds4Claims provides litigation funding in De Soto, Kansas for: Pre Settlement Funding, Post Settlement Funding, Structured Settlement Funding, Commercial Lawsuit Funding, and Law Firm Funding.
There are numerous financial institution of which might choose to give you more than just how much you choose therefore you will default along with incur expenses. This means much more fees from people whenever you rotate over the bank loan. A cash advance for De Soto residents from Funds4Claims can help you reach financial freedom. We understand how valuable your time is, which is why our application process is quick and easy. Call Funds4Claims today at 855.386.3744 or fill out our simple online lawsuit funding application for fast litigation pre-settlement loan services. Funds4Claims lawsuit loan services in De Soto are established by the merits and strengths of your case and not your credit history. These companies will give you far more simply because they recognize they may help to make much more returning in the operation. Access merely just how much which you will want in addition to almost nothing more. Take the initial step to receive your litigation finance by contacting Funds4Claims today. Funds4Claims is recognized as a frontrunner in the lawsuit funding industry and are preferred by some of the most renowned law firms in the country.
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School Crafts
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Sheet of black/brown construction paper
Scraps of orange/yellow construction paper
Markers/Crayons/Pencil Colors/Pencil
Under 1 hour
Instruct students to:
Draw 3 equal size circles from construction paper.
Fold 1 circle 1/3 of the way down to form head.
Cut facial features and glue in place. (Circles for eyes, triangle for beak)
Cut 2nd circle in half to form 2 wings.
Feather curved edge by cutting slits approximately 1" from edge to center.
Glue head to the top edge of the 3rd circle.
Glue wings to side of 3rd circle with feathered edge to the inside.
Display by hanging or attaching to a surface.
Last Updated: 08/20/2013
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Soft Surface Floors
Carpets & Rugs
Carpets & Rugs at Eberhard Carpet One Floor & Home
Soft Surface Flooring has come a long way since the 60's and 70's. Before, rugs and carpet would wear and tear easily, and let's not get started on those colors. Surprisingly, many of those carpets still exist in homes, for many still believe that carpets haven't changed over the course of the years. Contrary to that belief, Eberhard Carpet One Floor & Home has a vast collection of technologically advanced soft surface floors.
Now, you may be asking yourself "How can a rug and a carpet become advanced?" Just like technology, our soft surface floors have upgraded. They have become softer, durable, and have even developed more color choices.
To learn more about our Soft Surface Floors read below:
Providing our customers with quality, long-lasting carpets is one of our specialties. We know exactly which carpet types will accommodate your desired setting, so we'll certainly be able to help you make the perfect choice.Learn About Carpet
Area Rugs
Homeowners love adding area rugs to their rooms because they can add extra style and even protect flooring underneath from moisture and dirt.Learn More


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Movie REviews
REviews by scripture
reviews by alphabet
About the CRitic
It's been a while since a mainline movie was rated NC-17.
And this one carries that rating for very good reason.
So be warned, all you genteel people out there:
the people in "Shame" have no shame.
They are not only not shy about their sexuality, they are unceasingly showcasing it.
They are not only not bashful, they are downright exhibitionist.
They are not nobly nude, they are starkly naked.
It's not about covenantal commitment between a man and a woman, it's about self-gratification with little regard and no love for the other.
Yes, for many gentle folk, it will not only prance up to the line of pornography, it will bend over bare-bottomed and show you much more than you care to see.
As far as sex on screen in contemporary cinema, it shoots you the moon.
And yet….and yet.
It deals with some of the darkest corners of sexuality in a shadowy area that few have dared to trod:
And it does so in such a way that is so searingly honest that we cannot help but be impacted.
You cannot watch this film and be unaffected.
And so this movie will, undoubtedly, garner Oscar attention, and you, gentle reader, will have to decide if you have the constitution to withstand its unblinking descent to the depths of depravity.
Brandon (Michael Fassbender) is an obviously successful New York City businessman, in one of those modern firms where employees work at their laptops and confer in quiet but intense meetings, as if this is serious and we have our competitors and we have to be on top of our game at all times.
The co-workers are young and savvy.
There's not much humor, or camaraderie, either, at least on the job.
After hours is a different story, maybe, but what happens at other places stays at other places.
But when
gets home to his nicely-furnished apartment, he lets out his libido.
He gets online and does porn over the Internet, the interactive kind, where the models will do things on a webcam and say things into your headset that you wouldn't want your Mother to see or hear.
But even that's not enough.
has to "take care of himself" frequently afterwards---in the shower, in the bedroom, and yes, even in the men's room at work.
He'll pay for an "escort service" to send someone to his place, and he'll pay her, and afterwards, send her away, still not satisfied.
He'll go to a club or a bar and meet someone, and maybe even couple furtively in a dark alley, but he doesn't "date" for long and he doesn't bring them home.
Because he really can't stand to be "in relationship."
That would involve his emotions.
And all that's involved for him is his runaway fantasy world of strange women as sex objects.
The minute they stop being strangers, he's no longer interested.
Who will deliver him from his own determined self-dissipation? Well, his sister comes to visit, rather unexpectedly, and needless to say, this puts a big crimp in his continual need for personal privacy.
Sissy (Carey Mulligan) is so open about her own nudity and crying need for affection that at first we wonder if this is some kind of really taboo incestuous relationship, but it's not quite like that, either.
