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I'm totally excited to be a guest blogger for the Tour de Greenbelt 2010!! I'll do my best to post fun, cool information about the Tour de Greenbelt on the Greenbelt's blog @ greenbelt.posterous.com and my own blog @ rocketgirlblog.com. I'm stoked to talk to some past participants, some local producers and to do some "Touring" of my own this year too (I missed it last year due to a trip)!!
Here is the first of many posts about the Tour de Greenbelt 2010!
Mini-interview with Peter Hart and the Tour de Greenbelt!
Who is Peter Hart and what does he have to do with the Tour de Greenbelt?
I'm the president of PHL & Associates ltd., a Toronto based event management company that specializes primarily in developing and producing fundrasing events for the not-for-profit sector. I've been involved in both recreational and professional cycling since 1988 when I became ride director of the 1st annual ride for heart, a position that I have retained to this day. I've also been race director for a number of world class, televised pro-am cycling events, and is currently working in his third year as tour director for Tour de Greenbelt.
On a personal note, I'm an avid recreational cyclist and rides two bikes; one is a beautiful carbon fiber Specialized Tarmac pro which I use for longer, faster rides, and the second a Steve Bauer turbulence mountain bike, a real classic from the 80`s that was state-of-the-art in its' day! I use it around town and when riding with friends who are either new to cycling or prefer riding shorter distances, bike paths…etc. I live in Newmarket so I can access literally 100's of km's of peaceful, scenic country roads within ten minutes riding from my house!
How is the Tour different this year from last year's Tour?
This year we are truly celebrating the concept of "meander!". We are focusing on three unique routes for each of the four days of the Tour de Greenbelt, so twelve routes in total. For the first time this year, we have 100% traffic free routes that range in length from 1-10km, or more if riders choose to do more loops. These rides have been designed specifically for families with small children that are not yet comfortable cycling on the open road, and they actually get to choose their own distance! The Nokiidaa trail in York region and the vineyard of Henry of Pelham in Niagara are two examples of these short routes; both are spectacular!
For novice cyclists or those wishing to really take their time and enjoy a liesurely ride, we have four "meander" routes averaging 20-30 km in length. These are relatively flat rides through beautiful countryside that are "user-freindly" to even a brand new cyclist who just bought their bike on Thursday!
The "long haul" routes tend to be 40-55 km in length, and are perfect for those who maybe ride once or twice a week and have a few miles on their bike already this year. While being a bit more challenging than the meander routes, as Tour de Greenbelt is not a race but rather a relaxed, recreational ride through the Greenebelt. Participants can take their time, stopping frequently to enjoy the many "experiences" that we are offering this year!
Name 3 exciting things you can see on the Tour de Greenbelt this year!
There are so many!
1. The "dirty hands project" at Henry of Pelham winery in Niagara. Short route cyclists (and others if they choose to) will experience a personally guided tour of the vineyard where adults and children alike will have the opportunity to cycle through the vineyard, stopping to taste wine (and grapejuice for the kids!) Right beside the very grapes that it came from! Feel the soil, squeeze the grapes, enjoy the unique experience…hence the name "dirty hands" project!
2. Corn maze at Coopers Farm in Durham region! Kids (both big and small!) Will have the unique opportunity to navigate an actual living corn maze as one of the "greenbelt experience" stops along the route! And this year for the first time we are offering "the greenbelt express" which is a free pick-up service for any local produce, wine, baked goods…etc purchased along the way! Our Greenbelt express van will follow the last rider and pick up all of the name-tagged items that have been purchased and bring them back to the finish area, where they can be claimed with the other half of the claim tag by the purchasers, then loaded into their vehicles for the trip home!
3. An apple orchard in Niagara, where tour participants will be able to take a break along their ride and pick some fresh apples to take home later that day! Plus there will be hayrides for kids as well as assorted crafts and children's games, so great fun for all ages!
Which Tour route are you most excited for?
I love them all! There are a few favourites though; the Jordan valley ride in Niagara is absolutely stunning in its beauty and serneity…all lush green valleys and winding country roads, and traveling right through the incredibly quaint and lovely town of Jordan. Another is the York region long haul route which winds through the Greenbelt and at one point circles around musselman's lake in Whitchurch Stouffville…lakeside riding at it's best! Another is the Durham meander route that passes by several of the Durham regional forest tracts…widely acknowledged as some of the best trail systems in Canada!"
The Tour de Greenbelt is put on by the Friends of the Greenbelt Foundation and Mountian Equipment Co-op
Please visit www.tourdegreenbelt.ca for more information or to register for this year's Tour!
See you on the roads!
Rocket Girl 2010


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SAN FRANCISCO (MarketWatch) - Most media stocks closed lower Friday, including Univision Communications, which disappointed the market with a weaker-than-expected second quarter profit and third-quarter outlook.
Spanish-language media titan Univision UVN, +16.67% fell $2.43, or 8.3%, to $26.79. It said after the market closed Thursday that its second-quarter profit declined 57% on a charge related to its investment in broadcaster Entravision Communications EVC, +0.78%. See full story.
Pixar Animation Studios PIXR, up 8% at $44.58, said after the market closed Thursday that its second-quarter profit declined 66% from the year earlier period, when it benefited from strong DVD sales of "Finding Nemo." See full story.
Washington Post Co. said Friday that second-quarter profit fell 7% and missed Wall Street's target, hurt by higher costs and weaker results from its newspaper publishing and television businesses.
Washington Post WPO, based in Washington, D.C., reported second-quarter net income of $78.8 million, or $8.16 a share, compared with $84.9 million, or $8.82 a share, in the same period in 2004. Revenue rose 10% to $897.6 million. Analysts polled by Thomson First Call expected the media company to earn, on average, $9.65 a share on sales of $899.8 million. Shares were down $64.30, or more than 7%, at $835.60.
Viacom said Thursday that second-quarter profit rose 6% on strong growth at its filmed entertainment and cable networks units. Viacom VIA VIA, +0.00% said net income was $762 million, or 47 cents a share, compared to a profit of $717 million, or 41 cents a share, in the year-ago period. Revenue rose to $5.9 billion from $5.4 billion. Analysts polled by Thomson First Call were expecting a profit of 46 cents a share on revenue of $5.74 billion. Shares were up 13 cents to $34 on Friday. See full story.

subset: commoncrawl


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Caliche High School has released its honor rolls for the first quarter of the 2018-19 school year. Students on the Regular Honor Roll have eared a minimum grade point average of 3.333. Those on the Superior Honor Roll have a 3.75 GPA or higher.
Regular Honor Roll
Ninth Grade
Baylonna Bivens
Olivia DiSimone
Brandyn Hill
Harley Koehler
Alexandria Kuskie
Zachary Ralston
Reegan Skeels
Coy Waitley
10th Grade
Amerli Reyes
Camden Sandberg
Savanah Terrill
11th Grade
Correy Derr
Erin Foster
Dylan Huss
Brandon Kuster
Emerald Lester
Deon Sanchez
Riley Stump
12th Grade
Tate Green
Joshua Kokes
Tristan Lambrecht
Jeremiah Wooldridge
Superior Honor Roll
Ninth Grade
Emma Breidenbach
Trisa Hernandez
Kyra Lambrecht
Haylee Marick
Kyser Martinez
Nevada Mitchell
Annalise Roth
Macie Smock
Ali Stromberger
10th Grade
Asa Bogie
Raegan Gerk
Riley Grauberger
Traevin Jones
Andrea Marick
Justin Pollart
Laryssa Schumacher
11th Grade
Chase Armstrong
Nevaeh Becker
Taylor Breidenbach
Howard Dedrickson
Braydon East
Ezekiel Johnson
Esben Kleist
Kristofer Meraz
Alexia Reyes
Brayden Wasson
Aly Young
Sabrina Zink
12th Grade
Talor Devie
Rachel Fryrear
Kenzie Grauberger
Alexia Hernandez
Cristen Houghton
Kaitlyn Houghton
Jamie Kielian
Erica Kolahi
Jason Kuster
Kinlie Lewis
Madalynn Painter
Brooke Stromberger
Adam Zink


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In the unfortunate event that aliens or zombies decide to invade our planet and social media becomes obsolete, you'll probably want to cancel your NapoleonCat subscription.
To do that, just go to the "Account" menu in the upper right-hand corner and click the "Billing" tab. Now, simply remove your credit card details by clicking the "remove card" link next to your card number and...it's simple as that.
Your account will remain active until the end of the current billing period. If we aren't able to charge your card again, your account will expire and we'll send you a goodbye message. And then we'll cry ;-(
If you wish to cancel your account while you're still on trial, you don't have to do anything. When your 14-day trial expires, your account will be deactivated automatically. And then we'll send you a goodbye message and cry twice as much.


