As property managers, we are charged with preserving and enhancing the owner's asset and serving the needs of our tenants. Getting recognition from our peers is the ultimate compliment and a true reflection of the hard work, energy and passion we bring to the job every day.
In June, the FBI Chicago Regional Headquarters had the good fortune of earning the International TOBY in the Earth Award category, BOMA's highest honor recognizing environmentally sustainable property management. The building won over six other outstanding, well-managed properties located throughout North America. It truly was a magical moment for the USAA Real Estate team when our property was announced during the BOMA International Conferencein Washington, D.C.
Make it a Team Effort. Every member of the USAA Real Estate management team participated in our entry. Our team was led by me and included the chief and assistant engineer, our property assistant, the journeyman engineer and the cleaning supervisor. We felt it was critical to have everyone involved and on the same page. Plus, we maintain that staff is more committed and will take more pride if they know from the onset that they're part of the process.
Establish a TOBY "Situation Room." To prepare for the onsite judges visit, we reserved a separate conference room that had everything we wanted to highlight and showcase, plus enough room for the judges and staff. On the conference room table, we neatly arranged binders containing cleaning records, supply purchases and MSDS sheets of products and cleaning equipment. Several pieces of cleaning equipment were positioned in the room; each had spec sheets attached that reflected low decibels and low HEPA filters.
The Clock is Ticking. Allow for sufficient time to complete the online application and prepare for the onsite visit. Gear up for a lot of hard work, but as noted above, involving the entire staff will help spread the work load and contribute to the sense of camaraderie.
Little Things Mean a Lot. We went out of our way to creatively and clearly showcase the FBI Chicago Regional Headquarters in the best possible way. For example, we systematically reviewed every judging category requirement and prepared binders that included every piece of information requested. Each binder had a clear table of contents and included a professionally-shot photograph that related to the contents. If your property does advance to the Regional and International competition, the judging gets much tougher and small details will play a major factor.
We think about sustainability every day in our building, and we promote the concept as much as possible to our tenants. In fact, our building was the first in the world to earn the Platinum Level LEED EB O&M from the USGBC. Earning the Earth Award justifies our commitment to the environment as well as our tenants. During each TOBY victory, we celebrated with our entire building.
Winning a major award has a positive effect on the entire company. I hope our TOBY will motivate other USAA-managed office properties to enter the process and compete. My staff worked hard throughout the process, and frankly I was relieved that their efforts really paid off in the end. What's more, the FBI was thrilled, since the Earth Award marked the first time any FBI property captured a TOBY award of any kind.
I'd welcome thoughts and comments from other BOMA/Chicago Building Members that have advanced through the local, regional and international TOBY competition. Your property can, too. Take the TOBY challenge and try.
This entry was posted in Property Management, Tenant Relations and tagged BOMA International Conference, BOMA/Chicago TOBY competition, Platinum Level LEED EB O&M, USAA Real Estate. Bookmark the permalink.


Do you have an idea for a great app? Want to focus on nutrition and fitness? We've created an example business plan to help you get the ball rolling!
Have you developed a unique app for nutrition and fitness? Need to get it out there? Creating a business plan will help you identify your market and streamline your finances.
To help you get started we've created an example business plan for a Health and Wellness Application based business. Our example focuses on a fitness app, but it will also work as a framework for other apps. Click the 'Download Tool' button to gain access to the word document.


Let's get this out there right in the beginning, I am totally NOT a dream whisperer. I haven't even really studied dreams enough to be considered an expert. HOWEVER, I had this realization yesterday and I just have to share. Dreams are powerful.
Last week I started a Bible Study by Havilah Cunnington "Eat, Pray, Hustle: Dream Chasing God's Way" and I have got to tell you my eyes are really being opened. Even more so, I'd say my heart is being stirred as old dreams are resurfacing and new dreams are occurring. I am excited for what's to come.
What Does the Bible Have to Say About Dreams?
19-25 Abraham didn't focus on his own impotence and say, "It's hopeless. This hundred-year-old body could never father a child." Nor did he survey Sarah's decades of infertility and give up. He didn't tiptoe around God's promise asking cautiously skeptical questions. He plunged into the promise and came up strong, ready for God, sure that God would make good on what he had said. That's why it is said, "Abraham was declared fit before God by trusting God to set him right." But it's not just Abraham; it's also us! The same thing gets said about us when we embrace and believe the One who brought Jesus to life when the conditions were equally hopeless. The sacrificed Jesus made us fit for God, set us right with God.
During this time having children was the "bee's knees" you know, the ultimate compliment to marriage. Abraham and Sarah longed for a family yet never lost sight of God's promise. The odds never were in their favor as they were both quite old. However when you have a God sized dream and a God that doesn't play by anybody's rules amazing things can and will occur. Sounds like permission to dream big to me!
How exciting is this?! I'm real fired up over here!
Let me say this in "Amanda-ease" (ha I have no idea… Just go with it) God promised Abraham and He kept His word! He fulfilled His promise or His COVENANT to Abraham. Because Abraham trusted that God's word was going to be fulfilled we all get to be blessed by it. Listen to me here, Abraham didn't live an infallible life. Abraham DID however trust in God.
As Abraham was busy growing his testimony – you know all the trials and tests that came his way God was busy pouring out His GRACE on Him. Come on!!! Are you as excited as I am? God didn't expect Abraham to be perfect in order to fulfill his greatest dream. He simply wanted Abraham to trust in HIM! Talk about a testament of faith. Because Abraham never lost sight of God and was obedient to Him, God fulfilled His promise.
Hallelujah!!! God is so good. Dreaming big, as big as the stars is fun, but I know that it will also require me standing long to. But thank you for this encouragement!! We are heirs to the promise!


text:
namespace ZooRestaurant.Web.Models.ViewModels.Users
{
    using System.ComponentModel.DataAnnotations;
    using Data.Models;
    using Infrastructure.Mapping.Contracts;

    public class UserViewModel : IMapFrom<User>
    {
        public string Id { get; set; }

        [Display(Name = "Име")]
        public string FirstName { get; set; }

        [Display(Name = "Фамилия")]
        public string LastName { get; set; }

        [Display(Name = "Поща")]
        public string Email { get; set; }

        [Display(Name = "Телефон")]
        public string PhoneNumber { get; set; }

        [Display(Name = "Потребителско име")]
        public string UserName { get; set; }
    }
}

redpajama_set_name: RedPajamaGithub


text:
Home >> Virginia
Top 10 Places to Visit in Virginia
Photograph Flickr by ShenandoahNPS, via Wikimedia Commons
#1 Shenandoah National Park
Located about 75 miles from Washington, D.C., Shenandoah National Park lies along the Blue Ridge Mountains in north-central Virginia. The park stretches for 105 miles along popular Skyline Drive. The national park features spectacular vistas, waterfalls, and over 500 miles of hiking trails. Park activities include hiking, camping, birdwatching, wildlife viewing, and rock climbing.
Photograph by Protoant, via Wikimedia Commons
#2 Virginia Beach
Located on the Atlantic Ocean at the mouth of the Chesapeake Bay, Virginia Beach is a popular resort city. It is known for its miles of beaches and recreation along oceanfront. Virginia Beach is home to many popular state parks. The beach city has the longest pleasure beach in the world. Virginia Beach is a popular family vacation gateway. Things to do and see include Virginia Beach Boardwalk, Virginia Aquarium & Marine Science, First Landing State Park, Chesapeake Bay Bridge-Tunnel, False Cape State Park, and Cape Henry Lighthouse.
Photograph by CoredesatChikai, via Wikimedia Commons
#3 Richmond
Photograph Flickr by Humberto Moreno, via Wikimedia Commons
#4 Colonial Williamsburg
#5 Arlington National Cemetery
Photograph by Otherspice, via Wikimedia Commons
#6 Mount Vernon
Photograph by Lluck002, via Wikimedia Commons
#7 Norfolk
Photograph by The old perfesser, via Wikimedia Commons
#8 Natural Bridge
Photograph by Moofpocket, via Wikimedia Commons
#9 Monticello
Photograph by Tomandjerry211, via Wikimedia Commons
#10 Luray Caverns

redpajama_set_name: RedPajamaCommonCrawl


At Buskirk Engineering, we design complete turnkey systems for all of your material densification needs. Being the first company to bring such a solution to market, we can have you up and running in under 24 hours once delivered, on site. We also offer a complete line of ancillary equipment as well. From Bulk Holding Tanks and Handling Conveyors. Counter Flow Pellet Coolers and everything in between.
Don't see something you're looking for, let us know. We can work around your vision.


text:
Q: Customized rules and can be changing very often -every week Our application has many business rules and it can be changing very often , even the parameters and prioritizationapplied on the rules can change very often consider -every week .
SHould i use drools workbench or customized UI