She's tremendously sad, vulnerable, needy, and lonely, and wants her brother to be there, emotionally, for her, but he just can't.
He can't make himself attach to anyone.
There's this girl at work that he likes, and who's young and attractive, so he tries to "date" like a normal person, but…he can't get himself interested.
He's so into his fantasy addictions that they have completely substituted for reality.
And clingy Sissy is left out, no matter how hard she cries.
Michael Fassbender is so intense and dedicated to this role that he completely convinces us that he is this pitiful character.
And Carey Mulligan is such a perfect counterpoint with her own compulsivities that we aren't at all surprised they're related.
And the angst of never being able to meet each other's needs is only a mirror-image of their desperate needy insatiable loneliness with the rest of the world.
This movie will send the viewer away reeling with its gut-level impact.
It will neither give any emotional quarter nor ask it.
But it will demand your wide-eyed attention.
Dr. Ronald P. Salfen, Interim Pastor, St. Stephen's Presbyterian Church,


text: The GDS Dodge Fuel Cap! Because there is nothing more aggravating when you pay good money for a new truck and on your first fill-up you realize you have no fuel cap. We decided to design a cap that the manufacturer should have provided. Ours is machined from aluminum, anodize with a diesel green color, and then laser etched 'DIESEL' into the top to make it stand out. To make sure you never lose your fuel cap, we put a magnet on the bottom so you can conveniently stick it to the inside of your fuel door while refueling but still maintaining aesthetic appeal. As functional as it is appealing, this cap will keep dust, water and debris out of your fuel system.


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Come down and pick your perfect Christmas tree for this seasons festivities.
Our fantastic selection of Devon grown Christmas trees are now available at the farm shop. There are top quality Nordmann, Fraser fir and Norway spruce trees.
Trees are potted and freshly cut. They vary from 3ft-10ft and we're on hand to help you carry it to your car or truck - or trailer! Just ask a member of staff.


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Astrologists agree that there is a fantastic chance that beneath the ice shield of Jupiter's moon, Europa, lies a vast global ocean where life may exist. My interpretation of Europa is a world of lilac, salty water where ocean-floor-heat-vents spew heat, oxygen, and sparkles of light up into the waters. Her deep oceans are filled will a diversity of life, many with the capability of bioluminescence, just as our deep ocean fish have. Thus, Europa's ocean is the complete opposite of ours – the light is at the bottom and the dark is at the top, for little sunlight reaches Europa, and her ice shield blocks any that does.
In this beautiful fluorescent world of light and color, lives a sentient race known as the Oonocks, an ancient race at least seventy to one hundred million years old. They are, for the most part, peaceful beings, who value family and honor above all else. They have no words such as divorce, greed, want or hate, for those things do not exist. The race consists of clans which number from several hundred members to several thousand. Each clan specializes in two or three fields, such as fishing, farming, shipbuilding, engineering, physics, chemistry, metal urging, aerodynamics and so forth. Each clan is governed by a royal family, usually a Lord and Lady. Together, the clans are managed by a King and Queen, who everyone pledges their loyalty and lives to. Every individual contributes something to the whole; those who do not partake is a specific area of study become soldiers; those too young to work attend school; and those too old teach the young. Although not a perfect society, it is pretty close.
No land exists above water on Europa, so the Oonocks are aquatic beings. They have large lilac eyes, and their blood is purple. They have three wings on each side of their body that glides them through their water world. Their body is muscular and strong, slender, tapering into a long tail with a feathery lilac end. Their hair is strings of pearl-shaped glowing lights which cascades down from the top of their head. Like most creatures of Europa, they are bioluminescent and glow a soft lilac color. Their expected life span is approximately twelve thousand years. And they are also blessed with a special power – the ability to transform into other creatures equal to or larger than themselves.
Approximately sixty-four hundred years ago, this peaceful existence of the Oonock world was destroyed when the reigning king betrayed his middle son, JeffRa, devastating him beyond repair. In retaliation, JeffRa tried to assassinate his older brother, Enok, who is the future king. Once the best of friends, the two brothers became mortal enemies and civil war broke out. JeffRa and his followers are hunted down and exiled to Ganymede, another of Jupiter's moons. There he became the leader of another race called Terrians, a barbaric society who embrace cruelty, greed, and war. From his place of exile, JeffRa continued to plot his brother's end, promising to never stop until ever Oonock in the universe was dead.
When JeffRa's invention destroys Jupiter and turns it into the gas ball it is today with its giant storms, King Enok and his mate, Queen Medaron, take twenty ships filled with Oonocks and leave Europa. Most go out further into the galaxy, but King Enok brings his three ships to Earth to live. Another two settle on Mars, which is Earth's sister planet, green with vegetation and covered with life. But JeffRa follows, consumed by his vow to eradicate all Oonocks, searching across our solar system for his hated brother. He makes the mistake of attacking Mars first, thus allowing King Enok to be warned of his approach. To save Earth and his Oonocks, King Enok sinks his city to the depths of the Pacific Ocean at night, in a blaze of fire and water. Thus the legend of Atlantis is born. There, beneath the cloak of darkness and water, the Oonock race remains, safe and unknown, for thousands of years. Several times over the millennia they emerge and try to live on land again, but for one reason or another, are forced back to the security of the deep, causing the creation of many Earthly legends. During this time they have no idea that JeffRa has crashed on Earth, and is desperately seeking his brother and wife.
