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So can us Canadians buy your wootacluar goodness yet? Come on, we're practically neighbors or something. Please? I'll let you borrow my flag when you travel overseas.
How about now?
I don't know aboot that.
man, ever since the Canadians helped our people in Iran all those years ago, they act like they're a seperate country or something.
yeah, what's up with that?
LOL. If we are all one big happy family what's up with the latex glove treatment at the border??
They give a Latex Glove Treatment at the border? [;)]
I wonder if the Quebecois have to cough in English?!?!?
Do I really need to bring up Celine Dieon again?
how about all that arctic cold they keep sending down here EVERY SINGLE WINTER?
Somebody ought to shut off the power to those big fans they blow the cold air down with.
Personally I blame canada.
Sheila: Times have changed
Our kids are getting worse
They won't obey their parents
They just want to fart and curse!
Sharon: Should we blame the government?
Liane: Or blame society?
Dads: Or should we blame the images on TV?
Sheila: No, blame Canada
Everyone: Blame Canada
Sheila: With all their beady little eyes
And flappin' heads so full of lies
Everyone: Blame Canada
Blame Canada
Sheila: We need to form a full assault
Everyone: It's Canada's fault!
Sharon: Don't blame me
For my son Stan
He saw the darn cartoon
And now he's off to join the Klan!
Liane: And my boy Eric once
Had my picture on his shelf
But now when I see him he tells me to fuck myself!
Sheila: Well, blame Canada
Everyone: Blame Canada
Sheila: It seems that everything's gone wrong
Since Canada came along
Everyone: Blame Canada
Blame Canada
Copy Guy: They're not even a real country anyway
Ms. McCormick: My son could've been a doctor or a lawyer rich and true,
Instead he burned up like a piggy on the barbecue
Everyone: Should we blame the matches?
Should we blame the fire?
Or the doctors who allowed him to expire?
Sheila: Heck no!
Everyone: Blame Canada
Blame Canada
Sheila: With all their hockey hullabaloo
Liane: And that bitch Anne Murray too
Everyone: Blame Canada
Shame on Canada
The smut we must stop
The trash we must bash
The Laughter and fun
Must all be undone
We must blame them and cause a fuss
Before somebody thinks of blaming uuuuuuuuuuuuuuuuuus
Of a still, cold night you can hear 'em up there . . . mutterin' . . . and sharpenin' their skates . . .
Sometimes you can even hear Shatner… sharpening his toupee…
testing his phaser again "damn, still doesn't work yet"
serves him right - he shoulda shopped ebay instead of priceline…
What's with all the Canuck bashin? You guys better keep an eye on your "good neighbors" to the north. Pretty soon we are going to load up our battle canoes with highly trained attack beavers. Let's see homeland security prepare for that!
Seriously though, all these replies and no actual answer to the original question.
You need to find a woot god. We are just a bunch of people who hijacked your thread.
The beavers have been conscripted and follow me now.
Mwah ahah ahhahahah haahahahaahha!
(and do you really think they'll ever charge 5 bucks to ship to canuckia?)
And if not, did you ever consider beaver chili?
Why give the cows all the glory?
Leave the beavers be, and stick to the Nutria.
the Nutria thought they could skip on the payments and conscripts - why else did you think we'd make them fine cuisine?
What about some nice NYC rats, I've seen some bigger than nutria. Nutria, aren't they from the south? Not going to be much help in the war with Canada, yeah cook the suckers! Those city rats would fight better anyway.


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51 low budget profitable business ideas for beginners startups in India 2019
Self-owned businesses are for those who don't like to work under a person or may avoid poking of a nose of various unwanted people and also enjoyed by flexible working hours. Now, the question arises that what business ideas may start? And is it worth risk taking?
Company Profile of Amazon – The world's largest online retailer
Find out more about Amazon.com, there Introduction, Background History of Amazon, Funding in Amazon India, Acquisitions made by Amazon, Competition from home-grown e-commerce companies, About the Founder of Amazon (Jeff Bezos)
18 Business ideas to Start in Canada
Check out this article for some amazing businesses to get an additional income. | Business ideas in Canada | Low investment and easy to start business ideas
Top 5 new medical stores to buy medicine online India
We all here are live witness to the new transition technique of buying goods online like clothes, electronics, furniture etc.
Company profile of Flipkart: One of the leading online selling stores
Flipkart is an online marketplace for ecommerce..In this article you find Background History, funding, major competitor and about the founder.
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Check out here some of the interesting ideas to earn money even from sitting at home. | Don't want to work outside or don't have enough time to go out, especially in case of housewives? Adopt ways to earn money from home. | Different Ideas or ways to earn money.
How can one get Big Bazaar franchise?
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Top 10 Retail Companies in India
Retail business referred as an process where consumable goods are sold to people known as consumers for settlement of their needs and wants.
Kirana Store: The small neighborhood shop in India
Kirana store is the small neighborhood shop in India. These kirana stores are also known as corner shops, C- store or convenience shop.
Participate in Amazon Quiz today and win Oppo R17 pro
Amazon has launched Amazon Quiz on it's Indian portal where they are giving away exciting prizes and amazon pay balance to all who participate and answer quiz questions right.

subset: commoncrawl


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Fujitsu could float Amdahl subsidiary
Merger with Siemens puts heat on computer manufacturer.
Fujitsu may offload its Amdahl subsidiary following the merger of its computer operations with Siemens, writes Peter Branton. The two companies last week announced a non-binding agreement to merge most...
More news
Spectre bugs likely to 'haunt us for a long time' as software alone can't fix all of them, warn Google researchers
Spectre vulnerability affects microprocessors able to carry out branch prediction
NCSC believes risks associated with Huawei equipment use in 5G networks are manageable, claims report
Huawei faces pressure over claims its equipment could be used by Chinese government agencies to conduct espionage
Machine-learning techniques often produce misleading or wrong results, researcher warns
Algorithms don't know how to say 'the data is not clear' or 'I don't know'
Apple reportedly planning a 16-inch MacBook Pro
Sixteen-incher would be the biggest MacBook since 17-inch MacBook Pro was binned in 2012
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text: solesupnyc 3 Posted December 12, 2014 (edited) SOLD!! preowned black Visvim deckhand jacket size 2 with gore-windstopper, one of the iconic item from visvim.collar is lamb leather, outer shell 100% cotton, lining 100% polyster.It is used but in excellent condition, worn less than 10 times, comes with original dust bag.The approximate measurement are:shoulder to shoulder = 17''pit to pit = 20''length = 25.5''arm = 28''nyc meetup welcomed. price 550 plus shipped and paypal fee. obo http://cdn.styleforum.net/f/f4/f416a15f_image.jpeg http://cdn.styleforum.net/1/17/17fd210c_image.jpeg http://cdn.styleforum.net/5/5a/5abec6d2_image.jpeg http://cdn.styleforum.net/6/6e/6e1b8b85_image.jpeg http://cdn.styleforum.net/f/f9/f914e6ae_image.jpeg http://cdn.styleforum.net/0/0f/0f042ba8_image.jpeg brand new, bought myself this birthday gift but the fur is a little too much for me. 2012 visvim deckhand size 2, paid $1500. comes with Original tags/extra button included, email me for more details. trying to get back something close to that price. [email protected]The Deckhand is Visvim's interpretation of the classic US navy deck jacket, constructed from a beige Egyptian cotton canvas outer, backed with Gore 'Windstopper' and lined with thick fleece for ultimate warmth and comfort.Made in Japan100% Windstopper CottonDeer Leather Chest DecalsRiri Zip Fastening & Button Up PlacketFur CollarTowel LiningElasticated CuffsVented Front PocketsMEASUREMENTS:Shoulder width 45cm / 18inBody width 50cm / 20inLength 70cm / 28inSleeve length 62cm / 24in price 1200 plus shipping and paypal fee obo best way to reach me is via email or text. I am also on styleforum.net and ebay. https://encrypted-tbn1.gstatic.com/images?q=tbn:ANd9GcSiwzNmS_RdkYewM9Cd3KPeGLsnPqPgM I have the tan one Edited January 16, 2015 by solesupnyc 0 Share this post Link to post Share on other sites


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Guitar Lessons & Classes in University
Find the perfect teacher or instructor in University for private guitar lessons in your home or in their studio.