redpajama_set_name: RedPajamaStackExchange


text:
« Rob's Movie Vault home page
The Times of Harvey Milk
The money shot comes about half an hour before the end of The Times of Harvey Milk. In response to the shocking murders of San Francisco Supervisor Harvey Milk and San Francisco Mayor George Moscone, the grieving masses of Milk's district march down Castro Street holding candles. 50,000 people, marching quietly. "Harvey would've loved it," says an associate in the film.
The sight is breathtaking and sadly ironic; it took Milk's death to bring so many together. Later, Dan White, Milk's fellow supervisor, who killed Milk and Moscone, was brought to trial and found guilty of voluntary manslaughter rather than first-degree murder. This time, the masses were not so calm; the resulting White Night Riots caused over a million dollars' worth of damage. Milk would not have loved this, and he would've been the first to try to quiet the rage.
"Harvey Milk" is an almost comically bland name, calling forth an image of a dorky man sipping a glass of harmless white stuff. Milk seemed to live his life in opposition to his name. He was a firebrand, though not so radical as to alienate the hetero whitebreads whose support his movement needed; he was a skilled politician, opening a rhetorical umbrella under which oppressed people of all types — blacks, Asians, women, the elderly, the disabled — could assemble and be acknowledged. The Times of Harvey Milk, which won 1984's Oscar for Best Documentary Feature, includes plenty of footage of Milk at work, and if he wasn't 100% sincere in his rhetoric, he sure stayed on-message pretty well. There's no reason to believe he wasn't sincere, though the movie touches lightly, if at all, on Milk's flaws (a temper, a certain rigidity).
You need to see the movie (and, perhaps, read Randy Shilts' The Mayor of Castro Street) to understand the true impact Milk had, not only locally but nationwide; you need to see the people who knew him and the people who never met him speaking fondly of his example and his achievements. I liked what I saw of Harvey Milk, but the movie itself, a typical early-'80s bit of talking-heads hagiography, hasn't aged very well. Aesthetically, it's not much, except for the pleasure of its narration, read by Harvey Fierstein in his familiar theatrical growl. The subject commands more respect and affection than does the movie itself.
Partly, the film falls victim to the inevitable structure of a story of an assassination: roughly halfway through the 88-minute running time, Milk is abruptly removed from the picture, and the remainder is about the aftermath and about Dan White. (A later documentary, The Brandon Teena Story, had the same problem.) We may rebel against this structure, feeling that Milk deserves more screen time than his opaque assassin. Why did White do it? The consensus is that he was unhinged and dangerously depressed — he'd resigned from his job, then wanted to change his mind, but Moscone wouldn't have it. (Mike Weiss' 1984 book Double Play does a thorough job of analyzing White's motives, which went beyond his lawyer's infamously ridiculous "Twinkie defense.") So White killed Moscone, then went to Milk's office and killed him too, because he felt Milk and Moscone were in cahoots to ruin White's career.
The problem with a movie about Harvey Milk is that it has to end with all this conjecture about the man who snuffed him out. What the movie doesn't tell you (but the extra features on the 2-disc DVD do) is that White, after serving five years of his seven-year sentence and being paroled in 1984, killed himself in 1985. So justice was served in a rough way; White had to live with himself, and he couldn't. Audiences for the film in 1984, though, were left with the knowledge that White was free out there somewhere. The movie ends with Milk's "hope" speech, but again, the structure works against that as well.
Reviewing the movie on eFilmCritic in 2005, Aaron West supposed it was "maybe not as relevant today as it was in 1984." Things, of course, have changed even since 2005; as I write this, California's gays are still reeling from the passage of the anti-gay-marriage Proposition 8. Watching Milk in action in this movie, I kept wondering if Prop 8 would've had a chance in hell if Milk were still alive. He'd have drawn people together, appealed to their sense of fairness, and reached out beyond the gay community to everyone who might otherwise have pulled the lever for Prop 8. It's all well and good to keep hope alive, but — as Milk himself says in the film — you can't live on hope alone.
Categories: documentary
But I wish the public could, in the midst of its pleasures, see how blatantly it is being spoon-fed, and ask for slightly better dreams.
- Iris Barry, Let's Go to the Movies, 1926
How can a movie review be written in the third person, as if it were an account of facts? If it isn't subjective, there's something false about it.
- Roger Ebert
A man watches a movie, and the critic must acknowledge that he is that man.
- Robert Warshow
If art isn't entertainment, then what is it? Punishment?
- Pauline Kael
If you have the right face saying the right words at the right time, you've got everything cinema has to offer.
- David Cronenberg
Letterboxd capsule reviews
Movie Vault Bookstore
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Hispanic Heritage Month: A Book List for Celebrating Culture!
Ana-Sofia DuLaney
We are celebrating Hispanic Heritage Month from September 15 to October 15! Let's use this time to celebrate the history and beauty of Hispanic culture all over the world.
Hispanic Heritage Week was first observed in 1968 under President Lyndon Johnson. However, in 1988, President Ronald Reagan extended the week into a month-long celebration and signed Hispanic Heritage Month into law. Each year, our presidents make official proclamations to begin the month. So whether you're of Hispanic or Latino origin or not, I encourage you to share these books with your children so they can learn the beauty of this culture, especially here in Miami!
Why is Hispanic Heritage Month in the middle of two months?
Interestingly enough, we celebrate Hispanic Heritage Month from the middle of September until mid-October to coincide with the independence of various Hispanic countries like Chile, Mexico, Costa Rica, El Salvador, Guatemala, Honduras, and Nicaragua.
Did you know Costa Rica, El Salvador, Guatemala, Honduras, and Nicaragua all share the same Independence Day of September 15?
How can you bring Hispanic heritage into your home?
What's better than books that tell of Hispanic and Latinx people and culture?
I've compiled a list of books that are great for different age groups, contain a mix of English and Spanish, and are both fiction and nonfiction. So, I hope you will agree these books touch upon culture, people, families, and the diversity of various groups of Hispanics. I own all these books and love them! I have conveniently linked all the books here so you can easily purchase them from our Amazon Storefront just by clicking the photo of the book. All books are under $15 and on Amazon Prime! FYI… You can find tons of other great items for Moms by Moms on our Amazon page!
I Love Saturdays y Domingos by Alma Flor Ada, illustrated by Elivia Savadier
We are a multicultural family; my husband is Non-Hispanic American, and I am Hispanic American; so, this book really resonated with me. The book details a young child's visits with her grandparents on Saturdays and Sundays. On Saturdays, the lead character visits grandma and grandpa, and they have a European-American background. And then on Sundays, Domingos, she goes to visit her abuelita and abuelito, they are Mexican-American. It shows the beautiful differences in culture along with their similarities, especially their love for their grandchild. I loved reading it to Parker and in a way, comparing the ways his grandparents are different and equally special. I loved that the book used both English and Spanish in a way that helps expand their vocabularies. This book is recommended for ages 5-8 but my almost 3-year-old had no trouble with it.
Mango, Abuela y Yo or Mango, Abuela and Me by Meg Medina, illustrated by Angela Dominguez
This book comes in Spanish and English. I bought it in Spanish but either one works well because the story is so sweet. A little girl's grandmother moves in from her Latin American home country and immediately they realize there is a language barrier. The granddaughter and grandmother are desperately trying to communicate with each other and by working together, they find a way to teach each other words in English and Spanish. Using a fun new tactic along with the help of a fun parrot named Mango, the two create an even stronger bond. I love that the words were written in both languages so your child can learn or you can help teach them as you read and continue beyond Hispanic Heritage Month. The book is recommended for ages 5-8 but I think it works perfectly well for younger or older.
Frida Kahlo: The Artist Who Painted Herself by Margaret Frith, illustrated by Tomie dePaola
Who doesn't love Frida? Her paintings, her strength, her spunk. I love that this book because it is a biography with a twist of fiction. A young girl writing a school report on Frida narrates this book for a fun new perspective. It combines real reproductions of Frida's artwork along with original illustrations and fun comments from the narrator. It's a quick-moving look at Frida's life, her childhood, her struggles, and her amazing creativity and uniqueness. Frida's creative personality comes to life and the book helps us understand how this incredible artist is an inspiration to love oneself and why exactly it is that she painted so many self-portraits. This book was solely in English and is recommended for ages 5-9.
My Name is Gabriela/Me llamo Gabriela by Monica Brown, illustrated by John Parra
This book was particularly special to me because I am Chilean. This biography of Gabriela Mistral by Monica Brown is an absolute beauty. Mistral was a Chilean poet and a teacher and she became the first Nobel Prize-winning Latina woman in the world. She has inspired adults and children alike. This book is both eye-catching and beautiful to read as the words seem to move with the pages with the beat of poetry. Mistral traveled the world and shared her passion for storytelling. It was a lovely tribute to a woman who taught us the power words can have and that we must always follow our dreams. The story is bilingual with English and Spanish side by side without losing its majesty. Recommended for ages 4-10, it can be for younger readers as well.
Turning Pages by Sonia Sotomayor, illustrated by Lulu Delacre
This book is Sonia Sotomayor's children's autobiography, her story of becoming the first Hispanic and Latina supreme court justice. Perfect for Hispanic Heritage Month. What a beautifully illustrated book and the story is even better. Sonia Sotomayor's life was full of difficulty, including her diagnosis of diabetes and the passing of her father. However, she overcame her struggles and she found inspiration, support, and contentment in… guess what? BOOKS! She writes beautifully to inspire young children to love books as she does. Sotomayor teaches us all about overcoming adversity and being driven to make a positive impact on our great country. Recommended for ages 4-8 years old. It comes in either English or Spanish, we loved it in English.
Yes! We Are Latinos: Poems and Prose About the Latino Experience by Alma Flor Ada and F. Isabel Campoy, illustrated by David Diaz
A book for the older readers, this collection of poems and short stories are fictionalized profiles of Latinx American characters along with historical information of where they came from. The stories open a window into the wealth of Hispanic Americans that exist in our country, making for a beautiful read for Hispanic and non-Hispanic readers. From African ancestry, Asian ancestry, Sephardic, the book touches nearly any combination of Latinx you can think of and gives a glimpse into the real history along with what it's like to live in different cities in the United States. This book is recommended for ages 10-13 years, but I think the book is great for adults, as well!
My Name is Gabito/Me llamo Gabito: The Life of Gabriel Garcia Marquez by Monica Brown, illustrated by Raul Colon
Another Monica Brown book, My Name is Gabito uses incredible imagery to capture the talent of Colombia's Gabriel Garcia Marquez. His powerful imagination is beautifully depicted in this multilingual book that goes from English to Spanish seamlessly. Marquez, nicknamed Gabito as a child, was one of the greatest writers of our time. This picture book follows his childhood in Colombia to his life more recently, prior to his 2014 death. A very inspiring story of a man full of imagination that saw beauty in the world. 4-10 years old is the recommended age but also works for younger and older age groups.
I truly encourage you to delve into some books with your children this Hispanic Heritage Month. So, I hope this list makes it easier for you to choose and may you celebrate the diversity and beauty of Hispanic heritage not just for a month, but every day.
Do you have any books you'd recommend? I'd love to hear your favorites too. Drop a comment below!
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Ana-Sofia is a Miami native that chose to never leave her hometown. She attended FIU and is an attorney that graduated from the University of Miami School of Law. Ana-Sofia finally became more than friends with her childhood neighbor after over 10 years, and now they are happily married! She was a proud dog mom before she became a human mom, and after the birth of their now four-year-old son, Ana-Sofia has been lucky enough to work from home. In August 2021, their family grew by one more and they welcomed a new baby boy to the bunch! She loves traveling with her family but also loves staying home curled up on the couch. An avid Dave Matthews fan, Ana-Sofia is also a self proclaimed extroverted introvert, grammar freak, Harry Potter obsessed, Heat and UM cheering, and gardening loving mama that hopes she's doing right by her sons while trying to reduce her ecological footprint. Follow her on Instagram as she tries to live each day fully @TheAnaMachine or her gardening journey on @GardeningInJeans
Work-Life Balance: How to Manage It as a Working Mom
Body Positivity in the New Year
Vision Boards: 6 Steps to Creating One That Rocks
Becky Salgado September 14, 2020 At 4:56 pm
Thanks for curating this list!
Ana-Sofia DuLaney September 15, 2020 At 11:14 am
I hope it's useful and the kids and you can enjoy some interesting reads!!
BE A MIAMI MOM COLLECTIVE INSIDER
Guide To Miami
Cierra - January 31, 2023 1
February 2023 Local Family Friendly Events & Activities If you are looking for a guide of family events and activities in the 305 to keep...


text:
CJ Giri may step in to resolve Judicial Council deadlock
By Himalayan News Service
Chief Justice Kedar Prasad Giri is likely to take steps to resolve the deadlock cropped up in the functioning of the Judicial Council next week. Giri, also the ex-officio chairperson of the JC, has initiated process of dialogue for solving the problem.
CJ Giri has scheduled to hold talks with the rift parties and called the government not to take stance on the issue. A Supreme Court source revealed that the Chief Justice has fixed separate schedules of meetings with the minister for law and justice and senior advocates in order to settle the dispute. "We will be able to find out a solution by the next week if everything goes as expected," the source told this daily. The JC members have also suggested
that the Chief Justice play his role as the head of the institution and calling the government to give up its wrong stand.
"Yes, the Chief Justice is scheduled to hold meeting with the senior lawyers and the Nepal Bar Association representatives next week," NBA President Bishwo Kanta Mainali confirmed. The lawyers' body demanded that Chief Justice intervene for resolving the crisis through its decision last week. The Judicial Council could not hold even a meeting in the last two months due to the rift between the Law Minister and the JC member which created a hurdle for the appointment of around four dozen judges in the judiciary.
Moti Kaji Sthapit said that it would be appropriate if the CJ, who is the head of the institution, intervened into the problem for the solution. "I am also waiting for his initiative as a right way to put an end to the problem," he added.