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He Cheap Toronto Raptors Jerseys has a chance to prove himself at the Senior Bowl in a couple of months. So I took him and threw him out of the club. Single filers: Married filingRead more
7 Days Travel Itinerary to Andaman Nicobar Islands
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My First Trip to Mumbai – Day 1
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Things to do at Ananthagiri Hills Vikarabad
Nearby to Hyderabad the best sightseeing place with refreshing greenery, amazing weather and adrenaline pumping mountains is the only one Ananthagiri Hills Vikarabad. It's a mesmerizing place to visit in the monsoon season and pushRead more
5 Best Mouth Watering Fast Foods in Hyderabad.
Do you love fast foods? bored of the know places and tastes around your area? explore the unknown places that serve the best at fast foods in Hyderabad. Today i am telling you the bestRead more


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I don't consider myself a particularly squeamish person. I can watch gory movies and not bat an eye. Sweeney Todd? No problem, just give me a bucket of popcorn and a coke and I'm ready for a good night in front of the TV. Heck, as a historical writer with many stories set during the world wars, I even write gory scenes from time to time. But watching and writing are very different than witnessing first hand. That, I have a little doubt about.
1914. Ideas, weapons, and countries collided in the fields and woods of Europe in what would be known as the Great War, and later WWI. The call came for boys and men to take up arms for king and country. But what of the women? Roll bandages, serve soup, knit mittens, send care packages. All noble and much needed jobs for such a horrific time. But Vera Brittain wasn't content to sit home and darn socks. "The disadvantages of being a woman have eaten like iron in my soul." So with that sentiment in mind, she left her studies behind in dear old Oxford, and joined up for the voluntary aid detachment, otherwise known as VAD, and went to France and Malta to nurse wounded soldiers.
But a mere three weeks into nursing life quickly dimmed the noble and romantic ideas she had had of war. "It was very hard to believe that not far away men were being slain ruthlessly … The destruction of men, as though beasts, whether they be English, French, German or anything else, seems a crime to the whole march of civilization." Losing her beloved brother and fiancé cemented her to a lifetime of advocating for peace.
A Testament of Youth records her experiences from an innocent girl at school to the tragedy of war-torn Europe, and the beginning of her journey to pacifism. It is perhaps the best and most powerful first-hand account of what that horrible time truly did to ordinary people. I have a well-worn copy of the book on my bookshelf that I put to use during my WWI research. I highly recommend it to anyone looking for an eye opening view on the subjects.


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Jaguar Gets Fashionably Functional
High style collides with utter functionality in Jaguar's latest collection of sunnies for suave guys everywhere! From their streamlined rectangular and wayfarer-inspired silhouettes, to their slate blue and shimmery silver and bronze color palette, these shades scream masculine chic. Topped off with metallic accents, aviator-inspired bridges and wood detailing, they're beyond sleek. To boot, they… Read more »


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You never think the day will come when you'll be leaving your child home alone
From the very moment your baby is born, they are either snuggled in your arms, attached to your boob, wriggling on your hip or wedged in your bed! They are very rarely out of your sight and leaving your child home alone never enters your mind. As they get a bit older you go so far as to trap them on the same floor as you – with stairgates, and if you are anything like me, only realise that they don't actually need them once they are out of nappies and actually walking and talking! Somehow you leave them up just in case – you can see them, you know they are safe, everything is fine.
Once you become a mum it becomes almost impossible for you to leave the house without your baby, unless you make a thorough plan way in advance. Who will babysit for you? Should you feed/bath/dress them all before the babysitter arrives or not? Do you put them to bed before the babysitter turns up and hope to God that the baby doesn't wake up and notice that you have abandoned them? You work all of this out and are quite exhausted, then just as you make it to the door they start to scream the house down as you attempt to leave. On top of the exhaustion, you feel guilty and tearful yourself. Essentially when your children are very small they don't want you to leave them. This, however, does not last forever.
Leaving your child home alone for the first time
There comes a time when your 'baby' would love for you to give them some space and starts to crave more independence. For me, the thought of leaving my eldest child at home alone went against everything that I had felt and done up to that point. After all, I wouldn't be able to see her with my own eyes!
Before I left her home alone we went through what she would do in an emergency. I'm also lucky in that we have a lovely neighbour who we have a good relationship with and who would be at hand should she be needed. My daughter also has a mobile phone. All of this added together meant that both my daughter and I felt happy.
The first time I actually left her at home by herself felt very weird. Would she be ok? Would there be a fire? Would she cut herself with a sharp knife? Would the ceiling fall down? All of these nagging thoughts went through my mind and I was only gone for ten minutes! It turns out that she sat and watched T.V and that was all. When I got home she was in exactly the same place as she was when I left, although she did ring me to ask if she could have an apple, so I guess it was slightly weird for her too.
What are the positives of leaving your child home alone?
After leaving my daughter home alone the first time, we made it a regular thing but kept it to short periods of time in any one go. We then increased the length of time as she (but mostly me) got used to it. Although I felt the whole process slightly alarming, my daughter has really benefitted from knowing that she doesn't always need to depend on me and her dad, that she is capable of thinking for herself. Most importantly she knows that we trust her. As she changes from being a child to a young adult I hope as parents we can continue to give her the best opportunities in order for her to become a confident and independent adult. However, right now the thought of my daughter learning to drive and going clubbing scares me half to death but then again removing the stairgates once gave me the same feeling!
Useful Links:
What The Law Says About it – Gov.UK
RELATED: It's okay to wrap your kids in cotton wool-just not too tight


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Cover for company directors
New to the UK: Personal Guarantee Insurance provides financial security and peace of mind for individuals, usually Directors who have given Personal Guarantees for all types of secured and unsecured Company borrowings.
What does PGI cover?
PGI covers guarantees given in respect of all secured and unsecured lending to include; bank overdrafts / secured loans, Invoice Finance, Asset Finance, Property Finance and unsecured loans (Peer to Peer). The insurer will pay out up to 80% of your liability to the lender.
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The guarantor is protected in the event of the company being unable to payback its lender. Cover for Secured Loans is 80% in Yr 1 and thereafter. Unsecured loans cover is 60% year 1; 70% year 2; 80% year 3 and thereafter.
Cover is available for existing guarantees or as additional finance is taken out and is reviewable annually.
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The annual premium is based on the applicants individual requirements and on the level of the cover required. For a quick, convenient and simple online quote Click Here . Alternatively call us to discuss your requirements.
Please note Nationwide Asset Finance does not offer advice or services in this area of insurance.


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Nigel Buchanan | TIME Magazine
Nigel Buchanan was selected by TIME Magazine's Design Director, Chrissy Dunleavy, to create 3 full-page illustrations for the annual "Person of The Year" issue. Nigel was honored to be chosen for Time's most important and prestigious issue and created portraits of Donald Trump. Robert Mueller, and Moon Jae-In in his distinctive style. All three were shortlisted for the 2018 Person of […]
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The multi-talented Yuta Onoda is known for his ability to illuminate fictional worlds. His cover illustration for Nahoko Uehashi's "The Beast Player" has been accepted into the Society of Illustrators 61 book and exhibition. The novel is a YA fantasy about a girl with the special power to communicate with magical beasts, who tries to […]
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Last week, with the passing of Stephen Hawking, Daniel Baxter posted his tribute to who the Gaurdian described as "scientist's brightest star." The portrait was shared by artists, industry leaders like the Workbook, but also by many celebrating the life of Hawking, showing the power in portraiture. Daniel's uniquely styled portrait reminds of how there […]
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Work Happy Personal Projects
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text: Pinito (or little Pine Tree) is the name of a group of restaurants in Acapulco, specializing in ample portions of deliciously prepared meals in the Mexican style. Entrées are priced at about $75 pesos, and come with salad or spaghetti on the side. The house specialty is quail, but the "sábana Pinito" – a thin slice of beef covered with melted Manchego cheese – is a favorite. The "colchón" is a grilled chicken breast, which can be ordered plain or with the melted cheese as well. Their Caesar Salad, served with or without chicken, is ample and delicious. If you want anchovies, though, you will have to bring them with you. This Caesar comes simply with lots of parmesan cheese and croutons. Beers are inexpensive, especially when you buy them by the bucket. The waiters are attentive and very friendly. You can get in and out quickly, if you are in a hurry, but it is more relaxing to linger over a pleasant lunch or dinner.