King Enok and Queen Medaron try again to have their Oonocks live above. Far from humans, they build a new city, called Third City, on top of a high plateau in Peru (Puma Punku). There they live in happiness and peace, safe behind their stone walls. After several hundred years, that peace is once more shattered when they hear the sound of marching feet. JeffRa and his army of Terrians have found them.
The two brothers, King Enok with his Oonocks and JeffRa with his Terrians, fight a great war. Both sides suffer great losses. JeffRa is defeated, but the result is the Oonocks must once more return to the ocean depths. King Enok is gravely injured and Queen Medaron is in a deep state of depression over the loss of her only son, whom JeffRa killed. After returning below, they learn the horrifying truth of JeffRa's attack: the release of a biological weapon that will cause all future Oonock newborn children to die within the first few years of their life. It also causes infertility, thus dooming the Oonock race. With no way to reproduce and no way to return home, they face a dismal future. Thus they exist, knowing each day that they when they die, the great Oonock race will be no more. But then something marvelous happens. Numerous centuries later, Queen Medaron discovers she is pregnant with a female who will be the next Queen. The Oonock race is a matriarchal society, so nothing is more important than this child surviving. But because of JeffRa's curse, the unborn female cannot survive as an Oonock. To ensure her survival, the decision is made that she will be born on land as a human, and will never know who or what she really is. Thus begins book one, EUROPA: Awakenings.


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- Research
- Open Access
Non-inflammatory tumor microenvironment of diffuse intrinsic pontine glioma
© The Author(s). 2018
- Received: 7 June 2018
- Accepted: 7 June 2018
- Published: 28 June 2018
Diffuse intrinsic pontine glioma (DIPG) is a universally fatal malignancy of the childhood central nervous system, with a median overall survival of 9–11 months. We have previously shown that primary DIPG tissue contains numerous tumor-associated macrophages, and substantial work has demonstrated a significant pathological role for adult glioma-associated macrophages. However, work over the past decade has highlighted many molecular and genomic differences between pediatric and adult high-grade gliomas. Thus, we directly compared inflammatory characteristics of DIPG and adult glioblastoma (GBM). We found that the leukocyte (CD45+) compartment in primary DIPG tissue samples is predominantly composed of CD11b + macrophages, with very few CD3+ T-lymphocytes. In contrast, T-lymphocytes are more abundant in adult GBM tissue samples. RNA sequencing of macrophages isolated from primary tumor samples revealed that DIPG- and adult GBM-associated macrophages both express gene programs related to ECM remodeling and angiogenesis, but DIPG-associated macrophages express substantially fewer inflammatory factors than their adult GBM counterparts. Examining the secretome of glioma cells, we found that patient-derived DIPG cell cultures secrete markedly fewer cytokines and chemokines than patient-derived adult GBM cultures. Concordantly, bulk and single-cell RNA sequencing data indicates low to absent expression of chemokines and cytokines in DIPG. Together, these observations suggest that the inflammatory milieu of the DIPG tumor microenvironment is fundamentally different than adult GBM. The low intrinsic inflammatory signature of DIPG cells may contribute to the lack of lymphocytes and non-inflammatory phenotype of DIPG-associated microglia/macrophages. Understanding the glioma subtype-specific inflammatory milieu may inform the design and application of immunotherapy-based treatments.
- Diffuse intrinsic pontine glioma
- Tumor-associated macrophage
- Tumor-infiltrating lymphocyte
- Glioma
- Glioblastoma
- Immune microenvironment
Diffuse intrinsic pontine glioma (DIPG) is a devastating tumor that arises in the ventral pons, chiefly during childhood, and is universally fatal. Currently, the standard of care is radiotherapy, which offers temporary stabilization or reduction of symptoms and extends median survival by approximately 3 months [21]. Overall median survival remains 9–11 months with less than 10% of children surviving beyond 2 years [7, 20]. Over the past decade, comprehensive efforts to characterize the genomic and epigenomic features of DIPG and other pediatric tumors have revolutionized our understanding of this disease [29]. Pediatric high-grade gliomas are genomically distinct tumors when compared to adult glioblastoma (GBM) [47]. A highly recurrent mutation in genes encoding histone-3 (H3K27M) is found in ~ 80% of all DIPG and also the majority of thalamic and spinal cord gliomas of childhood [2, 9, 11, 22, 44, 45, 48, 50, 53, 54]; these H3K27M mutant gliomas were recently reclassified as "diffuse midline gliomas, H3K27M mutant subtype" [23]. Broadly, analysis of immune infiltrates of pediatric central nervous system tumors suggests the immune compartments are less predictive of tumor grade or prognosis, compared to adult gliomas [37]. For example, pediatric GBM samples exhibit a muted immune functional phenotype compared to pediatric pilocytic astrocytomas and ependymomas [15], but the immune state of DIPG has not been similarly analyzed to date. DIPG exhibits a unique mutational profile and transcriptional state [27, 29], raising the question of a similarly unique immune microenvironment. As interest in the clinical application of immunotherapeutic approaches for DIPG grows, it is critically important to understand how potential differences in the DIPG immune microenvironment could influence treatment efficacy. For example, the presence or absence of lymphocytes would inform the consideration of strategies such as checkpoint inhibition.