Popular Guitar Teachers near University
Check out some of our most popular teachers near University including Corona, Rancho Cucamonga, Fontana, Redlands and San Bernardino.
About Milton J.Close
View Full Profile
I've been teaching guitar for over four years. I've previously been a part of two bands during my formative years and recently have been a session guitar player for four upcoming indie rock, alternative, and R&B/Soul albums.
For children and beginners, I use the Mel Bay publication Children's Guitar Chord Book as well as Hal Leonard's Guitar Method series. I also apply basic music theory that includes learning the Circle of Fifths and basic reading of sheet music.
For intermediate guitar players, I utilize the Hal Leonard Guitar Method series to teach Power Chord Formation and Delivery, Fingerpicking, Pentatonic Scales, Reading Tabulature, and tackling some of classic rock and jazz's favorite tunes.
For guitar lessons at my studio, I leverage Guitar Pro to project and display tabs and play-alongs with ease. For in-home lessons, I use the software for select lessons as we begin song selection and play-alongs.
I can't wait to rock out with you as you find your inner ...
About Milton
My name is Milton Carl Jackson II and I teach vocal, guitar, piano, and musical theatre lessons in the Inland Empire area.
I help those young, middle-aged, and not-as-young tap into their own soul through music so that they may help others find theirs as well. I believe that in order to entertain others with your voice or your instrument, one must first be able to entertain oneself - all the while having fun doing it! As a recent graduate of the University of California, Davis with a double major in Music History & Ethnomusicology and African-American & African Studies; a former co-host of the popular hip-hop radio show ATF Radio: All Things Fresh on KDVS 90.3 FM; a former Music Director of The Afterglow of UC Davis; and a recent graduate of American University with a Master's of Education in Business Education
If you're looking for a teacher who will motivate and inspire you not only to learn your instrument of choice, but also how to perform it (whether on the big stage or in your living room for the family) and record it as well, I'm the perfect teacher for you. Learning to sing, play, and act beautifully doesn't have to be difficult -- we'll work on finding your voice or your style, all while having fun along the way!
I teach in-studio, in-home, and online lessons, so we can be flexible with the lesson type and schedule!
This is a perfect chance for you or your child who can't stop singing around the house or has dreams of shredding on the guitar or twinkling on the ivories to learn the fundamentals of music, start something new that keeps them or you focused, and have fun while doing it!
Feel free to contact me at show contact info for any questions and check out my student reviews and qualifications.
I look forward to meeting you and accompanying you or your child on your musical journey!!
· Piano · In home
· Piano · In studio
· Speaking Voice · In studio
Milton J.
Milton J.
About Joao M.Close
View Full Profile
Our lessons will be laid-back, encouraging, and productive. We will work together to help you achieve your goals and gain a well-rounded music education, regardless of age or musical background. Students who study guitar with me will not only learn songs they love to play, but will learn proper technique, sight-reading, music theory, and ear training. I tailor each lesson based on the musical interests and goals of the student. I aim to give the students a solid foundation in technique and theory in order to help the student feel comfortable with the guitar and to make progress at a steady rate.
About Joao
I lived in Brazil, Portugal, Netherlands, Germany, and in Boston before moving to New York. Moving around so much has brought me in contact with all sorts of people and cultures and has allowed me to grow as a musician and a teacher.
My teaching philosophy draws from different traditions: Socratic teaching method, Buddhist techniques of intense focus and self-kindness, Frederich Wieck school of lyrical expression, and the detailed science of Body Mapping.
As a graduate of Berklee College of Music, I place great emphasis on relaxed technique, ear training, and the use of contemporary repertoire along with classical pieces.
My first degree was in English and French linguistics and literature. I later ded
Eventually, I decided it was time to do a rigorous program at one of the best music schools in the world so that I could perform and teach music at a high level. I graduated from Berklee College of Music with degrees in Performance and Film Scoring.
I teach online and normally in my Brooklyn studio. I am currently in Orange County, CA while at University of California - Irvine.
Some of the highlights of my time in New York:
I was awarded an arts grant from the New York Department of Cultural Affairs and Lower Manhattan Cultural Council for my performance and composition work.
I was commissioned by the organization MyFace to develop and implement a program for artistic expression through music, movement, and storytelling for children and adults with facial differences.
I worked as an accompanist and music director for shows and dance classes at Alvin Ailey American Dance Theater.
Whatever your goals are with music, I can help you get there! Get in touch any time to tell me about your goals and aspirations.
· Piano · In home
Our son has studied piano with Joao for the past four years. He started with Joao when he was six and continues to this day. He loves Joao. Joao is a patient, intuitive and adaptive teacher that provides incredible instruction, in any music genre. He honed in on our son's musical interests early on, making learning piano fun and interesting. We often say, Joao is not just teaching our son piano, he is giving him a music education, introducing him to an array of amazing pieces from different genres. He's a rare talent both as a teacher and musician. We feel incredibly lucky to have found him. You will too!
· Piano · In home
· Piano · In home
Joao M.
Joao M.
About Omar G.Close
View Full Profile
I've been teaching music for 15 yrs on guitar, piano, bass, cello and bass. My students have ranged from from 5yrs to 75yrs of age. I have seen many student go on to join orchestras, bands, broadway shows or simply continued their education to a university or college. Being able to read music profeciently has given me the opportunity to teach off many method books but like to use Alfred Method, Mel Bay and Frederick Noad Method books. I teach many styles ranging from classical, jazz & blues, rock, contemporary and pop. I do stress and recommend all my students to learn how to read music and usually by our second or third lesson they will playing simple songs. I am here to help you make music!