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ruslan dress Off-the-shoulder midi dress
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If you are a diabetic or pre diabetic, you can choose a balanced lifestyle to control blood glucose levels following warning signs of diabetes. Changed lifestyle means you are offering to avoid certain bad diabetic foods as much as you can because these foods can topple your blood sugar levels. As such you should basically accept life style changes. The main theme of this article is to furnish a brief description of foods to control blood glucose levels.
As some foods can bear a massive impact on blood glucose levels you need to know precisely what foods to avoid in Toto. Here are some diet foods to exclude in your diet meal plan.
\"diet Meal\"
Artificial sweeteners: It is pretty difficult to avoid sugar in your diet to zero level. Still, it is advisable to avoid sweeteners like refined sugar as much as you can to increase control of blood glucose in the blood stream.
Sweet Chocolates: You should avoid chewing candy chocolates as they contain high sugar mix. Consuming these can increase sugar levels in your body.
High carb grains: Grains high in carbohydrate should be kept off. The foods rich in carbohydrates can increase the glucose levels in the blood. In this list, you have the white bread, refined rice, pasta, and pastry as bad diabetic foods to avoid.
Vegetables high in starch: Usually all vegetables are good for health. However, there are some vegetables high in starch component which should be cut off from eating. In this regard, you may avoid consuming much of sweet beets, carrots, potatoes, peas, and butter beans.
Fruits rich in sweet: As fruits contain minerals and vitamins they are highly recommended for diabetics to control blood glucose levels. Usually fruits are good fiber sources. However, fruits like mango, banana, jackfruit, strawberry, pineapple are not good for controlling blood glucose.
Fatty dietary foods: Milk is a healthier food but skimmed milk is good for reducing fat intake in the blood. Cheese and yogurt containing whole fat should be avoided. Butter cakes are very bad food for diabetics.
Processed foods: Though processed diet foods are added with preservatives they have very poor fiber content needed for diabetics to reduce blood sugar in the blood stream. So, any food packed in solid form or in liquid form should be totally avoided.
Be sure, if you can avoid these foods you can keep the blood glucose levels under control and no medicine is needed.Avoid These 7 Foods to Control Blood Glucose Levels
Controlling blood sugar is really an art. If you want to be safe you should know the description of diabetes foods to eat AND diabetes foods to avoid to lower blood sugar levels.
Varadharajan R is the author of this article. This article can be used for reprint on your website provided all the links in the article should be complete and active.


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Because WE ARE THE MICROGLOBE!
Music 4 The Microglobe #41 – August 2016
Here is finally the August episode of my radio show music 4 the microglobe. This is episode 41, syndicated to BLN.fm with German presentation and cuebase.fm and XLTRAX in English presentation.
Music 4 The Microglobe #40 – July 2016
Happy to present the July episode of my radio show music 4 the microglobe- This is already episode 40 and will be syndicated to BLN.fm with German presentation and cuebase.fm and XLTRAX in English presentation.
Music 4 The Microglobe #39 – June 2016
Football fever and terror threats, should we cheer or should we cry? Mijk van Dijk gives you music 4 the micro globe, music for all people, regardless of nation, race, religion or sexual orientation. Deep, profound, sentimental and powerful
Music 4 The Microglobe #38 – May 2016
Music 4 The Microglobe is back with presentation in German language on BLN.fm after a 3-months-break due to BLN.fm moving their studios. Mijk's show will be aired as usual on every third Thursday of the month at 18:00h CET.
Music 4 The Microglobe #37 – April 2016
Thunder & sunshine, night & day, music 4 the microglobe in April is as diverse as the April weather. With my favourite Techno tunes of the last month
Music 4 The Microglobe #36 – March 2016
music 4 the microglobe is back with another episode of dark and moody music in times of unrest and tension. Also introducing my first release in 2016, a mix for Superstrobe's The Future on BluFin Records.
Music 4 The Microglobe #35 – February 2016
"When the world is running down, you make the best of what's still around." That's not my rhyme, but what The Police sang back in 1980 on their Zenyatta Mondatta album.
Music 4 The Microglobe #34 – January 2016
Somehow 2016 feels vicious so far: public icons die at the age of 69, war and terror everywhere, the gaps are widening between the rich and the poor, North and South, East and West
Music 4 The Microglobe #33 – December 2015
Mijk van Dijk presents the Xmas 2015 edition of his radio show music 4 the microglobe on BLN.fm, cuebase-fm and XLTRAX.com, as well as on Mixcloud and hearthis.
Music 4 The Microglobe #32 – November 2015
Just a couple of days after the hostile and inhuman terror attacks on Paris, Mijk van Dijk presents music 4 the microglobe on a moody note. He starts with a classic from Paris, for Paris, "Acid Eiffel" by Choice aka Shazz and Laurent Garnier from 1993
Music 4 The Microglobe #31 – October 2015
Mijk van Dijk presents the October 2015 episode of his very own radio podcast "music 4 the microglobe", again with lots of favourites from his recent DJ sets plus a world premiere: Mijk's musical statement to the recent refugee crisis: "Music 2 Survive".
Music 4 The Microglobe #30 – September 2015
Mijk van Dijk's radio show music 4 the microglobe turns 30.House and Techno is a music, spoken and understood by all who understand the groove of Jack.


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Edwards Site Sux
One look at John Edwards' campaign website and it's clear: these people need to hire Oliver Willis. A new and essentially meaningless poll puts Edwards near the front of the pack among Demo contenders.
Outlook Cloudy, Try Again
Dave DeWitt of TarHeelDaily dissects Carolina's woes and sees them continuing. But Mark Simpson-Vos of InsideCarolina says things aren't so bad. Meanwhile SI's Seth Davis predicts the Heels will miss the tourney. Guess that's why they play the games.
One of Those Nights
Sydney began a run of serial vomiting at about 3:30 AM. Luna decided this would be a good opportunity to pee on a nice rug. Not quite as exciting as the Night of the Squirrel (below), fortunately.
Uninvited guests crash holiday quiet
By Edward Cone
News & Record
On the day before New Year's Eve, my wife told me she would come home if I cleaned the poop off the dining room table. She was on the phone at my mom's house, to which our family had repaired in the small of the night upon returning from our holiday travels to find that a squirrel had come down our chimney like St. Nick, only with less appealing presents.
We had rolled up the driveway just before midnight on Friday after enduring four states' worth of hellish traffic on I-95, not to mention the Denny's in South Hill, Va. The kids, 7 and 9, were so tired that they went straight to bed, even as their parents stood slack-jawed in the kitchen, trying to figure out who or what had trashed the place.
It didn't take Sherlock Holmes to deduce that our visitor was a squirrel, and it didn't take long for the squirrel itself to make an appearance, emerging in a hurry from beneath a desk and disappearing into the back of the house. For the record, I would like to stipulate that the noise I made was much more of a yell than a scream. My wife may disagree, but she was standing in the driveway at the time and so is not a completely reliable source.
At just about this moment, as we were trying to figure out where the squirrel had gone, our son got out of bed, staggered forward so as to position himself over the white hallway carpet, and began to vomit profusely. It was as if he knew that no holiday story is truly complete without a child throwing up. He blamed the food at Denny's, we thought he had a bug, but either way we still had a squirrel on the loose, so the poor kid didn't get much sympathy.
Where was that squirrel, anyway? Right next door, as it turned out, under our daughter's bed, onto which it leaped when the excitement in the hallway reached a certain pitch. Sydney was not at all pleased to be sharing her bed with a rodent, a sentiment she vocalized loudly enough to scare the nasty thing away again.
That settled it: round one had gone to the squirrel, and by 12:30 we had made our tactical retreat to my mother's house. Now it was Saturday morning, and Sherry, the nice lady from Critter Control, had cornered our uninvited guest in an upstairs bedroom and gently removed it from the premises, cooing to it as she held it in her leather gloves to prevent it from going into shock; my wife, already in shock, was on the phone bargaining with me over the poop-removal conditions necessary for her return.
That's when I saw the second squirrel. It was hiding behind the shutters in the dining room, but its fuzzy tail was just sticking over the top. I told Lisa to call Critter Control and tell them to turn the truck around. Sherry came back. The second squirrel made a magnificent leap onto the dining room table and tore up the stairs to the same bedroom where the other one had taken refuge (it really is the nicest room in the house). We had to chase that bad boy around the room for 15 minutes, but eventually he wound up in a cage and was deported to Kernersville.
The news of the second squirrel was not warmly received by my wife, and learning that this one was a male and the first one a female did not make her any happier. "That means they could do all the disgusting things two animals can do in addition to all the disgusting things one can do on its own," she observed, still sitting at my mom's kitchen table. The suggestion that mating season was preferable to birthing season gave her only modest solace.
Eventually Lisa accepted the fact that the squirrels had been exorcised and she returned home, where she set about a cleaning campaign of a scope and ferocity that I, who would have happily brushed the filth off the couch with my hand and watched football, could hardly comprehend. By yesterday she didn't even jump when I pretended to see a squirrel in the house.
If anybody's got an old Brunswick Stew recipe, though, she'd probably take it.
© News & Record 2001
Edward Cone is a News & Record columnist.