The All New, All Daring Delusional Honesty!: The Daily P.O.P. and Me - Together!
The Daily P.O.P. and Me - Together!
I'm taking a bit of a break from my normal writing routine to let everyone know that I'm getting a new spotlight for some of my mighty missives, over at the terrific Daily P.O.P. site. First up is an ode to the "dark" Spider-Clone, the man called Kaine, vis-a-vis "Grim Hunt," the storyline that has returned Kraven the Hunter to menace the Amazing Spider-Man!
So go over there already! And if you're over here because of my article over there--welcome! Hope you find something you like in my archives, and more articles to come!
Hi Gary, just saw your article at Jameson's site. One word, BRILLIANT!
Man, I Can't Even Get Arrested!


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Create and update new content about BTCH in this section.
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How to add 360 degree effects in video One of the needs that will become increasingly important in the pipeline work of a composer , will be to know how to work with scenes of the video in 360 , adding effects and compositing directly on the display 360.
The video 360 it will be the brand new trend , the new frontier mainstream leisure , new technology that are coming out there are getting ready for the new frontier of 360 , spectacled Oculus cinemas of the new frontier .
Mikey Borup shows us how to add effects to movies 360 .
In the video tutorial explains how to : add a watermark , make the clean plate , compositing , color correction , lens flare , adding the element of a 3D sphere .
The process Sky-box Studio , you will see how easy to make the process as if you were working on a plate " classic " .


text:
Artera colică stângă este o ramură a arterei mezenterice inferioare.

Anatomie
Această arteră are un traseu spre stânga în spatele peritoneului și în fața mușchiului psoas mare. După un traseu scurt, dar variabil, se împarte într-o ramură ascendentă și una descendentă. Tulpina arterei sau ramurile acesteia traversează ureterul stâng și vasele spermatice interne stângi.

Ramura ascendentă traversează prin fața rinichiului stâng și se termină, între cele două straturi ale mezocolonului transvers, prin anastomozare cu artera colică mijlocie; ramura descendentă se anastomozează cu artera sigmoidă superioară.

Din arcurile formate din aceste anastomoze ramurile sunt distribuite către colonul descendent și partea stângă a colonului transvers.

Semnificație clinică
Artera colică stângă poate fi ligată în timpul intervenției chirurgicale abdominale pentru eliminarea cancerul colorectal.  Acest lucru poate avea rezultate mai slabe decât conservarea arterei.

Imagini suplimentare

Referințe

Legături externe

 Lotti M. Anatomie în raport cu colectomia stângă
 sup&infmesentericart
 Anatomy photo:39:05-0105  - "Intestinele și pancreasul: ramuri ale arterei mezenterice inferioare"
 Anatomy image:8585
 Anatomy image:8658

Anatomie umană
Aparatul cardiovascular
Arterele abdominale

redpajama_set_name: RedPajamaWikipedia


text:
Dependent Pass– Who Is It For?
Married Employment Pass holders in Singapore who choose to have their spouses and children live with them in Singapore.
Key Features of Singapore Dependant's Pass (DP)
Only lawful spouses and children younger than 21 years old can apply for this pass.
DP application must be sponsored by the employer of the EP holder.
DP period is tied to the duration of the EP and will expire at the same time as the expiry date of the EP it is tied to.
Holders of Dependent Passes who wishes to work in Singapore can apply for an independent EP or work permit.
Dependent Pass holders can also start their own business by incorporating a company or sole proprietorship and then apply for a Letter of Consent to work.
A child who lives in Singapore on DP can get educated in Singapore's international or public schools.
Eligibility Criteria and Requirements for Dependent Pass Application
The spouse /parent who acts as sponsor must be a holder of either the Employment Pass (EP), Personalised Employment Pass (PEP) or S Pass.
The sponsor must earn at least SGD6,000 per month.
Documentary evidence of marriage and birth certificate must be provided in English.