About OMAR
*** Lesson Details ***
As a teacher I require all students to set time aside to practice. For without it, it is only wasting your time, your money and my time. I don't expect a total dedication to it but I do want to see some effort put into it. A little practice can go a long way and the more you do it, the faster you will progress and the more you will enjoy it. We will first start off with what you want out of music and the level you are at and go from there. Student can bring their own music either thru tabs, music sheet or just by ear but one thing I do expect is a student to learn how to read music notation. We will go thru the regular warm up exercises, material practiced during the week and any new music for the following week. Correct techniques in how to hold your instrument or voice procedures will be addressed. Music theory will also be part of the lesson as well as ear training. At the end of either 3 to 6 months you will have learned the basics of music and will be playing songs for you or someone else to enjoy. I will make it a total relaxing and enjoyable atmosphere but some dedication has to put on the students' part.
*** Studio Equipment ***
Dedicated music room, guitar both classical and electric, piano, cello, upright bass and electric bass guitar
*** Travel Equipment ***
I provide my own instrument. I can also provide music stand, small amplifier if needed and music material for the first few lesson until student provides for him/her self. Student must have own instrument.
*** Specialties ***
My way of teaching would have to be that they must learn how to read music while learning how to play an instrument.
· Classical Guitar · In studio
· Piano · In home
· Singing · In studio
Omar G.
Omar G.
About Jacob P.Close
View Full Profile
Scales, Chords, Riffs, Tricks, Technique, Music Theory.
About Jacob
Jacob was born and raised in Palm Springs; California. Growing up with parents who had very different musical backgrounds, which led him to want to live in world full of music. At the age 10, he picked up his first guitar and began to take his first piano lessons and a year later then picking up the first pair of drumsticks. He developed a self-taught technique for drum set, while being mentored by his older brother, Sean, (endorsed by Aquarian Drumheads, and former student of LA Music Academy and University of Oregon) traditional jazz artist, Allen Goodman. (Goodman has performed with many famous jazz artists in his during his drumming career and was mentored by Buddy Rich. He wrote the intro to the George of the Jungle cartoon series, and is now playing with many jazz artists at Vickys of Santa Fe in Indian Wells, California
In 2009, he performed Ron Nelsons Danza Capriccio with the Cathedral City High School Symphonic Band and world renowned saxophonist Kenneth Tse at the Walt Disney Concert Hall. That event was followed by a performance tour of the United Kingdom with the Cathedral City High School Symphonic Band.
In 2011, he ventured off to France and Germany with the CCHS symphonic band for his second performance tour with the group. Playing at many venues including, Heidelberg Castle in Germany and Chateau Vaux-le-Vicomte, outside of Paris, France. He has been on national television performing at the Pacific Life Holiday Bowl in 2007 and in the Hollywood Christmas parade in 2010 with the CCHS Marching Band. He has performed with various groups within the community throughout the years.
In 2012, began studies at the Art Institute of California Inland Empire studying Audio Engineering and received his Bachelor's of Science in the subject in December of 2015. During the time of his undergraduate studies, Jacob was working with various local artists at local venues around the Inland Empire and Los Angeles. Eventually joining the band, "Eva and the Vagabonds" in 2015 and worked on the recently released EP as a recording and mastering engineer. With the band he has played at Dreamworks, SOKA university, American Parlor Songbook, and DVAC. Now, Jacob attends Cal State San Bernardino studying for a teaching credential in Career and Technical Education. As well, Jacob now works on American Parlor Songbook on NPR as an audio engineer, working with various actors, comedians, and artists from LA and all of over the United States.
*** Lesson Details ***
Beginner to intermediate for Guitar and Drum Lessons
Professional Style Studio Recording
*** Studio Equipment ***
Fender Stratocastor HSS
Ovation Acoustic Guitars
Epiphone EJ-200 CE
DanElectro NOS '59
Grestch Custom Set 10", 12", 14",20"
Grestch Energy 10' 12', 16', 22'
Tama Snare Artwood All Maple 14"
Sabin SR2 Crash 16"
Sabian Hi-Hats 14"
Sabian Dry Bell Ride 21"
Paiste Rock Crash 14"
Paiste Thin Crash 12" and 14"
Vator Drumsticks
Alesis Multimix8/iO4
PreSonus FireStudio
MXL 604 Pencil Condenser
Blue Spark SSL Condenser
SM 57's
MXL A-55 Kicker Dynamic
Audio Technica ATH-M45 Headphones
M-Audio BX5 Monitars
Alto Professional PA Speakers
M-Audio 49 Venom Synth VA/Midi
Arturia MicroBrute
Logic Pro 9, X
Main Stage 3
Protools 10
Max MSP
Macbook Pro
*** Travel Equipment ***
Epiphone EJ-200 CE
MacBook Pro
*** Specialties ***
Recording Services!
All genres of music!
· Drum · Online
· Acoustic Guitar · In home
· Drum · Online
Jacob P.
Jacob P.
About Joe S.Close
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I have been teaching guitar for almost twenty years and have been playing for almost twenty three. It is by far one of my favorite things in life...and I love passing my knowledge and expertise onto others!
About Joe
I take teaching seriously, but definitely try to make it enjoyable for the student. I studied classical guitar in college, but was playing everything from Van Halen to Jazz to Country & Blues before that. Currently, I'd consider myself a flat out musical mutt...enjoying anything and everything. The love of guitar has also expanded to a multitude of instruments that I record, gig, & teach with.
· Ukulele · In home
· Guitar · In home
· Ukulele · In home
Joe S.
Joe S.
About Arnold M.Close
View Full Profile
Hello I am Arnold, I am a musician who has been involved in music for over 8 years. Ive been playing for churches and doing events where I lead for about 6-8 years. Ive been playing guitar for over 8 years, and have vast knowledge on different techniques as well as musical theory knowledge. I am very passionate about what I do and have patience to teach kids as well as adults. I look forward to working with you and making you become the best you can be.
About Arnold
Arnold M.
Arnold M.
About Keith H.Close
Hello, my name is Keith Harrison. I specialize in Custom Tailored Beginning and Intermediate Acoustic and Electric Guitar Fundamentals to advanced playing skills.
My goal is to help you start making great music and improve your skills quickly in a fun and easy to understand way. Lessons are based on YOUR personal goals and music interests. Learn the basic foundations and fundamentals then we dive right into your favorite songs and styles and focus on what interests you most.
Learn finger chords, bar chords, chord inversions, songs, scales, tricks, tips, licks and riffs, pitch, harmony, ear training, how to solo and improvise all up and down the fretboard and more.
I can also help you with singing, songwriting, recording, music gear and many other aspects of the music business.
Whether your goal is to learn the basics, make a living as a guitar hero, or any level in between, I'm happy to share my music passion, skill and experience to help you get exactly where you want ...
About Keith
Career Musician, guitarist, songwriter, vocalist, bassist, private instructor, recording producer/engineer, and screenwriter.
I have had the pleasure of making my living doing what I love and pursuing my passion as a professional musician for many years and I take great pride in helping my students succeed and fulfill their passions for music.
Credits include multiple songs with national airplay, number one song on LA radio 94.3 COOL FM, Featured song on KROQ's Kevin and Bean Christmas CD, Top seller in two categories on
· Guitar · In studio
· Guitar · In home
· Guitar · In home
Keith H.
Keith H.