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Equipboard's gear archive is collaboratively built and maintained by fellow music lovers like yourself. Anyone can submit gear and equipment to artists. Joining Equipboard also allows you to create your own list to track the gear you have and want, and follow artists like Chris Robertson to never miss gear updates.
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Deve Gowda backs Maoists, disrespects slain jawan
Hassan, Apr 20: In a startling incident, former Prime Minister Deve Gowda came out in the support of the Maoists while he was on a visit to express condolences to the family of a CRPF jawan killed in the Dantewada Maoist massacre.
The JD(S) supremo was in Hassan on Monday, Apr 19 to meet and convey his condolences to the family of a CRPF jawan Shivappa, who was one among the 76 security personnel butchered in a Maoist ambush in Chhattisgarh on Apr 6.
Speaking to media personnel, Deve Gowda batted for the Naxalites, who he believes "fight for the rights of the common man".
The former PM also criticised the government's way of handling the Maoist issue and pressed for an all-party meet on the issue.
"The government should make efforts to understand the problems of the Naxalites and what they are fighting for. Instead of this, the government is trying to solve the issue with bullets, which won't work," Gowda said.
Several leaders even in the government have spoken defending the cause of the Maoists and Naxalites. However, the former Prime Minister's statement comes as a shock due to two reasons.
For one, the fact that the statement comes from someone of the stature of Deve Gowda, who once held the post of Prime Minister. And secondly, the situational context in which Mr Gowda made the statement hailing the Moaists' cause while on a visit to the home town of a slain jawan makes the remarks disrespectful and inappropriate.
OneIndia News


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Sustainable Nantucket's Farm to School Program is hiring for the Summer College Apprenticeship position!
The Sustainable Nantucket Farm to School Program is looking to hire one individual to be the Farm to School Apprentice for Summer 2016
This paid position will enable one individual to participate in food production, local food advocacy, and education through experience in the Farm to School (F2S) Program enhancing their agricultural, educational, leadership and outreach skills.
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To apply please send a resume and cover letter to Kathleen at email@example.com


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Posted: Sep 02, 2018
Granville, Ohio - DePauw shot a tournament-low 304 in the second round and finished with a 615 to win the Lynn Schweizer Invitational by 13 strokes over Transylvania.
Rachele Miller was the tournament medalist with a 148 including a 72 in the second round with three birdies. Anna Foley and Larisa Luloff each shot 155 to tie for sixth, while Allison Miller tied for eighth with a 158. Tarinni Sameer Kakar rounded out the lineup with a 159 to tie for 12th.
The Tigers return to action on September 7-8 when they travel to Olivet's Kyle Campbell Invitational at The Medalist GC in Marshall, Mich.


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One day last week, I was wandering around Facebook and found this article on 3-D printing. You can read the article for the details, but someone has come up with a copier that works just like I write!
It layers, using malleable substances like plastic or metal. Think phyllo dough or papier mache.
Using this technology, a company called Bespoke is creating artificial limbs you can run through your dishwasher!
Anything you can think up can be 3-D printed, as long as you can draw the design. I'm sure it won't be too long before they can give one of those 3-D copiers a holographic image and press the COPY button.
This technology also encourages mom and pop businesses. Let's say you make odd looking lamps and don't want to sell them to IKEA to mass produce. Buy your own 3-D copier, set up a page on the internet and create on demand. Doll houses, Prefab walls you simply attach to another wall…
This fantastic concept kept me up for three nights.
I'm not out to create cookie cutter books, but for me, writing is about layers. If I can get the dialog down, the rest comes rushing in and gets added a layer at a time. I posted something for crit the other day, and Amber asked me, "What about the smells?"
Woops, Good catch, Amber!
The other direction it took me was what if someone gave the copier a design for a magic box? And what if they printed several boxes and just set them out on a table at a park? Book plot here we come!
My grandmother was born before cars were common. She got to see us go to the moon and much more. For our generation and our children's, technology could potentially make the world my twenty year old knows now completely obsolete in only a few years time.
And the best part is I get to write, to live and work in the creative world, which makes me a little bitty part of things as wonderful as a 3-D copier!