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Role of protein arginine methyltransferase 5 in group 3 (MYC-driven) Medulloblastoma
Nagendra K. Chaturvedi ORCID: orcid.org/0000-0001-7037-898X1 na1,
Sidharth Mahapatra1,2 na1,
Varun Kesherwani3,
Matthew J. Kling4,
Mamta Shukla4,
Sutapa Ray1,
Ranjana Kanchan2,
Naveenkumar Perumal2,
Timothy R. McGuire5,
J. Graham Sharp4,
Shantaram S. Joshi4 &
Don W. Coulter1
BMC Cancer volume 19, Article number: 1056 (2019) Cite this article
MYC amplification or overexpression is common in Group 3 medulloblastoma and is associated with the worst prognosis. Recently, protein arginine methyl transferase (PRMT) 5 expression has been closely associated with aberrant MYC function in various cancers, including brain tumors such as glioblastoma. However, the role of PRMT5 and its association with MYC in medulloblastoma have not been explored. Here, we report the role of PRMT5 as a novel regulator of MYC and implicate PRMT5 as a potential therapeutic target in MYC-driven medulloblastoma.
Expression and association between PRMT5 and MYC in primary medulloblastoma tumors were investigated using publicly available databases. Expression levels of PRMT5 protein were also examined using medulloblastoma cell lines and primary tumors by western blotting and immunohistochemistry, respectively. Using MYC-driven medulloblastoma cells, we examined the physical interaction between PRMT5 and MYC by co-immunoprecipitation and co-localization experiments. To determine the functional role of PRMT5 in MYC-driven medulloblastoma, PRMT5 was knocked-down in MYC-amplified cells using siRNA and the consequences of knockdown on cell growth and MYC expression/stability were investigated. In vitro therapeutic potential of PRMT5 in medulloblastoma was also evaluated using a small molecule inhibitor, EPZ015666.
We observed overexpression of PRMT5 in MYC-driven primary medulloblastoma tumors and cell lines compared to non-MYC medulloblastoma tumors and adjacent normal tissues. We also found that high expression of PRMT5 is inversely correlated with patient survival. Knockdown of PRMT5 using siRNA in MYC-driven medulloblastoma cells significantly decreased cell growth and MYC expression. Mechanistically, we found that PRMT5 physically associated with MYC by direct protein-protein interaction. In addition, a cycloheximide chase experiment showed that PRMT5 post-translationally regulated MYC stability. In the context of therapeutics, we observed dose-dependent efficacy of PRMT5 inhibitor EPZ015666 in suppressing cell growth and inducing apoptosis in MYC-driven medulloblastoma cells. Further, the expression levels of PRMT5 and MYC protein were downregulated upon EPZ015666 treatment. We also observed a superior efficacy of this inhibitor against MYC-amplified medulloblastoma cells compared to non-MYC-amplified medulloblastoma cells, indicating specificity.
Our results reveal the regulation of MYC oncoprotein by PRMT5 and suggest that targeting PRMT5 could be a potential therapeutic strategy for MYC-driven medulloblastoma.
Medulloblastoma is the most common malignant pediatric brain tumor, accounting for nearly 20% of all childhood brain cancers [1]. Current therapies of medulloblastoma have improved patient survival to about 70% and include surgical resection, radiation therapy, and chemotherapy [2]. Medulloblastoma has biological/genetic heterogeneity with 4 major molecularly distinct subgroups including wingless (WNT), Sonic Hedgehog (SHH), Group 3 and Group 4 [3,4,5]. Group 3 medulloblastoma often exhibits MYC amplification or overexpression and has the worst prognosis of the 4 medulloblastoma subgroups with < 50% survival. MYC-driven medulloblastomas have high metastatic potential and are often resistant to even multimodal treatments [6,7,8]. Thus, understanding the mechanisms of MYC-driven tumor progression/recurrence and integration of molecular-targeted therapies are critical to identifying novel and effective therapeutics for these high-risk patients.
Epigenetic deregulation has emerged as a key driver in medulloblastoma tumorigenesis, particularly alterations in histone modifying enzymes such as histone methyl transferases [9, 10]. Furthermore, Group 3 and Group 4 medulloblastomas present with high levels of histone H3-lysine 27 tri-methylation (H3K27me3) due to altered activity of the H3K27 methyltransferase and H3K27 demethylases [11, 12]. Post-translational methylation of histone may occur at lysine (K) or arginine (R) residues. Past studies have focused more on histone lysine methylation than histone arginine methylation. However, growing evidence supports the importance of arginine methylation by protein arginine methyltransferases (PRMTs) in cancer progression. Particularly, the overexpression of PRMT5 has been correlated with poor prognosis in a variety of cancers [13].
PRMT5 represents a member of PRMT family proteins that methylate histone and non-histone proteins to regulate gene expression and cellular development [14]. PRMT5 symmetrically dimethylates the arginine residues of histone proteins H4 (S2Me-H4R3), H3 (S2Me-H3R8) and H2A, and thereby regulates chromatin structure to support transcriptional repression [15]. PRMT5 over-expression in cancers is thought to epigenetically silence tumor suppressor and cell cycle genes [16]. In addition, PRMT5 is known to post-translationally methylate certain oncogenic transcription factors (non-histone proteins) such as p53, NF-κB (p65) and MYCN [17,18,19,20].
Recently, PRMT5 was found to associate with aberrant MYC function in various cancers including brain tumors such as glioblastoma and neuroblastoma [20,21,22,23]. However, the role of PRMT5 and its association with MYC in medulloblastoma have not been explored. Based on these observations, we hypothesized that PRMT5 is a novel regulator of MYC expression whose inhibition may serve as a novel therapeutic strategy in MYC-driven medulloblastoma.
Cell lines and culture
The human medulloblastoma cell lines Daoy (HTB-186), D-283 (HTB-185) and D-341 (HTB-187) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HD-MB03 (ACC-740) human medulloblastoma cell line was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). ONS-76 (IFO50355) human medulloblastoma cell line was obtained from Sekisui-XenoTech (Kansas, USA). These Cell lines were authenticated by their respective companies using short tandem repeat profiling. All cell lines were tested for mycoplasma contamination using MycoSensor PCR Assay Kit (Santa Clara, CA, USA). In this study, Daoy and ONS-76 were used as SHH medulloblastoma subgroup cell lines without MYC-amplification, whereas, D-341, HD-MB03 and D-283 were used as Group 3 medulloblastoma cell lines with MYC-amplified status. All these cell lines were cultured and maintained using Eagle's minimal essential medium (EMEM) or RPMI-1640 media supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA) in a humidified incubator at 5% CO2 and 95% air atmosphere at 37 °C. The experiments were performed using no more than 10 passages for each cell line. The cell lysate of human normal brain cerebellum was purchased from BioChain Institute Inc. (Newark, CA, USA).
Patient data acquisition for PRMT5 expression and survival
The R2 Genomics Analysis and Visualization Platform (www.r2.amc.nl) was used to investigate PRMT5 mRNA expression and its correlation with patient survival across medulloblastoma subgroups using publicly available datasets. The expression of PRMT5 mRNA in medulloblastoma was analyzed using a total 491 medulloblastoma tumors (5 independent cohorts) and 9 normal cerebellum samples. The survival analyses with respect to PRMT5 expression in medulloblastoma patients were performed using a separate cohort of 612 medulloblastoma samples from Cavalli (763 samples) dataset.
siRNAs and inhibitor
Both control (Scrambled, sc-37,007) and PRMT5 siRNAs ((sc-41,073) were purchased from Santacruz Biotechnology (Dallas, TX, USA). Each siRNA was dissolved in RNase-free water at 10 μM stock concentration and stored at -20 °C. The PRMT5 inhibitor EPZ015666 was purchased from Selleckchem Company (Houston, TX, USA). This inhibitor was dissolved in DMSO at 10 mM stock concentration and stored at -20 °C.
siRNA knock-down and transfection
Control (scrambled) and PRMT5 siRNA (a pool of 3 target-specific 19–25 nt siRNAs with 50 nM) were transiently transfected into medulloblastoma cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Following 72 h of transfections, cells were subjected to downstream analyses using western blotting and MTT assay.
Cell growth assay
To examine the effects of PRMT5 inhibition on medulloblastoma cell growth, twenty thousand cells of each medulloblastoma cell line were plated in 96-well plates 24 h before the experiment. Then, these cells were transfected with PRMT5 siRNAs or treated with PRMT5 inhibitor for 72 h according to the experimental plan and the growth of these cells was determined using an MTT assay as described previously [24].
Apoptosis and cell cycle analyses
The effect of PRMT5 inhibitor to induce apoptosis in medulloblastoma cells at 72 h, was determined using an Annexin-V:FITC flow cytometry assay kit (BD Biosciences, San Jose, CA, USA) following the manufacturer's instructions. For cell cycle analysis, the control and PRMT5 inhibitor-treated medulloblastoma cells for 24 and 48 h, were fixed with 75% ethanol and stained with propidium iodide using a propidium iodide flow cytometry kit (Abcam, Cambridge, UK).
Cycloheximide chase and co-immunoprecipitation experiments
To determine protein stability, medulloblastoma cells were treated with 50 μg/ml cycloheximide (Sigma Aldrich, St. Louis, MO, USA) following siRNA transfection for 72 h. Following transfection, cell lysates from the indicated time points of cycloheximide treatments were subjected to western blotting.
For co-immunoprecipitation, 500 μg protein lysate was precleared with 50 μl of protein A-Sepharose beads (Cell Signaling Technology, Danvers, MA, USA) for 1 h at 4 °C. Immunoprecipitation was performed in the presence of 8 μg of the indicated primary antibodies at 4 °C overnight. Immune complexes were captured by adding 50 μl of protein A-Sepharose beads and rotated at 4 °C for 2 h. After the supernatant was discarded, protein A-Sepharose beads were washed with PBS and lysed in 1x Laemmli buffer and then subjected to western blotting.
The expression levels of indicated proteins in medulloblastoma cells were determined using western blot analyses as described previously [24]. The primary human antibodies for cMYC (sc-40), PRMT5 (sc-376,937), histone H3 (sc-8654) and β-Actin (sc-130,301) were purchased from Santacruz Biotechnology (Dallas, TX, USA). H4R3me2s (61188) and H3R8me2s (ab130740) antibodies were from Active Motif (Carlsbad, CA, USA) and Abcam (Cambridge, UK), respectively. Immunoreactivity was detected using appropriate peroxidase-conjugated secondary antibodies (Jackson Lab, ME) and visualized using an ECL detection system (Pierce, IL).
Methanol-fixed HD-MB03 cells on glass cover slips, and an antigen-retrieved medulloblastoma tumor section were washed with PBS and blocked in 1% BSA in PBS for 30 min. The tumor cells were then co-incubated with PRMT5 (rabbit, 1:100) and MYC (mouse, 1:100) antibodies overnight at 4 °C. Following three washes with PBS, the cells were further co-incubated with fluorochrome-conjugated anti-rabbit (Alexa-488) and anti-mouse (Alexa-647) secondary antibodies (Invitrogen, Carlsbad, CA) for 1 h at room temperature. The cells were then washed three times with PBS and the cover slips were mounted on glass slides and visualized under confocal microscope. DAPI was co-incubated with the secondary antibodies to facilitate the visualization of the nuclei. Confocal images were taken using a Zeiss LSM 5 Pascal confocal microscope (Carl Zeiss, Oberkochen, Germany) using a 40x objective in the UNMC Confocal Microscopy facility.
Immunohistochemical analyses in patient samples
Frozen samples of normal cerebella and medulloblastoma tumor specimens were collected from the Children's Hospital and Medical Center, Omaha and the University of Nebraska Medical Center after Institutional Review Board (IRB) approval. Normal cerebellum specimens were obtained from patients at autopsy. All normal and tumor samples were from the pediatric age group.
Normal cerebellum and medulloblastoma tumor sections were deparaffinized with xylene and rehydrated with water. Antigen retrieval was performed using citrate buffer at 95 °C for 20 min. Sections were treated with 3% hydrogen-peroxide for 30 min to block peroxidase activity. Sections were blocked using 5% goat serum with 0.3% Triton-X-100 in PBS and incubated with PRMT5 (1:100) and MYC (1:100) rabbit-antibodies (Abcam, Cambridge, UK) overnight at 4 °C. Next day, primary antibodies were washed with PBS three times and incubated with appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Following three washes with PBS, detection was performed using a DAB Peroxidase Substrate Kit (Vector Labs, Burlingame, CA, USA) followed by counterstaining with hematoxylin. Sections were mounted in Paramount solution and visualized under an EVOS FL Auto Imaging System (Life Technologies, Carlsbad, CA, USA). Staining intensity was scored from 0 to 3, where signal detected at 10X was 3+, at 20X was 2+, at 40X was 1+, and no detection was 0. The percentage positive cells was scored from 1 to 4 scale, where < 25% scored 1, 25–50% scored 2, 50–75% scored 3; and > 75 scored 4. Composite score (0–12) was derived from the staining intensity and % positive cells.
All experiments were repeated at least two times and the mean and standard error values calculated. Differences (p-value) were calculated using independent Student t-tests or analysis of variance (ANOVA) and p-values < 0.05 were considered significant. The IC50 values of inhibitor EPZ015666 for each medulloblastoma cell line were determined using GraphPad Prism V6 software (provided in Table 1).
Table 1 IC50 of EPZ015666 in medulloblastoma cell lines (MTT assay 72 h)
PRMT5 expression correlates with MYC in primary medulloblastoma and cell lines
The aberrant expression of PRMT5 has been associated with a variety of cancers including glioblastoma and neuroblastoma. In addition, PRMT5 expression has correlated with MYC or MYCN protein in these cancers [20,21,22,23]. However, its expression and function in medulloblastoma have not been reported. These studies prompted us to examine the correlation between MYC levels and PRMT5 expression in medulloblastoma. We first examined the clinical relevancy of PRMT5 in medulloblastoma by analyzing its mRNA expression in 491 medulloblastoma (from independent 5 cohorts) and 9 normal cerebellum samples using the R2 platform (www.r2.amc.nl). Our analyses using these data showed a significant overexpression of PRMT5 in medulloblastoma compared to normal cerebellum tissues (Fig. 1a). We further analyzed the PRMT5 expression across medulloblastoma subgroups using a cohort that has maximum number (223) of samples with all 4 molecular subgroups. We observed significantly higher expression of PRMT5 in Group 3 (MYC-driven) medulloblastoma compared to other 3 subgroups (Fig. 1b). We next compared PRMT5 expression against patient survival. To this end, we performed survival analyses with respect to PRMT5 expression, using 612 medulloblastoma samples from the Cavalli (763 samples) dataset. Our survival analyses showed that high levels of PRMT5 expression correlated with poor survival of medulloblastoma patients, a pattern recapitulated in Group 3 medulloblastoma patients (Fig. 1c and d). These data suggest that PRMT5 expression is not simply deregulated in medulloblastoma, but is also a poor prognostic marker, particularly in Group 3 (MYC-driven) tumors.
PRMT5 expression and correlation in primary medulloblastomas. a Boxplots showing PRMT5 expression in five non-overlapping cohorts (total n = 491) of medulloblastoma tumors compared to normal cerebellum (n = 9) controls. *Anova p < 0.05 vs. medulloblastoma. b PRMT5 expression in four (Group3, Group 4, SHH and WNT) medulloblastoma subgroups using Pfister (n = 223) cohort dataset. *Anova p < 0.05 vs. Group 3. Kaplan-Meier plots showing overall survival of patients (Cavalli 763 cohort) with medulloblastoma all subgroups (c) and Group 3 medulloblastoma (d) with respect to PRMT5 expression
We next analyzed the correlation between PRMT5 and MYC mRNA expression across medulloblastoma subgroups using Pfister (n = 223) cohort at the R2 genomic analysis platform. Results from this analysis showed that high expression of PRMT5 was strongly correlated (R-value = 0.531; p-value = 2.51e-05) with high MYC in Group 3 medulloblastoma. Although results showed some degree of correlation (R-value = 0.069–0.284; p-value = 0.111–0.606) of these genes in the other three medulloblastoma subgroups, none of these medulloblastoma subgroups showed a significant correlation between PRMT5 and MYC expression (Additional file 1: Figure S1). We further examined the correlation between PRMT5 and MYC expression at the protein levels by western blotting in non-MYC (Daoy, ONS-76) and three MYC-driven (D-283, D-341, HD-MB-03) medulloblastoma cell lines compared to normal cerebellum. We observed that PRMT5 protein levels were significantly (p < 0.01) higher in MYC-driven medulloblastoma cell lines compared to non–MYC medulloblastoma cell lines and normal human cerebellum cells (Fig. 2a and b). Stronger PRMT5 band intensity seemed associated with higher MYC expression in medulloblastoma cell lines. To further authenticate this correlation at the protein level, we examined the expression of PRMT5 and MYC by immunohistochemistry in Group 3 medulloblastoma tumor samples (n = 6) compared to normal pediatric cerebellum (n = 4). We found that the protein levels of PRMT5 and MYC were significantly (p = 0.004) higher with more than 75% staining in Group 3 medulloblastoma samples than normal pediatric cerebellum tissues (Fig. 2c). We observed very poor immunostaining of these proteins in normal cerebellum with less than '1' intensity score. We not only observed intensely high expression of PRMT5 and MYC protein in Group 3 medulloblastoma but also, a positive correlation with their predominantly nuclear co-expression. These results consistently suggest a positive correlation and co-operative role of the PRMT5-MYC oncogenic axis in poor prognosis medulloblastoma.
Expression and correlation of PRMT5 with MYC protein in medulloblastoma cell lines and primary tumors. a Western blotting of PRMT5 in medulloblastoma cell lines with and without MYC amplification compared to normal cerebellum. Actin was used as a loading control. b Comparison of PRMT5 expression levels normalized to Actin in non-MYC vs MYC amplified medulloblastoma cell lines. Significance, p < 0.01. c Representative immunohistochemical images showing PRMT5 and MYC expression and their localization pattern in normal pediatric cerebellum and Group 3 medulloblastoma tumor tissues at 20x magnification. Scale bar, 200 μm. The box plots on right side showing quantification of composite score-based intensity of MYC and PRMT5 staining in Group 3 medulloblastoma tumor specimen (n = 6) compared to normal pediatric cerebellum (n = 4)
PRMT5 knockdown leads to decreased MYC expression and cell survival in MYC-driven medulloblastoma cells
The strong correlation between PRMT5 and MYC expression prompted us to explore possible influence of PRMT5 on MYC function. To investigate the role of PRMT5 on MYC function, we determined the effect of short-interfering RNA (siRNA)-mediated knockdown of PRMT5 on MYC expression and cell survival in MYC-amplified medulloblastoma cell lines. We used two cell lines D-341 and HD-MB03 in this study as both lines have previously been reported as well-established Group 3 medulloblastoma cell lines with high MYC expression. Consistently, our western blot results further confirmed strongly higher expression of MYC protein in these two cell lines compared to other medulloblastoma lines (Fig. 2a). Using these two lines, we first verified the knockdown of PRMT5 protein expression after siRNA transfections and then assessed the consequences of knockdown. We found that knockdown of PRMT5 efficiently suppressed the expression of MYC protein in both cell lines by approximately 35–45%, compared to control scrambled siRNA (Fig. 3), suggesting an on-target effect of PRMT5. Concurrently, PRMT5 knockdown significantly reduced cell growth (Fig. 3) in both cell lines by approximately 40–45%, compared to control scrambled siRNA. We also observed that D-341 cell line showed relatively lesser knockdown of PRMT5 and MYC protein by 5 and 13%, respectively, compared to HD-MB03 cell line. However, the impact of knockdown on inhibition of D-341 cell growth was relatively (~ 5%) higher compared to HD-MB03 cell growth, indicating differential knockdown activity between two MYC-amplified cell lines. One possible explanation for this differential response in cell lines could be that they have different growth pattern in culture in vitro, as we observed that D-341 cells grow more slower with mixed spheroids and monolayer cells compared to mostly monolayer HD-MB03 cells.
PRMT5 knockdown in MYC-driven medulloblastoma cells. MYC-amplified medulloblastoma cell lines D-341 (a) and HD-MB03 (b) were transiently transfected with PRMT5-siRNA and control scrambled siRNA (SCR) for 72 h. Following transfections, cells were subjected to cell growth analyses using MTT assay and western blotting to determine the expression levels of PRMT5 and MYC proteins. The values given below each western blot are showing the densitometric quantification of each protein expression relative to the control SCR after Actin normalization. *, p < 0.05
Physical and functional interaction of PRMT5 and MYC in MYC-driven medulloblastoma cells