Glioma-associated microglia/macrophages (GAMs) have received interest as a potential therapeutic target. In patients with adult GBM, expression of specific immune-associated gene sets are correlated with overall survival [52]. Moreover, adult GBM-associated macrophages have been implicated in supporting tumor cell proliferation, invasion, and survival as well as tumor angiogenesis [13, 17]. Targeting GAMs with CSF1R antagonism has demonstrated efficacy in a number of adult GBM preclinical models by affecting GAM activation state [39], an effect mediated by the tumor microenvironment [41]. CSF1R antagonism also potentiates the effect of radiotherapy in adult GBM preclinical models [46]. While evaluation of the clinical potential of CSF1R- and other GAM-targeting therapies is ongoing [3, 5], it is clear that GAMs are a critical component of the glioma microenvironment. Moreover, GAM activation state has been identified as an important factor in effective immunotherapy treatment [43], so understanding their phenotype across different tumors may help guide clinical translation of immune-targeting therapies.
In DIPG, little is known about the state of microglia and macrophages: due to the sensitive location and diffusely infiltrative nature of the tumor, resection is not possible, and the biopsy of DIPG has historically been uncommon [4, 23, 29, 38]. We have previously demonstrated that human DIPG samples demonstrate substantial immunoreactivity for the macrophage/microglial markers CD68 and CD163 [6], illustrating that GAMs are a large fractional component of the DIPG microenvironment. Here, we isolate GAMs from primary DIPG and adult GBM, as well as microglia from normal pediatric cortex samples for whole transcriptome analysis. We demonstrate that while DIPG-associated macrophages exhibit some gene expression programs similar to adult GBM-associated macrophages, they express substantially fewer inflammatory cytokines and chemokines compared to adult GAMs. Proteomic analyses reveal that patient-derived DIPG cultures produce markedly fewer cytokines and chemokines when compared to patient-derived adult GBM cultures, a finding corroborated by primary DIPG tissue bulk and single-cell RNA sequencing data. We also observed minimal lymphocytic infiltration in both primary DIPG autopsy and diagnostic biopsy samples. Together, these data suggest that DIPG and DIPG-associated macrophages are less inflammatory than adult GBM and adult GAMs.
Materials and methods
Acquisition and processing of human tissue samples
All human tissue studies were performed with informed consent and in accordance with Institutional Review Board (IRB)-approved protocols. Autopsy and biopsy tissue samples were processed as previously described [26], with some modifications. Briefly, tissue was minced finely, enzymatically dissociated in a collagenase/dispase + DNAse solution, triturated, and filtered through a 100μm filter to obtain a single cell solution. Debris was removed by centrifugation using a 0.9 M sucrose gradient, and red blood cells were removed using ACK lysis. Live cells were quantified via trypan blue exclusion, and resuspended for fluorescence-activated analysis and sorting.
Fluorescence-activated cell sorting and analysis
Isolated single cells were resuspended in flow cytometry buffer (2% BSA, 10μM HEPES in HBSS without calcium/magnesium) at approximately 107 cells/mL and processed at 4 °C. Cells were blocked with mouse IgG isotype control (ThermoFisher) and rat IgG isotype control (R&D Systems) as appropriate. Cells were then incubated with the appropriate conjugated primary antibodies (FITC-anti-CD45, BD Pharmigen 555482; PE-Cy7-anti-CD11b, BD Pharmigen 557743; PE-anti-CD3, Biolegend 300308; APC-Cy7-anti-CD31, Biolegend 303120) for 1 h. Next, cells were stained using the LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen) according to the manufacturer's instructions. Immediately before analysis and sorting, DAPI was added to the flow cytometry buffer. Cells were sorted and analyzed on a BD FACSAria II or FACSAria Fusion. Cells were identified and gated for size, singularity, cell viability, and all surface-marker expression was gated with appropriate full-minus-one controls. Cells were sorted into cold catching medium and a fraction was removed for cell purity analysis. Isolated cells were lysed in TRIzol (Invitrogen), and stored at − 80 °C until further processing.
RNA sequencing
Total RNA was extracted by chloroform extraction into the RNA Clean & Concentrator Kit with DNAse (Zymo Research), and examined for quality using an Agilent Bioanalyzer 2100 platform. First and second-strand cDNA synthesis and SPIA amplification was performed with the Ovation RNAseq system V2 (Nugen) following the manufacturer's instructions, and the resulting cDNA was fragmented with a sonicator (Covaris) using the following parameters: duty cycle 10%, peak power 175.0, cycles/burst 100, time 5 min.
Libraries were prepared for sequencing as previously described [35]. DNA was end repaired using T4 polymerase, Klenow fragment, and T4 polynucleotide kinase. 3′ A-tailing was performed using Exo-Klenow. NEBNext Illumina Multiplex Oligo Adaptors (NEB E7335S) were ligated for 1 h at room temperature. Unligated adapters were separated by gel electrophoresis (2.5% agarose, 0.5X TBE) and ligated DNA was purified using a NucleoSpin Gel Clean-up Kit (Macherey-Nagel). Ligated DNA was PCR amplified using NEBNext Multiplex Primers and purified using AMPure XP beads (Beckman Coulter). Purified libraries were quantified using Agilent 2100 Bioanalyzer HS DNA and multiplexed in equimolar concentrations. Sequencing was performed using an Illumina NextSeq at 1 × 75 bp by Stanford Functional Genomics Facility. RNA sequencing data is available through GEO (GSE115397).