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The Hustler's Round Table brings together business owners, entrepreneurs, creatives and community leaders in a candid and intimate discussion about the art of the hustle. At this Columbus event, we will discuss the challenges and opportunities to building capacity with freelancers, contractors, full-time employees, interns and other types of employees. This event is free and open to the public.
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Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA
Contributed by Gregg L. Semenza, February 25, 2016 (sent for review January 8, 2016; reviewed by L. Eric Huang and Carol A. Lange)
Pluripotency factors, such as NANOG, play a critical role in the maintenance and specification of cancer stem cells, which are required for primary tumor formation and metastasis. In this study, we report that exposure of breast cancer cells to hypoxia (i.e., reduced O2 availability), which is a critical feature of the tumor microenvironment, induces N6-methyladenosine (m6A) demethylation and stabilization of NANOG mRNA, thereby promoting the breast cancer stem cell (BCSC) phenotype. We show that inhibiting the expression of AlkB homolog 5 (ALKBH5), which demethylates m6A, or the hypoxia-inducible factors (HIFs) HIF-1α and HIF-2α, which activate ALKBH5 gene transcription in hypoxic breast cancer cells, is an effective strategy to decrease NANOG expression and target BCSCs in vivo.
N6-methyladenosine (m6A) modification of mRNA plays a role in regulating embryonic stem cell pluripotency. However, the physiological signals that determine the balance between methylation and demethylation have not been described, nor have studies addressed the role of m6A in cancer stem cells. We report that exposure of breast cancer cells to hypoxia stimulated hypoxia-inducible factor (HIF)-1α- and HIF-2α–dependent expression of AlkB homolog 5 (ALKBH5), an m6A demethylase, which demethylated NANOG mRNA, which encodes a pluripotency factor, at an m6A residue in the 3′-UTR. Increased NANOG mRNA and protein expression, and the breast cancer stem cell (BCSC) phenotype, were induced by hypoxia in an HIF- and ALKBH5-dependent manner. Insertion of the NANOG 3′-UTR into a luciferase reporter gene led to regulation of luciferase activity by O2, HIFs, and ALKBH5, which was lost upon mutation of the methylated residue. ALKBH5 overexpression decreased NANOG mRNA methylation, increased NANOG levels, and increased the percentage of BCSCs, phenocopying the effect of hypoxia. Knockdown of ALKBH5 expression in MDA-MB-231 human breast cancer cells significantly reduced their capacity for tumor initiation as a result of reduced numbers of BCSCs. Thus, HIF-dependent ALKBH5 expression mediates enrichment of BCSCs in the hypoxic tumor microenvironment.
Breast cancer has the highest incidence of all cancers affecting women (1). It is also the leading cause of cancer-related death for women worldwide (2). Although progress has been made in treating early-stage breast cancer, there is no effective strategy to prevent or treat metastasis, which is the major cause of breast cancer-related mortality (3). Within breast cancers, a small subpopulation of cells, which are designated as tumor-initiating cells or breast cancer stem cells (BCSCs), have the unique property of generating daughter BCSCs, which are capable of infinite proliferation through self-renewal, and transient amplifying cells, which are capable of a limited number of cell divisions and give rise to differentiated breast cancer cells (4, 5). Only BCSCs are capable of forming a secondary (recurrent or metastatic) tumor (5, 6). As in the case of ES cells (ESCs) (7), the BCSC phenotype is specified by the expression of core pluripotency factors, including Kruppel-like factor 4 (KLF4), Octamer-binding transcription factor 4 (OCT4), SRY-box 2 (SOX2), and NANOG (8⇓⇓⇓–12). Delineation of the molecular mechanisms that regulate the BCSC phenotype is needed to better understand metastasis and design more effective therapies.
Intratumoral hypoxia is a common finding in advanced cancers. The median partial pressure of oxygen (pO2) within primary breast cancers is 10 mm Hg [1.4% (vol/vol) O2], compared with 65 mm Hg [9.3% (vol/vol) O2] in normal breast tissue (13). Hypoxia-inducible factors (HIFs) HIF-1 and HIF-2 are heterodimeric transcriptional activators, consisting of an O2-regulated HIF-1α or HIF-2α subunit and a constitutively expressed HIF-1β subunit (14). In hypoxic breast cancer cells, HIFs activate the transcription of target genes that play important roles in tumor growth, angiogenesis, metabolic reprogramming, motility, invasion, metastasis, and resistance to chemotherapy (15, 16). Recent studies have demonstrated that HIFs are required for the specification and/or maintenance of BCSCs in response to hypoxia (17⇓⇓⇓–21) or chemotherapy (22, 23), leading to direct or indirect transcriptional regulation of genes encoding the pluripotency factors NANOG, SOX2, and KLF4. In addition, HIF-1α is required for hypoxia-induced epithelial-mesenchymal transition (24), which is also linked to the BCSC phenotype (25). In contrast to WT cells, the capacity of mouse HIF-1α–KO (26) or human HIF-1α–knockdown (21) breast cancer cells to initiate tumorigenesis is markedly impaired.
Recent studies have indicated that the expression of pluripotency factors is also regulated by changes in mRNA stability. N6-methyladenosine (m6A) is the most prevalent modification of mRNA in mammalian cells, consisting of 0.1–0.4% of all adenosine residues, particularly at the beginning of the 3′-UTR near the translation termination codon, usually embedded within the consensus sequence 5′-RRACU-3′ (R = A or G; methylated adenosine residue is underscored) (27⇓–29). This reversible modification is catalyzed by a methyltransferase complex consisting of the proteins methyltransferase-like 3 (METTL3), METTL14, and Wilms tumor 1 associated protein (WTAP), the activity of which is opposed by two m6A demethylases, fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5) (27, 30). In human cells, thousands of mRNAs are subject to m6A modification (28, 29), which has been implicated in mRNA splicing, nuclear export, and translation (31) as well as microRNA processing (32). However, m6A modification has been most strongly linked to increased mRNA degradation (33). NANOG, SOX2, and KLF4 mRNA were reported to contain m6A modification in ESCs (29). METTL3 KO led to decreased m6A modification of NANOG mRNA, increased NANOG mRNA stability, and decreased ESC differentiation (29, 34). These studies indicated that m6A methylation of mRNA plays a critical role in regulating ESC pluripotency. However, the physiological signals that regulate the balance between methylation and demethylation have not been described, nor have studies addressed the role of m6A in cancer stem cells.
ALKBH5 is a dioxygenase that uses α-ketoglutarate and O2 as substrates in the m6A demethylation reaction (35). Even before its identification as an RNA demethylase, hypoxic induction of ALKBH5 expression was reported (36), but, at that time, the functional significance of this observation was not known. In the study reported here, we tested the hypothesis that exposure of breast cancer cells to hypoxia is sufficient to stimulate HIF-dependent ALKBH5 expression, leading to decreased m6A methylation of NANOG mRNA, increased NANOG mRNA stability, increased NANOG protein levels, and BCSC enrichment, thereby establishing hypoxia as a major physiological stimulus in the tumor microenvironment that directly connects regulation of m6A RNA methylation to the specification and maintenance of BCSCs.
HIF-Dependent ALKBH5 Expression Promotes m6A RNA Demethylation in Hypoxic Breast Cancer Cells.
Breast cancers are divided into three groups based on expression of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). We first investigated whether ALKBH5 expression is induced by hypoxia in nine human breast cancer cells that are ER+ (ZR75.1), ER+PR+ (MCF-7 and T47D), ER+PR+HER2+ (BT-474), HER2+ (HCC-1954), or triple-negative (MDA-MB-231, MDA-MB-435, SUM-149, and SUM-159) (37). These nine breast cancer cell lines were exposed to 20% or 1% O2 for 24 h, and RNA was isolated for reverse transcription and quantitative real-time PCR (RT-qPCR). ALKBH5 mRNA levels were significantly increased under hypoxic conditions by greater than 1.6-fold in SUM-159 and MDA-MB-435 cells and greater than twofold in MDA-MB-231 and MCF-7 cells (Fig. 1A).
To determine whether hypoxia-induced ALKBH5 expression was dependent on HIF-1α, HIF-2α, or both, we analyzed MDA-MB-231 and MCF-7 subclones that were stably transfected with lentiviral vectors encoding shRNA to inhibit the expression of HIF-1α (sh1α), HIF-2α (sh2α), or both HIF-1α and HIF-2α [double knockdown (DKD)], as well as a subclone expressing a nontargeting control shRNA (NTC) (21, 38). In contrast to the NTC subclone, hypoxia-induced ALKBH5 mRNA expression was abrogated in sh1α, sh2α, and DKD subclones of MDA-MB-231 (Fig. 1B) and MCF-7 (Fig. 1C). Immunoblot assays revealed that ALKBH5 protein levels were increased by hypoxia in NTC subclones of MDA-MB-231 (Fig. 1D) and MCF-7 (Fig. 1E), but not in HIF-knockdown subclones. Thus, hypoxia induces ALKBH5 expression in a subset of ER+ and ER− breast cancer cell lines in an HIF-dependent manner.
Next, we investigated whether increased ALKBH5 expression in hypoxic cells leads to changes in levels of m6A RNA modification in total cellular RNA pools. We hypothesized that m6A levels in RNA represent the net effect of methylation mediated by the METTL3/METTL14 complex and demethylation mediated by ALKBH5 and FTO. Thus, increased ALKBH5 expression should lead to decreased m6A levels in RNA. Exposure of NTC subclones of MDA-MB-231 (Fig. 1F) and MCF-7 (Fig. 1G) to 1% O2 for 24 h led to a significant decrease (>30%) in the percentage of m6A residues among total adenosine residues in RNA. In contrast, there was no significant change in total m6A levels in RNA when the DKD subclones were exposed to hypoxia.
To demonstrate that decreased m6A modification of RNA in hypoxic cells was caused by increased ALKBH5 expression, we generated subclones of MDA-MB-231 and MCF-7 that were stably transfected with a lentiviral vector encoding either of two different shRNAs targeting ALKBH5 (shALK1#1 and shALK#2). Compared with the NTC subclone, knockdown of ALKBH5 in MDA-MB-231 (Fig. 1H) and MCF-7 (Fig. 1I) cells significantly increased m6A levels in total RNA isolated from cells incubated at 20% O2, and ALKBH5 knockdown blocked hypoxia-induced RNA demethylation. Taken together, the data in Fig. 1 indicate that HIF-dependent ALKBH5 expression mediates m6A demethylation in total cellular RNA pools of hypoxic breast cancer cells.
Hypoxia Induces HIF- and ALKBH5-Dependent NANOG Expression.
We next analyzed the effect of hypoxia on expression of the core pluripotency factor NANOG in human breast cancer cells. In NTC subclones of MDA-MB-231 (Fig. 2A) and MCF-7 (Fig. 2B), hypoxia significantly increased NANOG mRNA levels, whereas the hypoxic induction was abrogated in the sh1α, sh2α, and DKD subclones. Hypoxic induction of NANOG protein expression was also abrogated in the HIF knockdown subclones (Fig. 1 D and E).
ALKBH5 knockdown by either of two different shRNAs also significantly impaired the hypoxic induction of NANOG mRNA expression in MDA-MB-231 (Fig. 2C) and MCF-7 (Fig. 2D) cells. Finally, immunoblot assays demonstrated that ALKBH5 knockdown led to decreased NANOG protein expression in MDA-MB-231 (Fig. 2E) and MCF-7 (Fig. 2F) cells. Decreased levels of NANOG mRNA and protein were also observed in ALKBH5-knockdown subclones at 20% O2, indicating that there is significant ALKBH5 activity under nonhypoxic conditions, which is further increased by hypoxia.
ALKBH5 Enhances NANOG mRNA Stability by Catalyzing m6A Demethylation.
To investigate the mechanism by which ALKBH5 regulates NANOG expression, we first explored whether hypoxia-induced ALKBH5 targeted NANOG mRNA for m6A demethylation. Total cellular RNA was immunoprecipitated with an antibody that recognizes m6A, and the immunoprecipitated RNA was subjected to RT-qPCR to amplify the beginning of the NANOG 3′-UTR, which contains a match to the m6A consensus sequence, 5′-RRACU-3′. Hypoxic exposure led to decreased levels of m6A+ NANOG mRNA in NTC subclones of MDA-MB-231 (Fig. 3A) and MCF-7 (Fig. 3B) cells. In contrast, expression of either of two different shRNAs targeting ALKBH5 led to increased levels of m6A+ NANOG mRNA under normoxic and hypoxic conditions. HIF DKD also increased m6A+ NANOG mRNA levels, although to a lesser degree, and blocked the hypoxia-induced decrease in m6A+ NANOG mRNA levels that was observed in NTC subclones of MDA-MB-231 (Fig. 3C) and MCF-7 (Fig. 3D). These data indicate that, under hypoxic conditions, NANOG mRNA is subject to m6A demethylation that is HIF- and ALKBH5-dependent.
Hypoxia induced decreased m6A modification of NANOG mRNA and increased total NANOG mRNA levels, which is consistent with increased degradation of m6A+ mRNA (29, 33). To measure NANOG mRNA stability, flavopiridol was used to inhibit global mRNA transcription as previously described (29), and the ratio of NANOG mRNA in flavopiridol-treated cells relative to vehicle-treated cells (F/V ratio) was calculated. Exposure of NTC subclones of MDA-MB-231 (Fig. 3E) and MCF-7 (Fig. 3F) to hypoxia increased the NANOG mRNA F/V ratio, indicating decreased degradation of NANOG mRNA. However, this effect of hypoxia was lost in ALKBH5 and HIF knockdown subclones, indicating that hypoxia enhances NANOG mRNA stability through HIF-dependent ALKBH5 expression.
Sequence analysis revealed three matches to the 5′-RRACU-3′ (methylated adenosine residue is underscored) m6A consensus sequence within the NANOG 3′-UTR. Reporter plasmids, which contained or lacked the entire NANOG 3′-UTR immediately downstream of luciferase coding sequences, were transiently transfected into MDA-MB-231 (Fig. 3G) and MCF-7 (Fig. 3H) subclones, followed by incubation at 20% or 1% O2 for 24 h and measurement of luciferase activity. In NTC subclones exposed to 20% O2, luciferase activity was reduced four- to fivefold by insertion of the NANOG 3′-UTR into the reporter, but hypoxia significantly increased luciferase activity, whereas this effect was lost in the shALK and DKD subclones (Fig. 3 G and H).
Next, the adenine residue embedded within the consensus sequence located closest to the translation termination codon in the NANOG 3′-UTR was mutated (5′-AAACU-3′ to 5′-AAUCU-3′ in mRNA), and luciferase assays were performed comparing WT and mutant reporters. Mutation of the adenosine residue resulted in constitutively increased luciferase activity in parental MDA-MB-231 (Fig. 3I) and MCF-7 (Fig. 3J) cells, suggesting that the mutation prevented methylation and thereby increased the stability of the luciferase-NANOG 3′-UTR fusion mRNA. These results suggest that the mutated adenosine residue was a target for methylation, which was consistent with the identification of this site by others using m6A immunoprecipitation and RNA sequencing in ESCs (29). Taken together, the results presented in Fig. 3 indicate that hypoxia-induced and HIF-dependent ALKBH5 expression increases NANOG mRNA demethylation, stabilization, and accumulation.
ALKBH5 Deficiency Impairs Hypoxia-Induced BCSC Enrichment.
We next investigated whether ALKBH5 plays a role in regulation of the BCSC phenotype. Two properties that identify populations of breast cancer cells that are enriched in BCSCs are the ability to generate clusters of daughter cells when cultured on ultra-low adherence plates (mammosphere assay) (39) and increased aldehyde dehydrogenase (ALDH) 1 activity, which can be quantified by flow cytometry using a fluorogenic substrate (40). Analysis of MDA-MB-231 (Fig. 4A) and MCF-7 (Fig. 4B) cells grown as adherent monolayers or as nonadherent mammospheres revealed significantly increased ALKBH5 mRNA expression in mammosphere cultures, which are enriched for BCSCs.