About Phoenix K.Close
View Full Profile
I have a huge passion for teaching and playing guitar. I also teach you a wide variety of music such as rock, metal, blues, etc. This helps you not get stuck with one style of playing. I can teach all ages. I'm very patient and I'll always work at your level, so I won't go too fast for you. I'm dependable and always on time. After studying with me you will have a good understanding of music theory, a complete understanding of the instrument, how to write your own music, and how to play multiple different genres of music.
About Phoenix
· Guitar · In home
· Guitar · In home
· · In home
Phoenix K.
Phoenix K.
About Robert W.Close
View Full Profile
Are you interested in learning to play the guitar? I have been playing the guitar for 15 years and I am currently studying music education in order to obtain my teaching credential. In our lessons together, we will cover the basics of guitar playing from scale and chord shapes to melodic lines and improvisation. I am currently accepting guitar students at both the beginner and intermediate level. Each of my students receives their own custom lesson plan that aligns with their interests and personal goals. Thanks so much for checking out my profile and I look forward to working with you!
About Robert
Music is my greatest passion in life and I am so grateful to be able to get up every day and enjoy what I do. To instill that passion in others is what I hope to achieve during my lessons. I make a custom plan for every student based on their previous experience and their personal goals for our time together.
I currently play with both a funk/soul band and reggae band where I showcase my talents on the bass guitar. I have been playing with various bands for 15 years, but I always have room to improve. I hope to instill the idea that learning an instrument is a life long journey
Robert W.
Robert W.
About Raul P.Close
I am able to teach all major notes, minor, and sharps on the guitar. Other skills sets I teach are tremolos, requintos, and all the scales from fret 1 to the 13 fret. I also teach the circle of all major, minor notes. I can also teach you how to utilize the base note on any note, I will also teach how to play a scale at the end of the note, Thank you,
About Raul
Raul P.
Raul P.
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From finding the perfect teacher to managing payments, scheduling and tracking your progress, we take care of all the details, so you can focus on learning. Learn More
Search for a teacher
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When you've found the perfect match, schedule your lessons online using the teacher's real-time availability.
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Yikes! Don't Confuse These 7 Tricky Spanish Words
As a beginner learning Spanish, it's easy to feel overwhelmed by new vocabulary. How do you keep track of all of the Spanish words and meanings? Not to mention all the false cognates that are out there! Fortunately, with practice, Spanish words and phrases will become like second nature to you. Just watch out for the tricky word pairs! For a helpful lesson, our friends at Lingolistic recently shared an article with us with some words that are particularly confusing for beginners. Here are … Read More


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The WROR Street Team is powered by:
Christmas in the City Winter Wonderland Saturday, December 15th, from 11 a.m. to 4 p.m. at the Boston Convention & Exhibition Center
Join the WROR Christmas crew for a Family Skate at Kendal Square Community Skating, 300 Athenaeum St, Cambridge MA Sunday Dec 16th from 2pm to 4pm. We will have games, music and Frosty! For more information, click here.
Bundle up and join the WROR Street team at Hale Family Fun Day on January 12th from 10am-12pm!
Please join Hank at Salvatore's Lawrence at Riverwalk on Thursday January 24th for "A Night of Laughs" to help raise funds for the production of the movie Tough Sledding...the story about the Gold Medal winning 2002 US National Sled Hockey Team that was coached by Bruins great Rick Middleton!


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Employers Punishing Women for Being Too Intelligent
Wait, are women seriously being punished for being too intelligent?
InsideHigherEd.com reports that a new study finds that good grades may not help women become successful after college. It appears that the grades of male graduates does not seem to matter as much, while female graduates may actually be punished for excelling in their studies. This study is a result of female students outperforming male students in both high school and college.
Author Natasha Quadlin did an "audit study" of 2,106 applications for various jobs that looked at the applicants' grades, gender and undergraduate major. Her research will be available in the April issue of "The American Sociological Review." A key finding of this study was that female job applicants were benefited more by only "moderate" academic achievement rather than higher levels. Comparing equally intelligent males and females, men were called to discuss matters further twice as much as women with about the same grades. In STEM, the odds are stacked up even higher against women, with men being favored at a ratio of 3 to 1.
In a related study, it has been found that male and female candidates are evaluated differently by employers. Males are valued for their competence and commitment, while women are valued on "perceived likeability." Since there are stereotypes held about intelligent women, it may make sense that women who only have moderate academic success are receiving more positive job market attention. Real gender equality would require judging all applicants in ways that can be measured consistently.
Are you looking for digital journalism training and experience? Are you a journalism major who wants to take your career to the next level? CMN's Digital Journalism course gives you real-time experience, intense feedback on your writing, exposure to journalism influencers and mentors, and a great place to display your work. You can get academic credit too. Check out the Digital Journalism Course here.
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The Bern is coming back in 2020.
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Dementia friendly screening of Easter Parade (1948)
About this Event
The David Lean Cinema, in partnership with the Croydon Dementia Action Alliance, are organising a series of popular music films throughout 2019. These Dementia Friendly screenings aim to make cinema more accessible by providing a fun and inclusive experience to enable people living with dementia, their families and carers to attend the cinema in a safe and welcoming environment. The lights are left on low, there are no adverts or trailers and the audience is allowed to move around or even sing along to any musical numbers if they'd like to.
Admission is free for people with dementia and their carers. Tea, coffee and biscuits will be available before the screening. The performance will start at 11am, with refreshments available from 10.15.
EASTER PARADE (1948), is a romantic musical starring Judy Garland and Fred Astaire and features songs by Irving Berlin. Fred plays a nightclub performer who hires a chorus girl played by Judy to prove that he can train anyone to be his partner.


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Should an organization that opposes abortion be required to tell women where they can seek an abortion? It's a question the U.S. Supreme Court will face this month as it considers a challenge to a California state law.
The law in question, known as the California Reproductive FACT Act, was signed by Democratic Gov. Jerry Brown in 2015. It requires licensed "crisis pregnancy centers"—many of which are run by Christian groups whose mission is to discourage pregnant women from getting abortions—to post signs or distribute notices informing clients that the state offers subsidized programs for family planning services, including abortion. The notice must include a phone number for the closest government social services office.
The National Institute of Family and Life Advocates, which runs roughly 200 pregnancy centers in California, sued the state over the law. It argues the law infringes on its freedom of speech by compelling the centers "to speak a government message and to promote government programs contrary to their pro-life beliefs."
The centers typically offer pregnancy tests and ultrasounds, and counsel women to carry their pregnancies to term by providing information on adoption and parenting.
"All of their speech is designed to encourage childbirth. The Act purposely hampers this advocacy at the very beginning of a pregnancy center's interaction with expectant mothers. While the centers exist to support childbirth, the Act forces them to point the way to ending unborn babies' lives," the institute says in a court filing. The group did not make anyone available for an interview.