Gene expression of patient-derived DIPG cultures and primary bulk DIPG tissue was published previously [11, 30, 35]. Single-cell RNA sequencing of diagnostic DIPG biopsies was also published previously [7]. All additional analysis was performed in R; FPKM plots were plotted in Prism (GraphPad).
Gene expression analysis
Reads were mapped to hg19 annotation using Tophat2 [24]. Transcript coverage of RefSeq gene annotations were performed using featureCounts [25]. To visualize sample similarity between samples, we performed a regularized log-transformation using the R function rlog from DESeq2 [28], and plotted the PCA using ggplot2. Differential testing and log2 fold change calculation was performed using DESeq2, with FDR = 0.1 and normalization calculated by number of assigned reads. Active transcripts were defined as genes with a mean FPKM of at least 5 across all samples. Volcano plot was made in R. Gene Ontology on differentially expressed gene lists was performed using the DAVID v6.8 web portal [18, 19]. Pre-ranked gene set enrichment analysis was performed with the publically available software platform [33, 49].
Human tissue culture
Authenticity of all cultures was routinely monitored and validated using short tandem repeat (STR) DNA fingerprinting, and tested regularly for mycoplasma. DIPG and adult GBM cultures were cultured as described previously [14, 35, 40]; briefly, all tumor cultures were maintained as neurospheres in Tumor Stem Media (TSM) consisting of DMEM/F12 (Invitrogen), Neurobasal(−A) (Invitrogen), B27(−A) (Invitrogen), human-bFGF (20 ng/ml) (Shenandoah Biotech, Warwick, PA), human-EGF (20 ng/ml) (Shenandoah), human PDGF-AB (20 ng/ml) (Shenandoah) and heparin (10 ng/ml) (STEMCELL Technologies). Human neural precursor cell cultures were maintained as neurospheres in DMEM/F12, Neurobasal(−A), B27(−A), human-bFGF, human-EGF, heparin, and LIF (Millipore).
Luminex assay
Neurospheres were dissociated into a single cell suspension, and resuspended at 200 k cells/mL in 2.5 mL TSM without growth factors. After 24 h, supernatants were collected, centrifuged and filtered through a 0.22μm filter to remove cells, aliquoted and immediately stored at − 80 °C. All cultures were collected in triplicate at different cell passages. Supernatants were analyzed on the Luminex 200 platform by the Human Immune Monitoring Center at Stanford University. Human 63-plex kits were purchased from eBiosciences/Affymetrix and used according to the manufacturer's recommendations with modifications. Briefly: Beads were added to a 96-well plate and washed in a Biotek ELx405 washer. Samples were added to the plate containing the mixed antibody-linked beads and incubated at room temperature for 1 h followed by overnight incubation at 4 °C with shaking. Cold and Room temperature incubation steps were performed on an orbital shaker at 500–600 rpm. Following the overnight incubation plates were washed in a Biotek ELx405 washer and then biotinylated detection antibody added for 75 min at room temperature with shaking. Plate was washed as above and streptavidin-PE was added. After incubation for 30 min at room temperature wash was performed as above and reading buffer was added to the wells. Each sample was measured in duplicate. Plates were read using a Luminex 200 instrument with a lower bound of 50 beads per sample per cytokine. Custom assay Control beads by Radix Biosolutions are added to all wells.
Immunohistochemistry and light microscopy
Primary tumor samples were processed as previously described [34]. Briefly, primary tumor samples were fixed in 4% paraformaldehyde-PBS overnight, and then transferred to 30% sucrose until tissue samples sank (2–3 d). Tissue samples were transferred to cryomolds and embedded in optimal-cutting temperature (OCT) compound (TissueTek). 10 μm cryosections were generated on a cryostat (Leica). Endogenous peroxidase activity was neutralized (Bloxall, Vector Laboratories) before samples were permeabilized (0.5% Triton X-100, TBS) and blocked (5% horse serum, Vector). Immunohistochemical labeling was performed for CD3 (Biolegend, 300302, 3 h RT). CD3 was developed with a peroxidase secondary (ImmPRESS VR anti-rabbit IgG, Vector) and ImmPACT AMEC Red Peroxidase Substrate (Vector). Tissue samples were counterstained with Hematoxylin QS (Vector, 45 s, RT), developed in a bluing solution (0.1% NaCO3/H2O, 1 min) and coverslipped with Fluoro-Gel with Tris buffer (Electron Microscopy Services). Samples were imaged on a Nikon Eclipse.
Immunofluorescence and confocal microscopy
Primary tumor samples were prepared as described for immunohistochemistry. Frozen cryosections were rehydrated in PBS. Antigen retrieval was performed with L.A.B. Solution (Polysciences, 5 min, RT). Sections were blocked for 1 h at RT in 3% NDS (Jackson ImmunoResearch) and stained overnight at 4 °C with rabbit anti-IBA1 (Wako, 1:1000). Secondary antibodies conjugated with AlexaFluor 488 were used at RT for 4 h to detect primary labeling (Jackson ImmunoResearch, 1:500). Sections were mounted in ProLong Gold Antifade Mountant with DAPI (Thermo Fisher). Mounted samples were images using confocal microscopy (Zeiss LSM710), and acquired Z stacks were flattened through maximum intensity projection (Zeiss ZEN).