To determine the effect of ALKBH5 depletion on the specification/maintenance of BCSCs, we exposed NTC and shALK subclones to 20% or 1% O2 for 72 h, then transferred the cells to ultra-low adherence plates, incubated them at 20% O2 for 1 wk, and counted the number of primary mammospheres that had formed (Fig. 4 C–F). We then dissociated the primary mammospheres, replated the cells on ultra-low adherence plates, incubated them at 20% O2 for 1 wk, and counted the number of secondary mammospheres (Fig. 4 G and H). Hypoxic exposure of NTC subclones for 72 h dramatically increased primary and secondary mammosphere formation at 1 and 2 wk after the hypoxic exposure was terminated, as previously reported (21). In contrast, ALKBH5 knockdown significantly impaired hypoxia-induced mammosphere formation and, in MCF-7 cells, significantly decreased mammosphere formation even when cells were not exposed to hypoxia (Fig. 4 F and H). Analysis of BCSCs by the ALDH assay gave remarkably similar results: ALKBH5 knockdown significantly impaired the hypoxia-induced enrichment of ALDH+ cells in MDA-MB-231 (Fig. 4I) and MCF-7 (Fig. 4J) cells and decreased the percentage of ALDH+ cells even when MCF-7 cells were not exposed to hypoxia. Thus, mammosphere and ALDH assays indicate that ALKBH5 expression is necessary for hypoxia-induced BCSC enrichment.
Overexpression of ALKBH5 Is Sufficient to Enhance NANOG Expression and BCSC Enrichment.
To determine whether ALKBH5 induction is sufficient to decrease the methylation of NANOG mRNA and thereby increase the BCSC population, we stably transfected MDA-MB-231 and MCF-7 cells with a lentiviral vector encoding human ALKBH5 or empty vector (EV), and exposed the cells to 20% or 1% O2. Under nonhypoxic conditions, ALKBH5 overexpression led to significantly decreased m6A modification of NANOG mRNA (Fig. 5 A and B), increased levels of NANOG mRNA (Fig. 5 C and D) and protein (Fig. 5 E and F), and increased percentage of ALDH+ cells (Fig. 5 G and H). In MCF-7 cells, ALKBH5 overexpression also potentiated the decrease in m6A+ NANOG mRNA levels (Fig. 5B) and increase in ALDH+ cells (Fig. 5H) that was observed in response to hypoxia. These data indicate that, under nonhypoxic conditions, overexpression of ALKBH5 is sufficient to demethylate NANOG mRNA, increase NANOG expression, and enhance BCSC enrichment, thereby phenocopying the effects of hypoxia.
Knockdown of ALKBH5 Impairs Tumor Formation and Decreases BCSCs in Vivo.
To analyze the effect of ALKBH5 deficiency on the tumor-initiating potential of breast cancer cells, we injected a limiting number of cells (1 × 103) from MDA-MB-231 subclones into the mammary fat pad of female nonobese diabetic/SCID/IL2Rγ-null (NSG) immunodeficient mice. Ten weeks after injection, 100% of the mice (n = 7) that were injected with NTC cells developed palpable tumors, compared with only 43% (n = 6 of 14) of the mice injected with ALKBH5-deficient cells (Fig. 6A). To further study the effect of ALKBH5 deficiency on the BCSC population in vivo, the tumors that formed after injection of NTC or shALK cells were harvested for analysis of NANOG expression and BCSCs. NANOG mRNA (Fig. 6B) and protein (Fig. 6C) levels were significantly decreased in tumors formed by ALKBH5-knockdown subclones compared with the NTC subclone. Both mammosphere (Fig. 6D) and ALDH (Fig. 6E) assays revealed a significant decrease in the BCSC population in tumors derived from injection of shALK compared with NTC cells. These results indicate that loss of ALKBH5 expression significantly impairs tumor formation and decreases the BCSC population in breast tumors.
The results presented here demonstrate that an essential physiological stimulus, i.e., hypoxia, regulates m6A modification of RNA in human cancer cells and that hypoxia induces the BCSC phenotype in part by increased demethylation of an mRNA encoding a core pluripotency factor. Although we focused on the regulation of NANOG mRNA stability, it is likely that the stability of other mRNAs is increased in breast cancer cells as a result of HIF-dependent and ALKBH5-mediated m6A demethylation. It is remarkable that exposure of MCF-7 or MDA-MB-231 breast cancer cells to 1% O2, which is comparable to the median pO2 in advanced breast cancers, decreased the m6A content of the total cellular RNA pool by as much as one third within only 24 h (Fig. 1 F–I). In addition, our studies have demonstrated a critical role for ALKBH5 in mediating NANOG expression and BCSC specification and/or maintenance within the hypoxic microenvironment of human breast cancer orthotopic tumors. ALKBH5 expression was induced by hypoxia in ER+ (MCF-7) and ER− (MDA-MB-231, SUM-159, and MDA-MB-435) cells, but not in other ER+ (T47D, ZR75.1, and BT-474) and ER− (HCC-1954 and SUM-149) cell lines. Further studies are required to determine whether FTO, the other known m6A demethylase, is induced by hypoxia in these latter cell lines.
We have previously shown that shRNA-mediated inhibition of NANOG expression in MDA-MB-231 cells results in a profound depletion of BCSCs (23). High NANOG mRNA (23) or protein (41) expression in primary human breast cancers is significantly associated with increased patient mortality. Other groups have implicated HIFs in the direct transcriptional activation of NANOG and other genes encoding pluripotency factors, both in human ESCs (42) and human cancer cells (17). We demonstrated that expression of ALKBH5 was necessary (Figs. 1–4) and sufficient (Fig. 5) to mediate a posttranscriptional induction of NANOG expression and BCSC enrichment. Taken together with prior studies, these results suggest that HIFs may coordinately regulate NANOG expression at the transcriptional and posttranscriptional levels in hypoxic breast cancer cells. Other HIF-regulated genes may also encode ALKBH5-regulated mRNAs, as dual regulation by HIFs and ALKBH5 provides a means to rapidly accumulate a target mRNA by coordinately increasing its production and decreasing its destruction.
A single multiprotein complex (consisting of METTL3, METTL14, and WTAP) mediates m6A modification of RNA, whereas two different proteins (FTO and ALKBH5) mediate demethylation, and it is not known whether FTO and ALKBH5 have distinct or redundant functions. Knockdown of ALKBH5 expression in hypoxic MCF-7 and MDA-MB-231 cells increased the m6A content of the total cellular RNA pool by as much as one third (Fig. 1 H and I), indicating that ALKBH5 plays a major role in regulating RNA methylation in human breast cancer cells.
Finally, it should be noted that, in addition to activating transcription of the RNA demethylase ALKBH5, HIFs also regulate the expression of two other O2-dependent dioxygenases that play key roles in breast cancer metastasis and stem cell maintenance (43, 44): JMJD2C, which mediates demethylation of histones (45), and TET1, which mediates hydroxymethylation of DNA (46). It is interesting that, in the absence of HIFs, the activity of these dioxygenases should decline under hypoxic conditions as a result of reduced substrate (i.e., O2) availability. However, HIF activity does not merely compensate for reduced O2 levels but significantly increases ALKBH5, JMJD2C, and TET1 activity in hypoxic breast cancer cells, leading to transcriptional and posttranscriptional changes in gene expression that promote the specification and/or maintenance of BCSCs. In addition to O2, these dioxygenases use α-ketoglutarate as a substrate. Further studies are warranted to investigate whether competitive antagonists of α-ketoglutarate that inhibit ALKBH5 (and possibly other dioxygenases) might be useful as anticancer agents that target BCSCs.
Materials and Methods
Cell Culture.
MCF-7, MDA-MB-231, MDA-MB-435, and T47D cells were cultured in DMEM. BT-474, HCC-1954, and ZR75.1 cells were cultured in RPMI-1640 medium. SUM-149 and SUM-159 cells were cultured in DMEM/F12 (50:50) media supplemented with hydrocortisone and insulin. All culture media were supplemented with 10% (vol/vol) FBS and 1% (vol/vol) penicillin/streptomycin. Cells were maintained in a 5% CO2 and 95% air incubator [20% (vol/vol) O2]. For hypoxia exposure, cells were placed in a modular incubator chamber (Billups-Rothenberg), which was flushed for 2 min at 2 psi with a gas mixture containing 1% O2, 5% CO2, and 94% N2.
Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. cDNA synthesis was performed using the High Capacity RNA-to-cDNA Kit (Applied Biosystems). qPCR was performed by using the following primers: ALKBH5, 5′-CGG CGA AGG CTA CAC TTA CG-3′ and 5′-CCA CCA GCT TTT GGA TCA CCA-3′; NANOG, 5′-TTT GTG GGC CTG AAG AAA ACT-3′ and 5′-AGG GCT GTC CTG AAT AAG CAG-3′; and 18S rRNA, 5′-CGG CGA CGA CCC ATT CGA AC-3′ and 5′-GAA TCG AAC CCT GAT TCC CCG TC-3′. For each sample, the ratio of NANOG mRNA:18S rRNA was determined.
Immunoblot Assay.
Whole cell lysates were prepared in modified RIPA lysis buffer (50 mM Tris⋅HCl, pH 7.5, 1 mM β-mercaptoethanol, 150 mM NaCl, 1 mM Na3VO4, 1 mM NaF, 1 mM EDTA, 0.25% sodium deoxycholate and 1% Igepal CA-630). Blots were probed with HIF-1α (BD Biosciences), HIF-2α (Novus Biologicals), ALKBH5 (Novus Biologicals), NANOG (Novus Biologicals), and Actin (Santa Cruz) antibodies. HRP-conjugated anti-rabbit and anti-mouse secondary antibodies were used and the chemiluminescent signal was detected by using ECL Plus (GE Healthcare). Densitometric analysis was performed by using ImageJ (NIH).
Lentiviral Vectors and Transduction.
Lentiviral vectors encoding shRNA targeting HIF-1α and HIF-2α were described previously (38). pLKO.1-puro lentiviral vectors encoding shRNA targeting ALKBH5 mRNA (clone ID, NM_017758.2–1625s1c1 and NM_017758.2–1176s1c1) were purchased from Sigma-Aldrich. pLenti-GIII-CMV lentiviral vector containing cDNA encoding human ALKBH5 was purchased from Applied Biological Materials (cat. no. LV073201). All lentiviral vectors were transfected into 293T cells for packaging. Viral supernatant was collected after 48 h and used for transfection as described before (38). Puromycin (0.5 μg/mL) was added to the medium of cells transduced with lentivirus for selection.
Measurement of Total m6A and m6A+ NANOG mRNA Levels.
Total m6A content was measured in 200-ng aliquots of total RNA extracted from MDA-MB-231 and MCF-7 subclones using an m6A RNA methylation quantification kit (cat. no. P-9005; Epigentek) according to the manufacturer's instructions. To measure m6A+ NANOG mRNA levels, m6A immunoprecipitation was performed as described before (47). A 1-µg aliquot of m6A antibody was conjugated to protein A-agarose slurry (Millipore) overnight at 4 °C. A 100-μg aliquot of total RNA was incubated with the antibody in immunoprecipitation buffer (50 mM Tris⋅HCl, 750 mM NaCl, and 0.5% Igepal CA-630) supplemented with RNase inhibitor for 3 h at 4 °C, RNA was eluted from the beads by incubation in 300 μL of elution buffer (5 mM Tris⋅HCl, 1 mM EDTA, and 0.05% SDS) with 4.2 μL of proteinase K for 1.5 h at 50 °C, and m6A+ RNA was purified by phenol/chloroform extraction and analyzed by RT-qPCR. The nucleotide sequences of the primers used for detection for m6A+ NANOG mRNA were as follows: 5′-ATG CAA CCT GAA GAC GTG TG-3′ and 5′-GAG ATT GAC TGG ATG GGC AT-3′.
Measurement of NANOG mRNA Stability.
Cells were exposed to 20% or 1% O2 for 24 h and then treated with vehicle or flavopiridol (Sigma-Aldrich) at a concentration of 0.8 μΜ for 3 h (MDA-MB-231) or 3.2 μΜ for 6 h (MCF-7), followed by RNA extraction and RT-qPCR as described earlier.
Luciferase Assay.
The luciferase coding sequence NANOG 3′-UTR reporter (cat. no. S807466) and luciferase coding sequence-only reporter (cat. no. S890005) were purchased from SwitchGear Genomics. Cells were seeded in a 96-well plate and transfected with luciferase-NANOG 3′-UTR reporter or luciferase-only reporter, and exposed to 20% or 1% O2 for 24 h. Luciferase activity was measured using a commercial kit (SwitchGear Genomics) and is presented as the activity ratio of luciferase-NANOG 3′-UTR to luciferase only. Adenine-to-thymine mutation of the luciferase-NANOG 3′-UTR reporter plasmid was performed by using the Q5 Site-Directed Mutagenesis Kit (New England Biolabs) according to the manufacturer's instructions.
Mammosphere Assay.
Cultured cells were trypsinized, whereas tumor tissue was minced and digested with 1 mg/mL of type 1 collagenase (Sigma) at 37 °C for 30 min to prepare single-cell suspensions, which were seeded in six-well ultra-low attachment plates (Corning) at a density of 5,000 cells per milliliter in Complete MammoCult Medium (Stem Cell Technologies). After 7 d, the cells were photographed under an Olympus TH4-100 microscope, and primary mammospheres (diameter ≥70 μm) were counted. For secondary mammosphere formation, primary mammospheres were trypsinized, plated at a density of 5,000 cells per milliliter, incubated for 7 d, and counted.
ALDH Assay.
Cells were exposed to 20% or 1% O2 for 72 h and harvested for Aldefluor assay (Stem Cell Technologies). Single-cell suspensions (5 × 105 cells in 1 mL of assay buffer) were prepared as described earlier and incubated with BODIPY-aminoacetaldehyde (1 μM) for 45 min, followed by flow cytometry analysis.
Orthotopic Transplantation.
Protocols were approved by the Johns Hopkins University Animal Care and Use Committee and were in accordance with the NIH Guide for the Care and Use of Laboratory Animals. A total of 1,000 breast cancer cells were injected into the mammary fat pad of 6–8-wk-old female NSG mice in a 1:1 suspension of Matrigel (BD Biosciences) in PBS solution. At 10 wk after injection, mice were examined for the presence of tumors, which were harvested for analysis.
Statistical Analysis.
Data are presented as mean ± SEM. Differences between two groups or multiple groups were analyzed by Student t test and ANOVA, respectively. Fisher exact test was used for analyzing tumor initiation capacity. P values <0.05 were considered significant.
We thank Karen Padgett (Novus Biologicals) for providing antibodies against ALKBH5, HIF-2α, and NANOG. This work was supported in part by American Cancer Society Grant 122437-RP-12-090-01-COUN, the Cindy Rosencrans Fund for Triple Negative Breast Cancer, and the China Scholarship Council (C.Z.). G.L.S. is an American Cancer Society Research Professor and the C. Michael Armstrong Professor at the Johns Hopkins University School of Medicine.
- ↵1To whom correspondence should be addressed. Email: gsemenza{at}jhmi.edu.
Author contributions: G.L.S. designed research; C.Z., D.S., H.L., J.W.B., H.Z., and I.C. performed research; X.H. contributed new reagents/analytic tools; C.Z. and G.L.S. analyzed data; and C.Z. and G.L.S. wrote the paper.
Reviewers: L.E.H., University of Utah; and C.A.L., University of Minnesota Masonic Cancer Center.
The authors declare no conflict of interest.
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Anonymous, 02 Jun 2014This has got to be the most boring keynote ever.and i bet you watched from start to finish
'Continuity' feature is underrated but actually will change a lot of things for how people do things.
This is ridiculous for me that Apple won't allow Windows users to watch the live stream. It's like Google allowed Android-only devices to watch the I/O stream, which certainly is not true.
Maybe Apple don't have money to pay for the bandwidth?
for the nerds, well there are a lot of good stuff, like swift. been to some programming, boy those coding have to run i a lot of equation.
for the consumer end, its just the artistry, and simplicity of the user interface, the convergence of art and tech. it dosent need a degree holder to use one of these
Anonymous, 02 Jun 2014This has got to be the most boring keynote ever.Well said
It's looking incredible so far. Unbelievable amount of new tools. OSX, iOS looking incredible. Swift looks fantastic.
That Epic graphics demo was impressive. Wonder what the repercussion for Android gaming is.
Looks like google has forced apple to open up to 3rd party application
Tats an amazing change
which kinda makes to urge to switch Iphone from android !!!
you are demonstrating how smart apple is.. but i can see ur iphone..ipad connected via USB? can u justify why?
Whiners everywhere. If you're not interested about Apple's event then why are you putting such comment in an Apple article? Go use your laggy cheap Android. No one forces you to watch the event anyway.
gam30toronto, 02 Jun 2014u talk to much, no one caresYou wre saying?
iOS 8.
Interactive Notifications. Pull downs and respond.
Birdseye view of tabs.
New Side Bar.
New email swipe feature.
Home button now gives access to recent callers.
Search function like Windows 8.1
Privacy. Whatever you type stays on your device.
Calls relayed to your iPad from your phone.
130 million new Apple users. Some switched from Android. Half of Apple's new Chinese customers moved from Android.
AnonD-1385, 02 Jun 2014So now Apple is forcing people to get a apple device just to see their upcoming products??? Ge... moreWell no grieves here. Who wants to watch another row of icons innovation event anyway.