Previous studies [20, 23] in neuroblastoma and glioblastoma have demonstrated that PRMT5 can physically interact with MYC and regulate its stability at the post-translational level. To confirm whether endogenous PRMT5 physically interacts with MYC protein in medulloblastoma, we performed a co-immunoprecipitation experiment in MYC-driven HD-MB-03 medulloblastoma cells using MYC and PRMT5 antibodies. Our results presented in Fig. 4a, showed the presence of PRMT5 in MYC-immunoprecipitated complexes from cell extracts. We further confirmed this interaction by detecting MYC in the reverse PRMT5-immunoprecipitated complexes (Fig. 4b). To further support of association between PRMT5 and MYC, we examined co-localization of these two proteins in HD-MB03 cells and a tumor specimen of a Group 3 medulloblastoma patient, using immunofluorescence-coupled with confocal microscopy. As shown in Fig. 4c, the merged immunofluorescent-staining of MYC and PRMT5 demonstrated a significant co-localization pattern in both HD-MB03 and primary tumor cells. The results further showed that both MYC and PRMT5 were predominantly co-localized in the nucleus, and this localization pattern was consistent with their immunohistochemical co-expression in Group 3 medulloblastoma primary tumors shown in Fig. 2c. Together, the results of co-immunoprecipitation and co-localization of MYC and PRMT5 suggest that endogenous PRMT5 forms a complex with MYC in medulloblastoma cells harboring MYC amplification. The observation of physical interaction between PRMT5 and MYC indicates a potential functional role of this novel protein complex in medulloblastoma.
Physical and functional interaction between PRMT5 and MYC. a HD-MB03 cell lysate was subjected to co-immunoprecipitation (IP) analysis using MYC antibody and control IgG, followed by immunoblotting with PRMT5 antibody. b HD-MB03 cell lysate was subjected to reciprocal co-immunoprecipitation using PRMT5 antibody and control IgG, followed by immunoblotting with MYC antibody. c Confocal images for the co-localization of MYC and PRMT5 in HD-MB03 cells and Group 3 medulloblastoma tumor tissue at 40x magnification. d Western blot analysis of MYC expression after 50 μg/ml CHX treatment following transient transfection of scrambled siRNA (SCR) and PRMT5-siRNA in HD-MB03 cells. e Densitometric quantification of MYC protein expression shown in "d"
To determine the consequence of this physical interaction on post-translational MYC stability and influence on MYC expression, we performed cycloheximide (CHX) chase experiments in PRMT5 knocked-down HD-MB03 cells and measured the half-life of MYC protein. As shown in Fig. 4d and e, MYC has a half-life of approximately 60 min after CHX treatment in cells transfected with control scrambled siRNA, whereas PRMT5 knockdown dramatically decreased its half-life to approximately 35 min. There was approximately 25 min earlier degradation of MYC protein in PRMT5 knocked-down cells compared to control siRNA treated cells. Together, these results suggest that PRMT5 physically interacts with and stabilizes MYC in medulloblastoma cells at the post-translation level.
Anti-medulloblastoma efficacy of a small molecule inhibitor of PRMT5
Given the potential for anti-neoplastic effects with PRMT5 knockdown, via reduction in cell viability and MYC expression, we investigated the therapeutic potential of PRMT5 inhibition using a recently developed potent PRMT5 inhibitor (EPZ015666) [25]. We first determined the growth inhibitory efficacy of EPZ015666 against three MYC-amplified (D-283, D-341, HD-MB03) and two non-MYC amplified (Daoy, ONS-76) medulloblastoma cell lines. Cells were treated with inhibitor (0.1–10 μM) in a dose-dependent manner for 72 h and growth of cells was assessed using an MTT assay. Our MTT results clearly demonstrated that EPZ015666 significantly induced the dose-dependent growth inhibition of all MYC-driven medulloblastoma cell lines at low micromolar potency with IC50 of ~ 1.5–2.5 μM (Fig. 5a, Table 1). However, there was minimal effect of EPZ015666 on growth inhibition of non-MYC amplified medulloblastoma cells even at higher doses, suggesting anti-neoplastic specificity of EPZ015666 to MYC-dependent tumors.
Therapeutic efficacy of PRMT5 inhibitor EPZ015666 in medulloblastoma cell lines. a MTT assay showing the dose-dependent effects of EPZ015666 (0.1–10 μM) on non-MYC (Daoy, ONS-76) and MYC-amplified (D-283, HD-MB03, D-341) medulloblastoma cell growth. The values represent the means ± SD from four wells of 96-well plates. The percentage of cell viability is relative to control vehicle-treated cells. *, p < 0.05; **, p < 0.01, ***, p < 0.001 (b) Annexin-V assay showing effect of EPZ015666 on apoptosis in HD-MB03 cells. *, p < 0.05; **, p < 0.01, ***, p < 0.001 (relative to '0' (control vehicle). c Cell cycle profile in EPZ015666-treated HD-MB03 cells. d Western blot analysis for the expression of the indicated key proteins in EPZ015666-treated HD-MB03 cells. The values given below each western blot are showing the densitometric quantification of each protein expression relative to the control after β-Actin normalization
We next determined the ability of EPZ015666 to induce apoptosis in a representative MYC-amplified medulloblastoma cell line HD-MB03. The results of the apoptosis analyses (Fig. 5b) using Annexin-V assay demonstrated a dose-dependent induction of apoptosis by EPZ015666 and showed consistency with MTT growth results. Since PRMT5 is known to act during the G1 cell cycle phase, we sought to investigate whether inhibiting PRMT5 by EPZ015666 reduced medulloblastoma cell growth by disrupting the cell cycle. The cell cycle results (Fig. 5c) using PI staining of DNA, demonstrated that treatment of EPZ015666 at 24 h and 48 h arrested medulloblastoma cells in the G1 cell cycle phase in a dose-dependent manner. Since we observed a doubling time of the HD-MB03 cell line of between 28 to 36 h, we examined the impact of EPZ015666 on HD-MB03 cell cycle at 24 and 48 h, close to the doubling time points. Lastly, using western blot analyses, we confirmed that treatment of HD-MB03 cells with optimum doses of EPZ015666 efficiently downregulated the expression levels of PRMT5 and its key target symmetric dimethylated histone H3 (H3R8me2s) and H4 (H4R3me2s), including suppressed MYC expression (Fig. 5d). Taken together, these results suggest that inhibiting the specific interaction of PRMT5 with MYC arrests medulloblastoma cell growth and favors apoptosis in MYC-dependent tumors.
Despite significant improvements in outcomes and overall survival of medulloblastoma patients with current therapies, patients with high-risk disease, particularly MYC-driven medulloblastomas still face a paucity of effective therapies [2]. The minimal improvement in survival of these high-risk medulloblastoma patients identifies a need for novel targeted therapeutic approaches against MYC-driven (high-risk) medulloblastoma. In addition to genetic abnormalities, deregulated epigenetic modifiers are frequently observed in these aggressive medulloblastoma tumors [9, 10]. The importance of epigenetic control in aggressive medulloblastoma underscores the need to identify and understand epigenetic regulatory mechanisms and their targets.
The evolutionarily-conserved PRMT family of enzymes is involved in a wide range of developmental and cellular processes. PRMT5 is the major type II arginine methyltransferase that silences gene transcription by symmetric dimethylation of arginine residues on histone proteins [15, 16]. PRMT5 is involved in the epigenetic regulation of chromatin complexes by interacting with a number of proteins including transcription factors [26]. Growing evidence suggests that PRMT5 expression and activity are dysregulated in various solid and hematological malignancies [16]. Recent studies found PRMT5 as a key epigenetic regulator in glioblastoma tumorigenesis. Interestingly, increased expression of PRMT5 positively correlates with high-grade glioma malignancy and is inversely associated with patient survival. In addition, high levels of MYC and PRMT5 correlate with glioma malignancy [21,22,23]. Further, PRMT5 is associated with MYCN (another member of the MYC family of transcription factors) in neuroblastoma cells and promotes its stability [20]. However, the role of PRMT5 and its association with MYC in medulloblastoma are unexplored. Here, we showed that PRMT5 is a novel regulator of MYC protein in medulloblastoma.
To address the role of PRMT5 in medulloblastoma, we first accessed expression of PRMT5 across medulloblastoma subgroups including Group 3 medulloblastoma patients and high-MYC expressing medulloblastoma cell lines. Our expression findings confirm that high levels of PRMT5 not only mirror MYC expression in the most aggressive medulloblastomas but also inversely correlate with poor outcomes in patients. This finding purports the clinical utility of PRMT5 as a prognostic marker for patients with more aggressive disease. Although numerous epigenetic abnormalities have been reported in medulloblastoma tumors, including expression of histone and DNA methyltransferases [27], the prognostic applicability of these markers remains unclear. Identification of prognostic markers like PRMT5 will contribute to developing novel therapeutic strategies for this disease.
Our data on PRMT5 knockdown in MYC-amplified medulloblastoma cells showed that PRMT5 can regulate the stability of MYC protein by physically interacting with it, suggesting MYC regulation by PRMT5 at the post-translation level. Further, we showed that knockdown of PRMT5 suppressed medulloblastoma cell growth by inhibiting MYC expression, suggesting a functional role of PRMT5-MYC interaction in medulloblastoma tumorigenesis. These are in agreement with a previous study by Park et al. [20], where they showed similar interactions in neuroblastoma. Since our results showed that PRMT5 and MYC co-expressed and co-localized predominantly in the nucleus, it is possible that PRMT5 may also regulate MYC expression at the transcriptional level. Further studies with the analyses of MYC association to the chromatin and promoter activity are required to explore the possibility of transcriptional regulation of MYC by PRMT5. In addition, it is highly likely that there are other mechanism(s) that could be involved in the PRMT5-mediated regulation of MYC. Such analyses would certainly be a topic for future studies.
PRMT5 is a key and emerging stemness factor for normal and cancer stem cells. Its role in stemness has been demonstrated in embryonic and neural stem cells [28,29,30]. Given that neural stem or cancer stem cells have profound impact on driving medulloblastoma tumorigenesis and recurrence, there might be role of PRMT5 in regulating self-renewal capacity of medulloblastoma tumor initiating cells. Recently, PRMT5 has also been shown to methylate a key stemness factor KLF4 in breast cancer [31]. Methylation of KLF4 by PRMT5 leads to stabilization of KLF4 protein, resulting in promotion of tumorigenesis. In a subsequent study, the authors developed a novel and potent PRMT5 inhibitor, WX2–43, that disrupts PRMT5-KLF4 interaction and suppresses breast cancer progression [32]. Further investigation of targeting unexplored PRMT5-KLF4 interactions in medulloblastoma might be another new strategy to develop therapy for MYC-driven medulloblastoma.
Given the role of PRMT5 in MYC-driven medulloblastoma cells, we further tested the therapeutic potential of targeting PRMT5 using a selective small molecule inhibitor, EPZ015666, against medulloblastoma cell lines. Our results demonstrated that EPZ015666 significantly inhibits proliferation and survival of MYC-driven medulloblastoma cells associated with G1-S cell cycle arrest. Our results also indicated that MYC-amplified cells show greater sensitivity to EPZ015666 compared to non-MYC amplified medulloblastoma cells, further supporting the role of PRMT5 acting in MYC-dependent manner. Molecularly, EPZ015666 significantly downregulated the expression of PRMT5 and MYC protein in MYC-driven cells. These data support our hypothesis of the potential for PRMT5 to serve as a therapeutic target in MYC-driven medulloblastoma and this warrants further, systematic evaluation in appropriate preclinical mouse models.
In summary, we have demonstrated for the first time that PRMT5 is a critical regulator of MYC expression in MYC-amplified medulloblastomas. PRMT5 and MYC expression are positively correlated in medulloblastoma cells. Mechanistic studies revealed that PRMT5 could elevate MYC expression and stability, enhancing medulloblastoma tumorigenicity. Our results using a PRMT5 inhibitor EPZ015666 highlight the PRMT5-MYC oncogenic axis a viable therapeutic approach for MYC-driven medulloblastoma. With evaluation of this approach in preclinical mouse models, we may take the first steps towards translating this discovery to the clinic.
All data generated or analyzed during this study are included in this article.
DNA:
FBS:
FITC:
Fluorescein isothiocyanate
IC50 :
Inhibitory concentration of inhibitor with 50% inhibition;
ml:
Milliliter
mRNA:
Messenger ribonucleic acid
MTT:
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MYC:
v-myc avian myelocytomatosis viral oncogene homolog
nm:
nt:
SDS:
siRNA:
Small interfering ribonucleic acid
μg:
Microgram
μl:
μm:
Micromolar
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Cavalli FMG, Remke M, Rampasek L, Peacock J, Shih DJH, Luu B, Garzia L, Torchia J, Nor C, Morrissy AS, et al. Intertumoral heterogeneity within Medulloblastoma subgroups. Cancer Cell. 2017;31(6):737–54.
Northcott PA, Buchhalter I, Morrissy AS, Hovestadt V, Weischenfeldt J, Ehrenberger T, Gröbner S, Segura-Wang M, Zichner T, Rudneva VA, et al. The whole-genome landscape of medulloblastoma subtypes. Nature. 2017;547(7663):311–7.
Taylor MD, Northcott PA, Korshunov A, Remke M, Cho YJ, Clifford SC, Eberhart CG, Parsons DW, Rutkowski S, Gajjar A, et al. Molecular subgroups of medulloblastoma: the current consensus. Acta Neuropathol. 2012;123(4):465–72.
Cho YJ, Tsherniak A, Tamayo P, Santagata S, Ligon A, Greulich H, Berhoukim R, Amani V, Goumnerova L, Eberhart CG, et al. Integrative genomic analysis of medulloblastoma identifies a molecular subgroup that drives poor clinical outcome. J Clin Oncol. 2011;29(11):1424–30.
Pei Y, Moore CE, Wang J, Tewari AK, Eroshkin A, Cho YJ, Witt H, Korshunov A, Read TA, Sun JL, et al. An animal model of MYC-driven medulloblastoma. Cancer Cell. 2012;21(2):155–67.
Hill RM, Kuijper S, Lindsey JC, Petrie K, Schwalbe EC, Barker K, Boult JK, Williamson D, Ahmad Z, Hallsworth A, et al. Combined MYC and P53 defects emerge at medulloblastoma relapse and define rapidly progressive, therapeutically targetable disease. Cancer Cell. 2015;27(1):72–84.
Yi J, Wu J. Epigenetic regulation in medulloblastoma. Mol Cell Neurosci 2018;87:65–76. doi: https://doi.org/10.1016/j.mcn.2017.09.003. Epub 2017 Dec 18.
Roussel MF, Stripay JL. Epigenetic drivers in pediatric Medulloblastoma. Cerebellum. 2018;17(1):28–36.
Dubuc AM, Remke M, Korshunov A, Northcott PA, Zhan SH, Mendez-Lago M, Kool M, Jones DT, Unterberger A, Morrissy AS, et al. Aberrant patterns of H3K4 and H3K27 histone lysine methylation occur across subgroups in medulloblastoma. Acta Neuropathol. 2013;125(3):373–84.
Alimova I, Venkataraman S, Harris P, Marquez VE, Northcott PA, Dubuc A, Taylor MD, Foreman NK, Vibhakar R. Targeting the enhancer of zeste homologue 2 in medulloblastoma. Int J Cancer. 2012;131(8):1800–9.
Yang Y, Bedford MT. Protein arginine methyltransferases and cancer. Nat Rev Cancer. 2013;13(1):37–50.
Wei H, Mundade R, Lange KC, Lu T. Protein arginine methylation of non-histone proteins and its role in diseases. Cell Cycle. 2014;13(1):32–41.
Stopa N, Krebs JE, Shechter D. The PRMT5 arginine methyltransferase: many roles in development, cancer and beyond. Cell Mol Life Sci. 2015;72(11):2041–59.
Shailesh H, Zakaria ZZ, Baiocchi R, Sif S. Protein arginine methyltransferase 5 (PRMT5) dysregulation in cancer. Oncotarget. 2018;9(94):36705–18.
Cho EC, Zheng S, Munro S, Liu G, Carr SM, Moehlenbrink J, Lu YC, Stimson L, Khan O, Konietzny R, et al. Arginine methylation controls growth regulation by E2F-1. EMBO J. 2012;31(7):1785–97. https://doi.org/10.1038/emboj.2012.17.
Jansson M, Durant ST, Cho EC, Sheahan S, Edelmann M, Kessler B, La Thangue NB. Arginine methylation regulates the p53 response. Nat Cell Biol. 2008;10(12):1431–9.
Wei H, Wang B, Miyagi M, She Y, Gopalan B, Huang DB, Ghosh G, Stark GR, Lu T. PRMT5 dimethylates R30 of the p65 subunit to activate NF-κB. Proc Natl Acad Sci U S A. 2013;110(33):13516–21.
Park JH, Szemes M, Vieira GC, Melegh Z, Malik S, Heesom KJ, Von Wallwitz-Freitas L, Greenhough A, Brown KW, Zheng YG, et al. Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells. Mol Oncol. 2015;9(3):617–27.
Yan F, Alinari L, Lustberg ME, Martin LK, Cordero-Nieves HM, Banasavadi-Siddegowda Y, Virk S, Barnholtz-Sloan J, Bell EH, Wojton J, et al. Genetic validation of the protein arginine methyltransferase PRMT5 as a candidate therapeutic target in glioblastoma. Cancer Res. 2014;74(6):1752–65.
Han X, Li R, Zhang W, Yang X, Wheeler CG, Friedman GK, Province P, Ding Q, You Z, Fathallah-Shaykh HM, et al. Expression of PRMT5 correlates with malignant grade in gliomas and plays a pivotal role in tumor growth in vitro. J Neuro-Oncol. 2014;118(1):61–72.
Mongiardi MP, Savino M, Bartoli L, Beji S, Nanni S, Scagnoli F, Falchetti ML, Favia A, Farsetti A, Levi A, et al. Myc and Omomyc functionally associate with the protein arginine methyltransferase 5 (PRMT5) in glioblastoma cells. Sci Rep. 2015;5:15494.
Chaturvedi NK, Rajule RN, Shukla A, Radhakrishnan P, Todd GL, Natarajan A, Vose JM, Joshi SS. Novel treatment for mantle cell lymphoma including therapy-resistant tumor by NF-κB and mTOR dual-targeting approach. Mol Cancer Ther. 2013;12(10):2006–17.
Chan-Penebre E, Kuplast KG, Majer CR, Boriack-Sjodin PA, Wigle TJ, Johnston LD, Rioux N, Munchhof MJ, et al. A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models. Nat Chem Biol. 2015;11(6):432–7.
Karkhanis V, Hu YJ, Baiocchi RA, Imbalzano AN, Sif S. Versatility of PRMT5-induced methylation in growth control and development. Trends Biochem Sci. 2011;36(12):633–41.
Klonou A, Spiliotakopoulou D, Themistocleous MS, Piperi C, Papavassiliou AG. Chromatin remodeling defects in pediatric brain tumors. Ann Transl Med. 2018;6(12):248.
Tee WW, Pardo M, Theunissen TW, Yu L, Choudhary JS, Hajkova P, Surani MA. Prmt5 is essential for early mouse development and acts in the cytoplasm to maintain ES cell pluripotency. Genes Dev. 2010;24(24):2772–7.
Chittka A, Nitarska J, Grazini U, Richardson WD. Transcription factor positive regulatory domain 4 (PRDM4) recruits protein arginine methyltransferase 5 (PRMT5) to mediate histone arginine methylation and control neural stem cell proliferation and differentiation. J Biol Chem. 2012;287(51):42995–3006.
Banasavadi-Siddegowda YK, Russell L, Frair E, Karkhanis VA, Relation T, Yoo JY, Zhang J, Sif S, Imitola J, Baiocchi R, Kaur B. PRMT5-PTEN molecular pathway regulates senescence and self-renewal of primary glioblastoma neurosphere cells. Oncogene. 2017;36(2):263–74.
Hu D, Gur M, Zhou Z, Gamper A, Hung MC, Fujita N, Lan L, Bahar I, Wan Y. Interplay between arginine methylation and ubiquitylation regulates KLF4-mediated genome stability and carcinogenesis. Nat Commun. 2015;6:8419.
Zhou Z, Feng Z, Hu D, Yang P, Gur M, Bahar I, Cristofanilli M, Gradishar WJ, Xie XQ, Wan Y. A novel small-molecule antagonizes PRMT5-mediated KLF4 methylation for targeted therapy. EBioMedicine. 2019;44:98–111.
The authors thank the Flow Cytometry and Tissue Science Core Facilities at UNMC for their assistance with these studies. The authors thank the State of Nebraska for their financial support of the UNMC/Children's Hospital Pediatric Cancer Research Program. The authors also thank the Pediatric Cancer Action Network (PCAN) and the Fred and Pamela Buffet Cancer Center grant (NCI-P30CAO36727) supported Core Facilities.
This work was fully supported by the State of Nebraska through the Pediatric Cancer Research Grant Funds (LB905) awarded to D. W. Coulter, MD. This funding had no role in the study design, data collection and analysis, interpretation of the data, decision to publish, or writing the manuscript.
Nagendra K. Chaturvedi and Sidharth Mahapatra contributed equally to this work.
Department of Pediatrics, Division of Hematology and Oncology, University of Nebraska Medical Center, Omaha, NE, 68198, USA
Nagendra K. Chaturvedi, Sidharth Mahapatra, Sutapa Ray & Don W. Coulter
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, 68198, USA
Sidharth Mahapatra, Ranjana Kanchan & Naveenkumar Perumal
Child Health Research Institute Cancer, University of Nebraska Medical Center, Omaha, NE, 68198, USA
Varun Kesherwani
Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, 68198, USA
Matthew J. Kling, Mamta Shukla, J. Graham Sharp & Shantaram S. Joshi
Department of Pharmacy Practice, University of Nebraska Medical Center, Omaha, NE, 69198, USA
Timothy R. McGuire
Nagendra K. Chaturvedi
Sidharth Mahapatra
Matthew J. Kling
Mamta Shukla
Sutapa Ray
Ranjana Kanchan
Naveenkumar Perumal
J. Graham Sharp
Shantaram S. Joshi
Don W. Coulter
NKC and DC designed the study. NKC, SM, SR, VK, MJK, MS, RK and NP performed the experiments and analyzed the data. TRM, JGS, DC, and SSJ contributed significantly to the interpretation of the data. NKC, JGS and SSJ wrote the manuscript. All authors read and approved the final manuscript.
Correspondence to Nagendra K. Chaturvedi.
Ethical consent for using patient tumors and normal tissues in this study, were obtained under a University of Nebraska Medical Center Institutional Review Board approved protocol (# 561–16-EP). The informed consent to participate was not obtained due to exempted nature of study.
The authors declare that they have no competing interest.
The expression correlation of PRMT5gene with MYCgene in medulloblastoma. These data were analyzed using a Pifster(n = 223) cohort at R2-Genomics platform.
Chaturvedi, N.K., Mahapatra, S., Kesherwani, V. et al. Role of protein arginine methyltransferase 5 in group 3 (MYC-driven) Medulloblastoma. BMC Cancer 19, 1056 (2019). https://doi.org/10.1186/s12885-019-6291-z
PRMT5
MYC protein
PRMT5 inhibitor
Experimental therapeutics and drug development