Gene-ontology terms associated with the top 50 genes contributing to principal component 1 (top) or principal component 2 (bottom)
GO Terms: Top genes down in PC1 (up in DIPG/aGBM)
GO Term
Cell adhesion
Extracellular matrix organization
Platelet degranulation
Peptide cross-linking
Neutrophil chemotaxis
Movement of cell or subcellular component
Platelet activation
GO Terms: Top genes down in PC2 (up in aGBM)
GO Term
Monocyte chemotaxis
Inflammatory response
Immune response
Neutrophil chemotaxis
Cellular response to interleukin-1
Chemokine-mediated signaling pathway
Positive regulation of ERK1 and ERK2 cascade
Cellular response to tumor necrosis factor
We next performed differential gene testing between our sample groups using DESeq2 (Fig. 3b-c). After filtering for significance (adjusted p-value < 0.1) and minimum gene expression (mean FPKM > 5 across all samples), there were 330 differentially expressed genes between DIPG and control (176 up, 154 down); 203 between aGBM and control (164 up, 39 down); and 160 between aGBM and DIPG (108 up, 52 down) (Additional file 4: Table S2). Macrophage response phenotypes have historically been separated into M1 "classical activation" and M2 "alternative activation" states, in which M1 macrophages may represent an anti-tumor response whereas M2 macrophages provide pro-tumorigenic functions, although this delineation is widely held to be insufficient to capture the diversity of macrophage phenotypes [31]. Previous reports have suggested that adult GAMs span the spectrum of M1 and M2 phenotypes [10]. We performed pre-ranked gene-set enrichment analysis on significant differentially expressed genes between DIPG-associated macrophages and cortical microglia and similarly found no significant enrichment for either M1 or M2 defined gene sets [32] (Additional file 5: Figure S3A-B), indicating that DIPG-associated macrophages do not fit neatly into an "M1" or "M2" classification.
Gene-ontology terms upregulated in adult GBM tumor-associated macrophages compared to DIPG tumor-associated macrophages
GO Terms upregulated in aGBM TAMs vs. DIPG TAMs
GO Term
Inflammatory response
Positive regulation of smooth muscle cell proliferation
Cellular response to interleukin-1
Positive regulation of transcription from RNA polymerase II promoter
Immune response
Cellular response to tumor necrosis factor
Chemokine-mediated signaling pathway
Positive regulation of gene expression
The data presented here indicate that DIPG-associated macrophages are strikingly less inflammatory compared to adult GBM-associated macrophages, expressing markedly lower levels of IL6, IL1A, IL1B, CCL3, CCL4, among other inflammatory factors. Concordantly, we find that DIPG tumor cells in culture, primary autopsy tissue, and pre-treatment diagnostic DIPG biopsy samples, are also non-inflammatory and lack expression of most chemokines and cytokines. In fact, one of the few factors expressed by DIPG is TGFB1, a known immunosuppressive growth factor. We also observe a lack of infiltrating CD3+ lymphocytes in primary DIPG tissue, suggesting that the presence of DIPG does not recruit tumor-infiltrating lymphocytes (TILs). Importantly, we observed this both in early post-mortem DIPG autopsy samples as well as pre-treatment diagnostic DIPG biopsy samples, suggesting that TILs are not a meaningful part of early or late DIPG pathophysiology.
Historically, macrophages have been classified as "M1" classically activated or "M2" alternatively activated phenotypes, although this classification system has been acknowledged to be insufficient to capture the complexity of macrophage responses [31]. Although DIPG tumor cells produce CSF1, a cytokine associated with the M2, pro-tumorigenic phenotype, we found that DIPG-associated macrophages do not fit neatly into an M1 or M2 classification [32]. Similar to reports in adult GBM [10], DIPG-associated macrophages appear to be in a tumor-specific activation phenotype related to the distinct tumor-derived chemokine milieu. In our analysis of DIPG-associated macrophage gene expression, we observed that these cells express increased levels of antigen-presentation genes such as HLA proteins. However, the comparative lack of production of pro-inflammatory chemokines (e.g. CCL3, CCL4) and absence of lymphocytes in primary tissue are consistent with the failure of DIPG-associated macrophages to trigger an effective anti-tumor immune response. Adding to the evidence for lack of an effective innate or adaptive immune response in pediatric high-grade gliomas, a previous study demonstrated the lack of NK cell infiltration into pediatric high-grade gliomas [16], although this study did not specifically investigate DIPG. An "immune cold" state of DIPG is also consistent with the lack of inflammatory cells in pediatric non-brainstem gliomas recently described [30].
The findings presented here are particularly relevant to the development of immunotherapeutic approaches to DIPG. Many current approaches in adult GBM include the use of checkpoint inhibitors [36], the effectiveness of which is linked to pre-existing CD8+ T cell presence [51] and mutational load [42]. Along with our observation that DIPG tumors contain very few infiltrating T-cells, DIPG exhibits a lower mutational burden compared to adult glioblastoma [47]. Thus, immunotherapy approaches in DIPG may be better served by focusing on inducing recruitment or introduction of immune cells to the tumor. One promising strategy involves the use of chimeric antigen receptor T (CAR-T) cells, which are designed to recognize tumor-associated antigens. We recently demonstrated striking preclinical efficacy of GD2-targeted CAR-T cell therapy in preclinical models of DIPG [34].