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text: Next Story > Photo Journal Pictures of the Week: May 9 – May 14 By WSJ Staff May 14, 2010 11:02 am ET In this week's photos, the young lone survivor of a plane crash recovers, a solider who served in Afghanistan is buried, boys play video games in Tokyo and more. To Read the Full Story Subscribe Sign In Previous Pictures of the Day Next Pictures of the Day Most Popular Videos The Secret to Why a Tesla Costs So Much (Hint: Batteries) What Are Stock Buybacks and Why Are There Calls to Restrict Them? ATF Agents Must Take Back More Guns Than Ever Before. Here's Why Why Your Apps Can Find You, But 911 Can't How to Avoid Getting Sick at the Office Most Popular Articles 'Long Shot' Review: Triggering a Revolution Apple's Executive Shake-Up Prepares for Life After iPhone Opinion: Ocasio-Cortez Heralds a New Political Era Kim Jong Un Purges Wealthy Elite and Critics of Outreach to U.S. Opinion: The FBI's Trump Panic


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table. Published at Tuesday, July 10th 2018, 11:16:13 AM by tubep.
Keep in mind that design elements with distinct verticality – whether or not they're all that tall in reality – do a great deal to define the space surrounding them. These colorful, stackable drawers provide verticality and the subsequent definition of space but, because of their cut-out design, they maintain a sense of openness.
Because homeowners don't need to worry about stereotypically cold flooring surfaces (think tile, slate, or concrete) with and underfloor heating system, they can choose those materials with very low VOC emissions. Additionally, underfloor heating helps with moisture control and inhibits mold and bacteria growth. Studies have shown underfloor heating to be a benefit to combatting many common household allergens.
Fast-forward to today. In a modern radiant heating system, heat is supplied by electric wires or hot water tubes that are buried under the floor. As the thermal radiation rises up and out of the floor, they warm up everything they touch, which items then radiate heat as well. While the air temperature in an underfloor heating system tends to remain pretty constant, you will feel and stay comfortably warm because the surfaces you touch are warm, which means they won't steal warmth from you.
Let's face it – no one wants to have to work around the architectural constraints of conventional forced-air heating systems, whether they be boiler baseboards, radiators, or even heating vents. One of the best parts about underfloor heating is that it's truly invisible – no evidence that it's there, except for the nice, even blanket of warmth exuding from the floor when you need it.
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Senior Clinical Research Associate
- Company
- X4 Life Sciences
- Location
- Berkshire
- Salary
- 40,000 - 60,000
- Posted
- 05 Apr 2017
- Closes
- 19 Jan 2019
- Ref
- Discipline
- Clinical Research, Clinical Research Associate
- Contract Type
- Permanent
Job Title: Senior Clinical Research Associate
This is an exciting opportunity for a Senior Clinical Research Associate to join a growing CRO based in Berkshire. This great opportunity can offer CRAs the opportunity to take a more of hands on role be the lead role on their trials.
The Company:
My client is a full service CRO, focused on a series of core therapeutic disciplines. They work globally and are seeking a highly capable Senior Clinical Research Associate, preferably with experience working across all phases of trials and multiple therapeutic areas.
Senior Clinical Research Associate Requirements:
- BSc degree in life sciences.
- 5 or more years monitoring in clinical research experience: experience in pharmaceutical or biotechnology companies but preferably in a CRO
- Therapeutic area: Oncology experience is highly recommended. Other TA experience should ideally encompass complex indications and/or rare disease and/or trials in ICU settings.
Why Should You Apply?
This is an exciting opportunity for any experienced Senior Clinical Research Associate that is looking to progress their career with a highly rewarding company offering between £37,000 - £42,000 plus a £5000 car allowance.
If this role isn't what you're looking for don't worry, at X4 Life Sciences we cover all permanent and contract positions with specialist industry sector teams. You can check out all of our roles on our website and sign up for job alerts so you're the first to know about a new opportunity.
About Us
X4 Life Sciences is a specialist recruitment business for the life sciences sector. We are part of the X4 Group, a multi-award-winning group of brands specialising in Life Sciences, Engineering, Construction and Technology, with over 10 years of experience in successfully placing mid to senior level contract and permanent professionals across the globe.
Our network of world class talent sets us apart from the rest and allows us to deliver skilled professionals to our pharmaceutical, biotech and medical device partners quicker than the competition.
Our aim is to help businesses grow powerful teams that deliver against exceptionally high standards and we have partnerships with leading life science companies all over the world and have made placements in over 15 countries across Europe and the US.


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Oakland Blinds
homeware, home deco
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homeware, home deco
Speedy turnaround for beautiful blinds . . .
We at Oakland Blinds in Stevenage are celebrating our 25th anniversary this year and in that time we have built up an enviable reputation for quality, service and good value for money.
We were founded in 1993, by David Jones; the business was then taken over by his son Alan in 2006.
Based at Stevenage Indoor Market (directly behind Wilkinson), our showroom carries hundreds of different samples including vertical, roller, wood, aluminium, roman, venetian, pleated/roof and conservatory blinds. Our showroom is open Wednesday, Thursday, Friday & Saturday from 8.30am - 5.30pm, and you'll also find a vast range of accessories here too, including vertical slats, hangers, weights and chains. All our blinds are made to measure and each enquiry receives a FREE measuring and quotation service.
At Oakland Blinds we are particularly proud of our speedy turnaround times, quotation service and FREE fitting. From the placing of the initial order to job completion, it is usually 2 to 3 weeks. However a week turnaround is possible subject to prior arrangement with our factory. At Oakland Blinds we value your custom so have now introduced a loyalty voucher scheme.
To arrange an appointment call
01438 314263 or 079700 34578
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33 Market Place, Stevenage SG1 1DH
01438 314 263View on Google Maps → Visit Official site →
BrightHouse is the UK's largest weekly payment retailer, selling home furnishing, appliances, and computing
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Children on playground.
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Ingrid M. Scott - Title I Facilitator
This is my 20th year in education. All 20 years have been in the Jasper County School District. I have been a 4th grade teacher, a 5th grade teacher, a 7th grade math teacher, a guided reading teacher, a guidance clerk, a technology coach, an instructional coach, and currently I serve as the Title I School-Wide Curriculum Facilitator.
I am a native of Hardeeville, South Carolina, but currently I live in Gillisonville, South Carolina. I graduated from Chatham Hall High School in Chatham, Virginia. I received an Economics degree from Williams College in Williamstown, Massachusetts. I earned a M.A.T. degree from the College of Charleston in Charleston, South Carolina.


text:
Dimple Golden Selection - Blended Scotch Whisky 40%
Haig Dimple
A gourmand and seductive blend presented in a beautiful faceted bottle.
Composition :
Alcohol: 40%
Origin :
With this product, we recommend :
William Grant & Sons
70cl bottle and its box
34.90 €
Compass Box Whisky
70cL bottle in box
54.20 €
70cl bottle + water...
43.95 €
Douglas Laing Co
70cl bottle in tube
56.10 €
Customers who bought this product also bought :
Destilerias Unidas
38.00 €
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43.00 €
BienManger paniers garnis
Gift crate with 24x5cl
79.00 €
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text:
Thai Duong limousine van for rent to Vinh Long Mekong Delta by Thai Duong Limousine
Ho Chi Minh city to Mekong delta by private van Limousine , taxi is very popular for all tourists, car rental from Ho Chi Minh city to Mekong delta or market on the river ( Floating market ) is one of the best choice for whom loving the real country life along the river side of Mekong delta.