text:
Draw Tip Tuesday: Be Less Precious About Your Drawings
Welcome to Draw Tip Tuesday!
Do you feel like each art journal entry need to be meaningful? Perfect? Pfff what a pressure!
Let's not be too precious about our drawings.
Sometimes, just drawing what's right in front of you can be very satisfying! Especially if you use several colors, the drawing can cheer you up.
Want more videos?
Subscribe to my Youtube Channel!
Draw Tip Tuesdays


text:
Sometimes life kicks you in the teeth. Then it smacks you on the head, thwacks you in the bum, and punches you in the gut.
I know, I know, "What doesn't kill you makes you stronger." (Unless, as Lee adamantly puts it, it F&*%ing cripples you.)
Well I would like a little less strength of character, and while you are at it, fewer character strengthening opportunities. At least for a little while.
Why am I ranting about life being evil and cruel? Let me tell you!
The last time we came to Denver it was on the heels of the death of my close friend, and the near death of Lee's mother.
This time, it was for a happy visit and a wedding. Oh, and the near death of Lee's mother.
Yes, Mom is back in the hospital, this time in the burn unit at University, having been pulled from a fire in her apartment. (Yes, she is the woman who was in the apartment fire at the senior facility that has been all over the news today.) She is not well. We will not know if she will pull through. The doctors say we should know more about her chances in the next 72 hours. She has burns on 30% of her body. Her quality of life will be incredibly diminished if she survives this. She will likely experience chronic pain. We will go see her tomorrow, we couldn't face it before going to the wedding today.
We don't know what happened. There are rumors being bandied about that the fire was started by a cigarette, but she was an avid anti-smoker who suffered from COPD. I had seen her earlier in the week, and as an ex-smoker myself, I can tell you there was no trace of the smell of cigarette smoke on her during our visit. I just can't see her picking up the habit now. So really, who knows how the fire started. I hope we can get some answers from the Fire Marshal.
It's just so sad. I am so sad for my husband, and for his mother, and for the people who were hurt trying to help her. I am so confused as to what happened, and what will happen now. I am worried about her. I am worried about him.
So I find myself, once again, facing tough choices and hard emotions. As my dearly departed godmother would say: It's an AFGO. (Another F&%$ing growth opportunity.)
Personally, I feel my friends and family have had enough of them this year.
Saturday, August 25, 2007
Sometimes life kicks you in the teeth. Then it smacks you on the head, thwacks you in the bum, and punches you in the gut.
Posted by Scylla at 11:05 PM
5 comments:
- Woman with a Hatchet said...
I'm so sorry. Wish I could offer something other than "I love you", but that's all I have that may help in some small way.
Hang in there.
- ellen said...
I can't believe you are having to go through this again with her being sick and all. Wow. Wow. I really am at a loss for words.
Please keep us all updated on how things turn out.
I hope you get to enjoy the wedding.
- Coni Nelson said...
Oh my goodness Misty and Lee, we are here for you, we love you and we are sorry for your AFGO...
- Amy said...
You and Lee are in our thoughts. I am so sorry. Please let us know if we can help in anyway.
- Red Flashlight said...
You have my condolences - I hope things settle down for everyone, soon.
Healing thoughts to you and yours.