The garden should be one of your favorite hangout places. Besides the plants that beautify your environment, you need to fence your garden. Professionals have great ideas on how to enhance the appearance of your home but this comes at a price.
If you have the creativity and skills to keep the fences firm you can make something beautiful. Don't worry even if you don't have the technical experience, there are simple ways to combine the poles and fence to enhance the appearance of your garden.
A garden fence defines the look of your home. Wood is obviously a fundamental in garden design; everyone has access to wood and fence but the design ideas matter more.
If your house has wood finishing, this is the best idea to implement in your garden. It will match with your wooden fence. It is all about accuracy in wood and wire measurements. Wire mesh protects your plant from rodents and chicken; the wood stabilizes the fence. The wood part is also about appearance. All you need is a harmer and few dollars to get the right wooden poles. It is a quick process, you can fix it in few hours over the weekend.
If you have a small part of your home as a garden, you don't need a professional to help you fence it. it is the best type of garden to fence and beautify your home. Most gardens are fenced with long poles and middle length, for this type of garden; you should make a low-level fence.
Irrespective of your preference, the garden fence idea will suffice your need. The garden fence idea is best if you don't have kids or pets free in the compound. However, it is the best fence idea to enhance design of your home.
Your garden fence doesn't have to be boring. You can decorate the fence with multiple plastic and bags that carry flowers. On this, you have to be creative with colors. It is all dependent on your preferences. Find out the specific colors you need for your fence and give it the best shot. Of course, garden is full of green plants; go for other bright colors to make it more beautiful. The colors should match with your wood fence and green leaves.
If your plants are still young and you don't intend to keep long plants, consider this DIY technique. It is the best for shrubs in the backyard or along your fence. Garden is for enhancing the natural theme in your home environment. For this fence design, you have to keep the woods compact.
If you like the low orientation fence, this will be the next best thing. Go for the best wood; should be soft and easy to drill because of the compact design. It is easy to make this fence.
If you love design and a touch of class, this is a great option. This is one fence that makes you proud to show your visitors around the compound. The wood design is compact but with an open top. The compact design at the bottom is for protecting the plans in the garden from pests or kids. It is also part of the design; you don't have to cover the whole garden gate, you should leave space to keep the excitement.
If you are tired of the wood theme, you can add color on your fence. Blue is a relaxed and homely theme that won't compromise on your style or home decoration. Blue is calm; there are no reflections during summer. The fence is classy; you can easily change the nature of your home with this colored fence. It is durable and easy to make. You will have a hard time convincing your friends it is a DIY fence.
Enough of the modern designs, here is a garden fence with traditional theme. Don't worry where to find the crooked poles; they can easily be sourced from different parts of the country as long as you research. They are also cheap. The patterns are not as technical as they seem on the photo. You can easily combine the patterns to make one big fence that match your specifications. The crooked styles make it unique and vintage.
The royal garden fence design is easy to make. It is a trendy fence that can be painted different colors to match your interests. White is the most popular color and you can do it by your own. The varying patterns on the top make it suitable for any home.
This garden enhances the front part of your home. Make sure the pattern is constant and the paint is quality to avoid cracks and fades that compromise eon the style.
Making your fence compact is not good enough. Add a drawing or any form of art work on the fence to make it unique. The tree is easy to draw. Believe it or not, this is a DIY technique. You can make your backyard more appealing than you imagine with this one drawing.
The patterns can vary based on your creativity but maintain the compact and design idea of a garden fence. The poles should be well shaped; pointed top part is part f the design, do not ignore.
Style is important in garden fence choice. Find an open area on your backyard that you can make a path to the garden and place a gate. A wooden gate is the most popular for garden but you should add glass on it to make it more appealing. The transparent and covered garden fence idea allows you to add any accessories including flowers. Screened door also protects either side of the fence from the elements.
If your garden idea involves vegetables and short-term fruits, you need this kind of fence. It looks imported as it and installed but it can be made locally. While professional guidance is crucial, you can do it by yourself. it is a combination technique when you have the whole picture in mind. The bottom part of the fence is covered by wire mesh to keep away pests and rodents from your vegetables. The wood pieces have to be accurately placed before screwing the nails.
You can be simple and still achieve the style you desire in your garden. The purpose here is to make the garden fence unique and simple as well as attractive. The garden fence must have a gate with simple hinges; if you can add rubber on the hinges for simple swinging when you go in and out. Logs make durable and beautiful fences. Logs ever disappoint with durability as long as you get the right quality. Fixing the fence is simple.
Garden fence can be in double form. The inside and outside can have different patterns but all made of wood. Space the wood pieces in symmetric balance. The fence should be straight; irrespective of how long your garden is, you strive to make the fence straight to achieve the design.
This is the best garden fence is you are after style and class. The wooden patterns can be constructed right in your home. I know you are doubting this but it is possible. It won't be easy to convince your colleagues you did this on your own but it is still practical. You can run wire mesh 3 or 4 times depending on length of the fence.
If you love the traditional themes, logs should be your focus in building a garden fence, set the patterns in order to allow space for movement. You can cover the pace between poles with wire mesh. The gate design is also unique; it has the royal theme.
Double wooden patterns make this fence design unique and popular. The brick color also give color to your home. Space the woods evenly for impressive outcomes.
You can use concrete but only to a certain level. Wire mesh dominate his fence design with wooden poles at equal distances like a roof.