How far from Ho Chi Minh city to Mekong delta? the Mekong delta normail takes over 03 hour to Vinh Long city from Ho Chi Minh and 2.5 hours to drive to Cai be floating market and more than 180 km from Ho Chi Minh city to Can Tho city with 3.5hours in driving.
Rent a private van Limousine Thai Duong from Ho Chi Minh city to transfer for floating market to Mekong delta is very easy and fast by Freeway and no traffic jam on the main road.
Bài viết liên quan
Service includes:
– Gasoline, all toll fees, parking fee, driver expenses.
– 24/7 customer support
– Experienced driver.
If your package tour in Ho Chi Minh City is one day tour to visit Vinh Long city , Let enjoy one day tour with our limousine Thai Duong van rental service and English speaking driver, you will have a great emotion of Mekong delta.
Vinh Long sprawls along the bank of the northernmost branch of the Mekong River. How good from Saigon has made Vinh Long city the most popular destination for day-trippers who hope to catch a taste of authentic Mekong delta life. Here visitors can take a ferry boat ( xuồng ) along the waterways, visit orchards and try the local famous food of Elephant's Ear Fish.
Take Limousine Van Thai Duong to Mekong Delta are suitable all year round but the best time is the monsoon from June to September.
Limousine Thai Duong Van rental rates for HCM City – Vinh Long, Mekong – HCM City:
|Type of car
|Transfer fee (USD/car/day)
|Van Thai Duong Limousine 10 seat
Exclusive of Entrance fee, English speaking tour guide: USD 30/day tour
Service Includes:
• English Speaking Driver
• 24/7 customer service
• New and air conditioned car
• Gasoline, Driver Allowance and Accommodations
• Toll fee and parking fee


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Home > Norfolk > Colonial Place
Colonial Place in Norfolk, va
Colonial Place is a residential neighborhood in Norfolk, Virginia. It is a peninsula bordered by 38th Street on the south and surrounded on three sides by the Lafayette River. Once upon a time dating back to the 1900s, Colonial Place real estate was very high-class. Today, Colonial real estate is a charming area that includes mostly single-family homes and a few apartment buildings, still emanating much grandeur. Many large homes front the water and Mayflower Road arches around the shore of the river. Colonial Place Historic District is listed on the National Register of Historic Places. Colonial Place real estate is a wonderful option for both established, and growing families. You'll find plenty of unique homes that hold a lot of local history.
To take a tour of the Colonial Place real estate market, connect with one of the experts at Chandler Realty. Buying a home isn't always simple, and we'll be there to help you determine what you can afford, which homes best suit your needs, and which properties are situated in an ideal location for your lifestyle.
Colonial Place Properties for Sale - Norfolk, VA
MLS® #10240441 New Listing
511 Connecticut Ave, Norfolk, VA, 23508
3 Bed • 1 Bath • 1,812 Sf
So Charming! This Appealing Home In Historic Colonial Place Will Charm You From The Beautiful Front Porch To The Amazing Fenced-in Backyard! Gleami...
MLS® #10240076 New Listing
503 Massachusetts Ave, Norfolk, VA, 23508
3 Bed • 2 Bath • 1,953 Sf
MLS® #10239781 New Listing
414 Delaware Ave #318, Norfolk, VA, 23508
2 Bed • 2 Bath • 1,025 Sf
Open Floor Plan With Vaulted Cathedral Ceilings. Very Well Maintained Third Floor Unit With Washer, Dryer, Fireplace, Ceramic Floors And More. Gate...
MLS® #10239779 New Listing
507 W 38th St, Norfolk, VA, 23508
4 Bed • 3 Bath • 1,700 Sf
Turn Key Ready, Custom Built Wine Cellar, That Could Be A Bonus Room, Or A 4th Bedroom. Granite, New Roof, New Foundation, New Soft Close Cabinets,...
MLS® #10239701 New Listing
618 Carolina Ave, Norfolk, VA, 23508
4 Bed • 2 Bath • 1,923 Sf
Gorgeous Norfolk Bungalow That Has Been Fully And Tastefully Updated. Enjoy Life From Your Front Porch In A Terrific Neighborhood Or Travel A Few B...
325 W 38th St, Norfolk, VA, 23504
3 Bed • 2 Bath • 1,200 Sf
Beautiful Cape Cod Home Near Odu, Interstate, And Downtown Norfolk Shopping. Great Starter Home With Large Backyard. New Paint, New Carpet, New Lam...
3915 Gosnold Ave, Norfolk, VA, 23508
4 Bed • 3 Bath • 3,426 Sf
Grand Historic "one Of A Kind" Home Lies On A Huge Lot Featuring Loads Of Mature Landscaping. The Detached Garage/carriage House Features A Darling...
515 Mayflower Rd, Norfolk, VA, 23508
4 Bed • 3 Bath • 2,716 Sf
Exquisite Shake/brick 4 Bedroom/ 2.5 Bathroom Home Overlooks The Lafayette River From An Almost Half-acre Beautifully And Professionally Landscaped...
501 Mayflower Rd, Norfolk, VA, 23508
4 Bed • 3 Bath • 3,000 Sf
Absolutely Gorgeous Home With Stunning Views Of The Lafayette River! Featuring Great Layout With Custom Built-ins, Slate Roof, Copper Gutters And ...
511 New York Ave, Norfolk, VA, 23508
4 Bed • 3 Bath • 2,600 Sf
Welcome To The Well-known And Grand "painted Lady" In Historic Colonial Place. This Renowned Home Sits On A High Lot And Offers Beautiful Curb Appe...
3713 Newport Ave, Norfolk, VA, 23508
4 Bed • 3 Bath • 2,200 Sf
Do Not Miss Out On This Colonial Style Home!! All The Charm With Today's Modern Conveniences. Fully Renovated 4 Bedroom 3 Full Bath Home In The He...
619 W 38th St, Norfolk, VA, 23508
5 Bed • 2 Bath • 1,648 Sf
Beautifully Rehabilitated Home In Sought-after Colonial Place! Boasting Five Bedrooms With Additional Space For Home Office, Nursery, Playroom, Add...
417 New Hampshire Ave, Norfolk, VA, 23508
3 Bed • 2 Bath • 1,933 Sf
Charming Brick Home Nestled On A Quiet Street Along Side The River In Colonial Place, Norfolk. 3 Bedrooms (one Downstairs, & A Bonus Office/playroo...
MLS® #10231560 Under Contract
5006 Newport Ave, Norfolk, VA, 23508
3 Bed • 2 Bath • 1,800 Sf
Terrific Tudor Styled Home Well Located In Historic Colonial Place On High Dry Lot. No Flood Insurance Required. Gracious Open Floor Plan. Kitchen ...
715 New Jersey Ave, Norfolk, VA, 23508
4 Bed • 2 Bath • 1,802 Sf
This Beautiful Home Is One Block From Knitting Mill Creek Which Leads To The Lafayette River In A Desirable Sought After Neighborhood Which Also Of...