Adult and pediatric high-grade gliomas are distinct disease entities, and differences between DIPG and adult glioblastoma extend to the immunological phenotype of the tumor microenvironment. In contrast to adult GBM, the immune microenvironment of DIPG is non-inflammatory and does not contain a significant adaptive immune component. These observations provide important considerations for the design of immunotherapeutic approaches for DIPG.
The authors gratefully acknowledge support from the Cure Starts Now Foundation and DIPG Collaborative, Chan Zuckerberg Initiative and the Silicon Valley Community Foundation, McKenna Claire Foundation, Unravel Pediatric Cancer, Stefan and Julia Roever Research Fund, National Institute of Neurological Disorders and Stroke (NINDS R01NS092597), Child Health Research Institute at Stanford, Anne T. and Robert M. Bass Endowed Faculty Scholarship in Pediatric Cancer and Blood Diseases, the Stanford Medical Scientist Training Program, and the Stanford Graduate Program in Neuroscience.
Authors' contributions
GLL carried out the primary tissue processing, flow cytometry and FACS experiments, RNA extraction and library preparation, gene expression analysis, primary tissue immunostaining, cell culture experiments. SN assisted in RNA gene expression analysis and interpretation. MGF and MLS performed biopsy single-cell isolation and RNA sequencing preparation and analysis. HV assisted with primary tissue immunostaining and interpretation of immunohistochemistry experiments. MM conceived of the study, participated in its design and coordination and supervised all aspects of the work. GLL and MM wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
No animals were used in this study. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
Consent for publication
Informed consent was obtained from all individual participants included in the study prior to any tissue collection.
Competing interests
The authors declare that they have no competing interests.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Authors' Affiliations
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Gunner Liam Bretherton, 20, faces dismissal from the Army after walking into a guitar shop with a valuable Gibson Les Paul just 12 hours after a flagship music store in Manchester had been ransacked by looters.
He was arrested when the owner of the shop became suspicious and checked the serial number on the instrument. He then locked him in the shop until the police arrived.
Bretherton, who serves with the Royal Regiment of Artillery, indicated that he had been prepared to admit handling stolen goods but when the charge was upped to burglary pleaded not guilty.
Army sources admitted that they were braced for a number of soldiers to be charged following this week's rioting as the majority are on summer "bloc leave" in August.
One suggested that it could be as a many as half-a-dozen. "I'm not sure that this will be the last soldier as a lot of young lads could easily get caught up in this," he said.
Manchester City magistrates court heard that Bretherton, from the 7th Parachute Regiment Royal Horse Artillery, regarded as the top Gunner unit, had been in the Army for three years and had no previous convictions.
He claimed that he had purchased the instrument, worth almost £2,000, shortly after it was stolen because he had an interest in guitars but had not realised that it was a left-handed.
Gareth Brandon, prosecuting, told the court: "His account was that he'd headed into Manchester having heard about the riots.
"It seems clear that it was the defendant's position all along to see what he could take. He has some interest in guitars and was very pleased to obtain this item.
"It was his account that he was present outside the shop being looted and paid £20 to one of the looters for this item."
Carol Mellor, defending, applied for bail, telling the judge that Bretherton, from Leigh, Wigan, was "certainly" not involved in the looting but had been prepared to plead guilty to the lesser charge of handling stolen goods.
"Even if it was handling he realises how stupid he's been because he's put his career at risk," she said.
"Even if he's granted bail I'm sure there will be an inquiry that could result in his discharge."
District Judge Khalid Qureshi rejected the defence, criticising the "cavalier attitude" with which Bretherton had tried to sell the guitar.
He said: "I find it hard to believe that someone with an interest in guitars just gravitated to a music store where a crime was being committed and ended up with a left-handed guitar by mistake.
"I don't suggest that you were one of the people who forced themselves in, that's not the crown's case. Someone who would do this is not someone who can be trusted with bail."
Bretherton's mother, Eileen, sobbed as he was sent down to the cells. His distraught father, Alan, yelled from the public gallery: "He's been a good lad all his life, he'd never been any trouble to us".
If the soldier is jailed will almost certainly face dismissal from the Army. A Ministry of Defence spokesman said: "All those who are found to fall short of the Army's high standards or who are found to have committed an offence under the Armed Forces Act are dealt with administratively up to and including discharge."
Bretherton is next due before the court on August 19.


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Friday, February 3, 2017
Today's Specials:
GUESTS: Two Posters to Help Your Staff Avoid Food Allergy Incidents
No one wants to be the restaurant that sets off a guest's food allergy. Here are two useful posters to hang in your eatery to help aid your staff in keeping guests safe and happy.
WATCH: Gordon Ramsay Really Doesn't Like Girl Scout Cookies [How Hollywood Sees Us]
There is little doubt perception of the celebrity chef includes a sense they are self-importance and arrogant. Turns out Gordon Ramsay is not the least bit interested in dispelling that belief in the appearance on Jimmy Kimmel's show.
Getting Trashed Later?