text:
Acquisition Unites Rival Surgical Robot Manufacturers
Source: New York Times ( click here to go to the source)
Featured firm in this article: Intuitive Surgical Inc of Sunnyvale, CA
Intuitive Surgical Inc. said yesterday that it had agreed to acquire Computer Motion Inc. for about $68 million in stock, ending two years of patent disputes between the rival makers of surgical robotic arms. Under the agreement, Intuitive Surgical will exchange one of its shares for roughly two of Computer Motion's. The deal values Computer Motion, which is based in Santa Barbara, Calif., at $2.30 a share, 40 percent more than Thursday's closing price of $1.65. Intuitive Surgical shareholders will own 68 percent of the merged company; Computer Motion shareholders the rest. The combined company will have its headquarters in Sunnyvale, Calif.


text:
Entertainment and Culture
Prize-winning author Joyce Carol Thomas dead at 78
By Associated Press Aug 20, 2016, 9:04pm CDT
Share All sharing options for: Prize-winning author Joyce Carol Thomas dead at 78
Joyce Carol Thomas.
STANFORD, Calif. — Joyce Carol Thomas, a prize-winning children's author and champion of multiculturalism, has died. She was 78.
Thomas died Aug. 13 at Stanford University Medical Center in California of cirrhosis of the liver stemming from a bad blood transfusion decades ago, according to her sister, Flora Krasnovsky.
Thomas was best known for her debut book, "Marked by Fire," an autobiographical novel set in her native Oklahoma that won the 1983 National Book Award and the American Book Award, which highlights diversity in literature. Other works included "Bright Shadow," "Broomwheat Tea" and "The Blacker the Berry."
Thomas was also a three-time nominee for the Coretta Scott King award for outstanding children's books by an African-American.
The author was a board member of the American Book Award's sponsor, the Before Columbus Foundation.
Les Grobstein, 670 The Score overnight host, dead at 69 The Chicago sports broadcasting icon died Sunday at his home, the station announced Monday.
Celine Dion cancels North American tour due to health issues
The singer received treatment for "severe and persistent muscle spasms" that previously prevented her from singing, and she is still recovering, according to the Saturday announcement.
By USA TODAY