MLS® #10230875 Under Contract
4204 Colonial Ave, Norfolk, VA, 23508
3,250 Sf
The Sale Is Part Of A 1031 Exchange. Charming 2 Bedroom Plus Sun Room That Could Be 3rd Bedroom Or Office, 1 Bath Duplex (4202/4204 Separate Addres...
MLS® #10227802 Under Contract
433 Rhode Island Ave, Norfolk, VA, 23508
3,400 Sf
Sold As Is! Have Someone Else Cover Your Mortgage With This Charming Duplex Featuring Classic Architecture & Modern Upgrades Nestled Deep W/n Desir...
4806 Mayflower Rd, Norfolk, VA, 23508
4 Bed • 2 Bath • 2,650 Sf
Solid, Well-loved Traditional Home. Shady Wooded Lot Affording Private Enjoyment Of Knitting Mill Creek From The Front Porch. Welcoming Foyer Leads...
MLS® #10225099 Under Contract
549 Mayflower Rd, Norfolk, VA, 23508
5 Bed • 3 Bath • 3,681 Sf
Picture Yourself Sitting On The Second Floor Balcony Sipping Your Morning Coffee And Enjoying River Views! Nestled In Beautiful Colonial Place, Thi...
426 Delaware Ave #227, Norfolk, VA, 23508
2 Bed • 2 Bath • 1,025 Sf
This Is A Deal. Property Always Is Maintained Well. Located In The Section Back Off Of Delaware. Second Level Living. Fresh And Ready For A New Own...
411 W 37th St, Norfolk, VA, 23508
5 Bed • 3 Bath • 2,300 Sf
Welcome Home! This Beautifully Maintained Home Features 2 Bedrooms Downstairs, 3 Bedrooms Upstairs, Living Room, Family Room, And Dining Room. Open...
4715 Gosnold Ave, Norfolk, VA, 23508
5 Bed • 4 Bath • 3,691 Sf
Stunning Grand Brick Colonial In Sought After Colonial Place. Elegant Foyer With Rich Wood Trim & Architectural Elements Around. Updated Kitchen W/...
3714 Newport Ave, Norfolk, VA, 23508
3 Bed • 2 Bath • 1,796 Sf
Easy To Show!this Historical Charm In The Heart Of Colonial Place Is Ready For The Perfect Family. Beginning With The Front Porch To The Deck Out ...
519 Pennsylvania Ave, Norfolk, VA, 23508
5 Bed • 3 Bath • 2,138 Sf
Great Opportunity To Own In A Well-established Neighborhood. Home Has First Floor Bedroom With Bath. Conveniently Located Near Odu And Colley Ave. ...
MLS® #10125324 Under Contract
4108 Colonial Ave, Norfolk, VA, 23508
2 Bed • 2 Bath • 1,994 Sf
Good Bones In Historic Area, Needs Some Tlc. Front Porch, Off Street Parking, Unfinished Basement Used As Sewing Room, Laundry And Storage.
Discover Colonial Place Homes For Sale
Colonial Place neighborhood is full of many one-of-a-kind homes that were built nearly a hundred years ago. Over the years, these homes have undergone careful restoration to ensure its beautiful origins for years to come. Homebuyers attracted to this area appreciate the elegance that is found on each property. Many of the homes have a traditional East Coast feel with brick exteriors, bay windows accented with shutters, pitched roofs, and perfectly pruned hedges and established grounds. You'll also find turn-of-the-century craftsman homes with generous porches, covered sunrooms, original hardwood floorings, elegant carpentry, and high ceilings. Depending on how ambitious you're feeling, some of these homes are an absolute steal when it comes to finding a great deal for a fixer-upper, as the bones of these types of homes have much potential. Some homes have been added to the Colonial real estate market over the last decade to resemble the classic structures of a bygone era. These single-family homes accommodate anywhere from 2 to 5 bedrooms and bathrooms, with floor plans that provide 1,200 to 3,000 square feet.
Homes at the high end of the market are either new builds or date back to the early 1900s. Big, brick, and elegant is what you'll find. Older homes have updated kitchens including new appliances, custom cabinets, and granite countertops. Bathroom renovations include upscale vanities and sinks, tiled shower and tub shower, and fixtures. If you're a growing family, you'll appreciate the pockets of extra space provided in enclosed side porches, oversized privately fenced backyards, and basements that have been fully encapsulated and waterproofed, which can be used for entertainment purposes or extra storages. What truly brings these homes to life is the wood-burning fireplaces that can be found in formal dining areas and bedrooms. These homes are larger and provide 3 to 5 bedrooms and bathrooms, with up to 3,000 square feet of space.
You'll get instant access to our listings and advanced search features that let you target only the best Coastal Virginia real estate.
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Life in Colonial Place
Life in Norfolk boasts a lifestyle that has easy access to an urban waterfront, offering beautiful views and uses. Residents can easily enjoy memorable harbor tours, public festivals, kayak launches, and plenty of interesting shopping. If you're into jogging or biking in the city, the Elizabeth River Trail is a 10.5-mile urban biking and pedestrian trail that tours the waterfront of the city.
Colonial Place is conveniently located close to the Old Dominion University, Monarch Way Shopping Village, Norfolk Naval Station, Colley Avenue shopping, and various cultural attractions. The Colonial Place Historic District encompasses over 1,000 contributing buildings, and sites throughout the residential parts of town. Other Area attractions include the Virginia Zoological Park, the Jeff Robertson Park, The Chrysler Museum of Art, and the Virginia Museum of Contemporary Art.
Schools nearby Colonial Place Homes For Sale:
• Larchmont Elementary School
• Camp Allen Elementary
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Low-intensity pulsed ultrasound promotes chondrogenesis of mesenchymal stem cells via regulation of autophagy
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Low-intensity pulsed ultrasound (LIPUS) can induce mesenchymal stem cell (MSC) differentiation, although the mechanism of its potential effects on chondrogenic differentiation is unknown. Since autophagy is known to regulate the differentiation of MSCs, the aim of our study was to determine whether LIPUS induced chondrogenesis via autophagy regulation.
MSCs were isolated from the rat bone marrow, cultured in either standard or chondrogenic medium, and stimulated with 3 MHz of LIPUS given in 20% on–off cycles, with or without prior addition of an autophagy inhibitor or agonist. Chondrogenesis was evaluated on the basis of aggrecan (AGG) organization and the amount of type II collagen (COL2) and the mRNA expression of AGG, COL2, and SRY-related high mobility group-box gene 9 (SOX9) genes.
LIPUS promoted the chondrogenic differentiation of MSCs, as shown by the changes in the extracellular matrix (ECM) proteins and upregulation of chondrogenic genes, and these effects were respectively augmented and inhibited by the autophagy inhibitor and agonist.
Taken together, these results indicate that LIPUS promotes MSC chondrogenesis by inhibiting autophagy.