Leave it to English-speaking world, but English has more than 3000 words for getting drunk. In fact, only words for sex and money outrank getting drunk. Some of our favs? Ramsquaddled, obfusticated, drunk as a thrush and wamble-cropped.
Premium Seats = Premium Prices
Attending Super Bowl LI is not cheap considering secondary tickets are currently selling for roughly $2,000. According to this infographic, premium tickets for premium sporting events will easily break a fan's budget.
Where's the Beef?
According to the Seafood & Vegetarian CTR, 59% of consumers eat meatless meals at least once a week. It's more proof that you need high-quality vegetarian or vegan options to keep guests satisfied.
GNC Rejected!
The NFL has rejected GNC's Super Bowl ad? But not because it's offensive or controversial. It's because GNC is listed as a "prohibited company" by the NFL and NFLPA over banned substances in some of their products. Check out the banned commercial here.
Why it matters to you: customers often distort what believe based on perception.
Wendy's has been stepping up their game in recent days. This Sunday, Wendy's is set to run their first ever Super Bowl ad and they tackle the false claims that their burgers are made from frozen beef. In the ad they poke fun at other enormous chains that do freeze their burger meat playing on the fact that they are "always fresh never frozen." The ad also displays a web address, Othr-Guyz.com where they continue to poke fun at a frozen hamburger products via an infomercial.
One key aspect that Wendy's is playing on involves customer perception. Many people believe what they perceive, in turn, that fresh beef tastes better; all of which Wendy's sets the record straight. Not only is the ad funny, but it also contributes to a storytelling process that reinforces their brand. According to Wendy's advertising agency, seven out of 10 consumers don't believe Wendy's exclusively uses unfrozen beef. Therefore these ads for the upcoming Super Bowl are much needed.
Why it matters to you: Give your staff a road map to follow for achieving their aspirations
The bartender's role is among the most revered in our industry and therefore it should be no surprise that many servers aspire to get behind the stick. Managers that realize this and provide substantive direction to their aspiring servers accomplish several things simultaneously. First, you improve performance through competition, while also to creating bench strength through cross training so you never have to look too far for your next great bartender.
With the staffing woes we all experience, taking steps to ensure your most impactful positions are staffed by people prepared to immediately perform, it will only serve to improve our execution and ultimately our sales. Give anyone that wants to grow on your team, the steps they must take to achieve their goal and ultimately you will improve your own life along with that of those ambitious members of your team.
Why it matters to you: how are you supporting your community through leadership?
Restaurants can offer a taste of freedom for many guests as well as owners. This is especially true for two men recently imprisoned for murders they didn't commit; now becoming business partners in a new Brooklyn restaurant. The two men met while in jail, where they both worked together for more than 20 years as jailhouse lawyers to acquit wrongfully convicted inmates, themselves included.
Neither of the men had any prior restaurant experience when they decided upon their new Jamaican style establishment. More importantly, they stressed the fact that they hire many employees who are having trouble finding jobs due to past felonies. "If your ego doesn't stop you from picking up a broom and a mop and you want to work, we got you." Building a community is an essential aspect of the restaurant business and rallying in support of people you sympathize with is the epitome of a purpose economy.


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The Household Cavalry Museum
The Household Cavalry Museum allows people the chance to see history in action as troopers work with their horses in the original 18th century stables. The museum can be found in the Horse Guards in Whitehall and the famous Household Division dates back to the 1750. The Queen's Life Guard go about their daily duties while onlookers stand behind a glazed partition enjoying the ceremonies and actions of this iconic military division.
There are exhibits that recall the history of the regiment from over 200 years ago, including rare uniforms, musical instruments, silverware by Faberge and decadent horse furniture. The added treat is being able to watch the inspection of the guards at 4pm every day. This was originally a punishment meant to last 100 years, imposed by Queen Victoria after she discovered the guard gambling and drinking, but Queen Elizabeth II decided to keep the tradition in place once the 100 years expired in 1994.
Tel: 0207 930 3070
Adult : £6.00
Child : £4.00
Senior Citizen: £4.00
- Photos
- Map


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Steps to Straight Teeth
Take the Free Smile Assessment
This first step is a quick and easy way to gauge whether or not Invisalign treatment will fit your needs.
It's free and can be completed in just a few minutes.
For the best treatment results, choosing an experienced doctor can make all the difference. Each doctor may vary when it comes to pricing, patient interaction and treatment recommendations. We suggest doing a little research and possibly meeting with more than one doctor to find the one who best fits your needs. Many doctors offer free consultations.*
Make sure all your questions are answered. Bring a printout of our Frequently Asked Questions and Questions for Your Doctor sections to help provide topics for discussion.
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Begin Treatment
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Then your doctor will map out a precise treatment plan, including the exact movements of your teeth and how long your treatment will be. You'll even get to see how your teeth will move and preview your new smile.
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Wear your Invisalign clear aligners 20 to 22 hours a day for the best results, taking them out to eat and to brush and floss.
Your progress
At every stage you'll be able to see your progress and imagine how far your new smile will take you.


text: Stress Free STD Testing understands that your life is busy and you don't always have time to test during the workweek. That's why we have Saturday testing available to our clients at many of our locations at no extra cost to you. No matter your schedule, we can help you find a testing appointment that is convenient for you. If you have any questions, or to get more information about Saturday testing hours and locations, call our care counselors today at 1-888-978-3435.