text:
Ever wondered about using that red lipstick you finally found after a couple years, smooshed in between the cushions of your couch? Or what about the old concealer you use out of desperation a couple times a year when you cant get to the mall to buy the one you normally use? Well, I hate […]Read More
Spring Clean Your Makeup: Out with the Old!


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Having problems with your Pentax K100D Digital Camera ?
My pentax 100d camera screen shows up blank when i want to take a picture..but it still takes pictures and they come out fine..im confused as to why the screen is not working..i can still see its menu and options..
Answers :
Repair Help & Product Troubleshooting for Pentax K100D Digital Camera
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My pentax 100d camera screen shows up blank when i want to take a picture..but it still takes pictures and they come out fine..im confused as to why the screen is not working..i can still see its menu and options..
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When I turn on the camera the LCD screen no longer shows the settings. I can still take pictures and it shows the image on the LCD screen, and if I hit menu screen shows. Did I push something I wasn't supposed to?
That is just a setting. It turns off the lcd to save battery or when you want to just use the optics for sightiing/focus etc. Should be one of the menus that show up when you hit the menu button. You should be able to set it off as it is now or have ... Sony Alpha DSLR-A100 Digital Camera
White Screen Ever since a few nights ago, my camera has been showing this white screen. The funny thing is, that I can see the menu and other options perfectly. But when I want to take a picture, the screen is pure white, and even the pictures I take are absolutely white, and sometimes with a light grayish circle in the middle. I don't know why this is and I just bought this camera around the fifth of january and it was working perfectly fine then. Please help me?
Hi everyone, I know many posts on blogs when i was looking to replace my casio lcd and every site i looked at for the replacement lcd was so high priced, and there was no way i would pay how much they wanted so i was going to buy a new camera. Then i ... Fuji FinePix A400 Digital Camera
I have a sony cybershot, model DSC-W55. when i press the power on button, the camera does not turn on as it usualy does. instead, i get a black screen with the word Access on it. (also, when i turn the camera on, the lenses keep going in and out more frequently than before.) so the screen Access screen is just there right now, but when i click buttons like menu or display it makes beeping noises and it says 'press power on and off again '. i can still view the pictures i've taken but can't seem
You can try contacting Sony - maybe through online chat if its available for free. From what you have described, it sounds like a hardware failure. Contact Sony, and if it is a hardware failure, they will replace it for free. Or, if you have bought i ... Sony Digital Cameras
Blank LCD screen on shooting mode, but does show settings. Will not take pictures. Replaying mode works, shows pictures taken before screen went blank. New batteries. What is going on??? Is it fixable? I bought the Canon PowerShot A570 IS. I don't know if it is me or the camera but my LCD screen doesn't stay on. I can take a phote, the photo will then display on the LCD for 2 seconds BUT then the LCD goes blank, all i can see are the menu options. If I take a pic while LCD is blank my pic's
The Display button manually turns the LCD display on or off to save batteries. If you accidentally had turned the LCD off, It should only take one press of the button to turn it back on and leave it that way. As such, it's not you but the camera. ... Canon PowerShot A570 IS Digital Camera