TEMPERATURE: Generally, heat is furnished from an outside source, for example a match or spark, and then the fire produces enough of its own heat to be self-supporting. If we reduce the temperature of your burning substance below it is kindling point, the fire in most fire pits will go out. Sometimes enough heat is going to be generated within substances, just like in a pile of oily rags, to cause them to broken into flames. This is named spontaneous combustion. Certain bacterias in moist hay could cause the temperature to rise rapidly, causing the hay to burn. These sources of heating cannot be ignored when considering open fire prevention and safety, and in deciding what to burn in your outdoor fire pit. OXYGEN: However are other chemicals that can follow fuels to produce heat, o2 is the most common. The need for air to sustain a fire in all fire pits is demonstrated by the fact that fuels warmed up in a vacuum will not burn. Sorry there will be no outdoor fire pits in space!
Toutlemaghreb - Fire pit sets outdoor lounge furniture the home depot. Crossridge gas fire pit is durably constructed of crossridge gas fire pit is durably constructed of steel with a natural slate table top the antique bronze finish will match with a range of colors and styles of outdoor furniture making it the perfect addition to your deck or patio. Outdoor fire pit ideas and designs coastal living. Channel the organic, barefoot vibes of the beach in your fire pit's surroundings at this santa cruz, california, home above , a gas fueled fire pit with steel logs sits on a stone patio bordered by tall grasses and a boardwalk inspired wooden walkway that reference the beach that's just a block away. 20 creative beach style outdoor living ideas dream home. 20 creative beach style outdoor living ideas why bring sand to the beach when you can bring the beach to you! here are 20 creative beach style outdoor living ideas back yard beach fire pit; so easy to do in the desert outdoor fire pit with sand, needs different bench cushions fire pit sand flagstone patio fire pit wall back. 12 stunning fire pit seating ideas to spice up your patio. 12 stunning fire pit seating ideas to spice up your patio share 0 share 1 0 tweet 0 pin 0 your backyard should be an escape to paradise many design ideas help you transform the space into a fabulous retreat one of the most popular ideas is that of a fire pit photo by molly wood garden design discover beach style patio design. Organic modern outdoor living beach style patio san. Patio beach style gravel patio idea in san diego houzz the curved wall backing the fire pit area makes the seating nook feel pleasantly nestled within the beach style p for outdoor fire pits what houzzers are commenting on: w kot added this to landscape design 2 yesterday. Fire pit sets: all season style backyard & garden. A wide, powerful fire pit that's easy to use and maintain, the coral coast 36 in clarksville campfire fire pit with free cover makes a welcome outdoor focal point for your yard, porch, or patio a stainless steel burner provides odorless, smoke free flames with a maximum 50,000 btu output. How to style a fire pit the home depot. Backyard fire pits are a great addition to any outdoor living space they will keep you warm in cool weather and add ambiance to those special gatherings this guide will show you three different ways to style a fire pit. Fire pit patio sets hayneedle. Shop our best selection of fire pit patio sets to reflect your style and inspire your outdoor space find the perfect patio furniture & backyard decor at hayneedle, where you can buy online while you explore our room designs and curated looks for tips, ideas & inspiration to help you along the way. Fire pit table sets you'll love wayfair. Fire pit patio sets there is something special about being able to gather your family around a fire pit on a beautiful night outdoors if you do not currently have the seating to accommodate everyone around a fire pit to enjoy conversations or s'mores, we have the fire pit table sets to complete the space. Shop fire pits. Amazon home shop by room discover your style shop by style home d�cor furniture kitchen & dining bed & bath garden & outdoor home improvement fire pits solo stove bonfire fire pit outdoor fire pit for patio & backyard less smoke so clothes won't smell modern stainless steel design great for outdoor, backyards, patio, camping.


text:
Q: How to fix error urllib.error.HTTPError: HTTP Error 400: BAD REQUEST? I have a script(test.py) to test some api, like this:
def get_response(fct, data, method=GET):
    """
    Performs the query to the server and returns a string containing the
    response.
    """
    assert(method in (GET, POST))
    url = f'http://{hostname}:{port}/{fct}'
    if method == GET:
        encode_data = parse.urlencode(data)
        response = request.urlopen(f'{url}?{encode_data}')
    elif method == POST:
        response = request.urlopen(url, parse.urlencode(data).encode('ascii'))
    return response.read()

In terminal I call:
python test.py -H 0.0.0.0 -P 5000 --add-data

The traceback:
Traceback (most recent call last):
  File "test.py", line 256, in <module>
    add_plays()
  File "test.py", line 82, in add_plays
    get_response("add_channel", {"name": channel}, method=POST)
  File "test.py", line 43, in get_response
    response = request.urlopen(url, parse.urlencode(data).encode('ascii'))
  File "/usr/local/Cellar/python/3.6.5/Frameworks/Python.framework/Versions/3.6/lib/python3.6/urllib/request.py", line 223, in urlopen
    return opener.open(url, data, timeout)
  File "/usr/local/Cellar/python/3.6.5/Frameworks/Python.framework/Versions/3.6/lib/python3.6/urllib/request.py", line 532, in open
    response = meth(req, response)
  File "/usr/local/Cellar/python/3.6.5/Frameworks/Python.framework/Versions/3.6/lib/python3.6/urllib/request.py", line 642, in http_response
    'http', request, response, code, msg, hdrs)
  File "/usr/local/Cellar/python/3.6.5/Frameworks/Python.framework/Versions/3.6/lib/python3.6/urllib/request.py", line 570, in error
    return self._call_chain(*args)
  File "/usr/local/Cellar/python/3.6.5/Frameworks/Python.framework/Versions/3.6/lib/python3.6/urllib/request.py", line 504, in _call_chain
    result = func(*args)
  File "/usr/local/Cellar/python/3.6.5/Frameworks/Python.framework/Versions/3.6/lib/python3.6/urllib/request.py", line 650, in http_error_default
    raise HTTPError(req.full_url, code, msg, hdrs, fp)
urllib.error.HTTPError: HTTP Error 400: BAD REQUEST

The data is {"name": "Channel1"}. I couldn't understand what is wrong. Please can someone give some tip or show whats's wrong?
When I call using curl, works:
curl -X POST -H "Content-Type: application/json" -d  '{"name": "Channel1"}' http://0.0.0.0:5000/add_channel

A: I solved the problem change the test script:

*

*The api was expected a JSON_MIME_TYPE = 'application/json', so I add a header in a request as follow bellow.

*The scrit was using a wrong encode because some text in JSON couldn't be encode in Unicode, Eg:"Omö" encode in ascii launch the exception UnicodeEncodeError: 'ascii' codec can't encode character '\xf6' in position 1: ordinal not in range(128). So I changed to utf8.

Here is the fixed code:
def get_response(fct, data, method=GET):
    """
    Performs the query to the server and returns a string containing the
    response.
    """
    assert(method in (GET, POST))
    url = f'http://{hostname}:{port}/{fct}'
    if method == GET:
        encode_data = parse.urlencode(data)
        req = request.Request(f'{url}?{encode_data}'
                            , headers={'content-type': 'application/json'})
        response = request.urlopen(req)
    elif method == POST:
        params = json.dumps(data)
        binary_data = params.encode('utf8')
        req = request.Request(url
                            , data= binary_data
                            , headers={'content-type': 'application/json'})
        response = request.urlopen(req)
    x = response.read()
    return x