KeywordsLow-intensity pulsed ultrasound (LIPUS) Mesenchymal stem cells (MSCs) Chondrogenesis Autophagy
Autologous chondrocyte implantation
Analysis of variance
- BMSCs
Bone marrow-derived mesenchymal stem cells
Bovine serum albumin
Type II collagen
Dulbecco's modified Eagle's medium
Deoxyribonucleic acid
Extracellular matrix
Fetal bovine serum
Fluorescein isothiocyanate
Glyceraldehyde 3-phosphate dehydrogenase
- IgG
Immunoglobulin G
Low-intensity pulsed ultrasound
- MSCs
Mesenchymal stem cells
- mTOR
Mechanistic target of the rapamycin
Phosphate-buffered saline
Ribonucleic acid
Standard deviation
Sodium-dodecyl sulfate polyacrylamide
SRY-related high mobility group-box gene 9
Transforming growth factor
Articular cartilage injury is a common complication of joint diseases like osteoarthritis. Due to its low innervation, poor blood supply, and low chondrocyte proliferation and migration, autologous cartilage repair capacity is very limited and can lead to irreversible joint dysfunction after injury [1].
The current therapeutic strategies of alleviating articular cartilage injury have unsatisfactory clinical outcomes. Although cartilage engineering can be a promising option [2], the poor regenerative capacity of chondrocytes precludes their use as the seeding cells [3].
Mesenchymal stem cells (MSCs) are highly proliferative self-renewing cells with multi-lineage differentiation ability and have become the most promising cell source for cartilage regeneration. In fact, autologous MSCs implanted into regions of defective cartilage differentiated into chondrocytes, indicating their potential for efficient cartilage repair [4].
However, MSCs have an inherently limited capacity of chondrogenesis, which in turn limits the therapeutic efficacy and outcome of MSC transplantation [3, 5, 6, 7]. Differentiation of MSCs into chondrocytes is influenced by the extracellular microenvironment, as well as several growth factors, primarily the transforming growth factor (TGF) [8]. However, little is known regarding the regulatory mechanisms of chondrogenesis.
Autophagy is a catabolic process that enables cells to recycle damaged proteins and organelles to ensure their survival during stress conditions [9, 10]. The autophagy-related genes such as Beclin1 and LC3 are essential for autophagosome formation in the MSCs [11, 12]. Studies show that autophagy plays an important role in MSC differentiation and regulates its therapeutic effects in inflammatory diseases [13, 14]. However, it is unclear whether autophagy also affects chondrogenesis of MSCs.
In recent years, studies have shown that mechanical stimulation affects the differentiation of MSCs [15, 16]. Low-intensity pulsed ultrasound (LIPUS) provides mechanical stimulation in the form of acoustic waves and has been used as an adjuvant physical therapy to treat musculoskeletal injuries. Recent studies show that LIPUS promotes cartilage repair, and stimulation of chondrocytes with LIPUS increases production of extracellular matrix (ECM) proteins like type II collagen (COL2) and aggrecan (AGG) [17, 18, 19]. In addition, LIPUS has been shown to facilitate TGF-β-mediated chondrogenesis of MSCs in vitro [20, 21]. These findings strongly indicate the potential of LIPUS in regenerating damaged cartilage via MSCs.
In this study therefore, we analyzed the effects of autophagy regulation and LIPUS on MSC chondrogenesis and found that LIPUS drives the chondrogenic differentiation of MSCs by inhibiting autophagy.
Material and methods
MSC isolation and culture
Bone marrow-derived MSCs (BMSCs) were isolated from 18 8-week-old male Sprague-Dawley (SD) rats as previously described [22, 23]. The experimental protocol was in accordance with the US National Institutes of Health's Guidelines of Laboratory Animal Use and approved by the Nanjing Medical University Ethics Committee of Nanjing Hospital. Briefly, the bone marrow was flushed out from the femur cavity with low-glucose Dulbecco's modified Eagle's medium (DMEM; KeyGEN, Nanjing, Jiangsu, China) containing 10% fetal bovine serum (FBS; KeyGEN). After centrifuging the BM cell suspension for 10 min at 1000 rpm, the fat and other debris were removed and the remaining cells were washed twice with PBS. The cells were re-suspended in DMEM and cultured in petri dishes at 37 °C under 5% CO2. After reaching 80–90% confluency, the cells were trypsinized and re-seeded at the density of 2 × 106 cells per dish. The MSCs were identified morphologically under a light microscope [13].
Immuno-phenotypic characterization of MSC
The MSCs were obtained from each rat and identified respectively. The primary MSCs were harvested using 0.25% trypsin (KeyGEN), washed twice in PBS, and centrifuged at 400g for 5 min at room temperature. After re-suspending the cells in the staining buffer at the density of 2 × 106/ml, 100-μl aliquots were incubated with FITC-conjugated rabbit anti-mouse CD90 and CD31 (Abcam, Cambridge, MA, USA) and unconjugated anti-CD44 and anti-CD45 (Santa Cruz, Dallas, TX, USA) for 15 min at 4 °C. The cells were washed once with ice-cold staining buffer and re-suspended in the buffer containing FITC-conjugated goat anti-rabbit IgG (Jackson, Philadelphia, Pennsylvania, USA) for 15 min at 4 °C. After washing again with ice-cold PBS containing 2% bovine serum albumin (BSA), the cells were acquired using a flow cytometer (FACS Calibur, BD Biosciences, SanJose, CA, USA). FITC-conjugated mouse IgG1 (R&D systems Inc., Minneapolis, MN, USA) was used as the isotype control for CD90 and CD31, and rabbit polyclonal IgG (Epitomics, Burlingame, CA, USA) for CD44 and CD45. The acquired cells were analyzed using WinMDI 2.8 software (The Scripps Institute, West Lafayette, IN, USA).
Induction of chondrogenic differentiation
The MSCs were differentiated to chondrocytes in a three-dimensional pellet culture system as previously described [20, 24]. Briefly, the second generation MSCs were harvested (approximately 2 × 106 cells) and pelleted by centrifuging at 300g for 5 min. The undisturbed pellet was cultured in chondrogenic medium (KeyGEN)—DMEM containing 10% FBS, 50 units/mL penicillin, 50 mg/mL streptomycin, 0.1 μM hexadecadrol, 0.1 mM Vitamin C, 50 μg/mL ascorbate 2-phosphate, 0.35 mM proline, 1 mM pyruvate, 10 ng/ml TGF-β3, 50 mg/mL ITS Premix, 6.25 μg/mL insulin, 6.25 μg/mL transferrin, 6.25 μg/mL sodium selenate, and 5.35 μg/mL linoleic acid—at 37 °C under 5% CO2. Control MSC pellets were re-suspended in basic medium (DMEM with 10% FBS). The culture medium was changed every 3 days until the pellets were harvested. The MSCs were cultured in chondrogenic medium for 10 days before analyses.
LIPUS stimulation and autophagy agonist and inhibitor treatment
The tubes containing the differentiated MSCs were placed on the transducer (HT2009-1, Ito Corporation, Tokyo, Japan), and LIPUS waves of varying intensities (20 mW/cm2, 30 mW/cm2, 40 mW/cm2, or 50 mW/cm2) were transmitted through the bottom of the tube coated with a coupling agent as previously described [20]. The cells were treated once a day for 10 days at the on–off ratio of 20%, and irradiated with 3 MHz for 20 min in a humidified 37 °C incubator with 5% CO2. To determine the role of autophagy on the chondrogenic effects of LIPUS, the cells were incubated with the autophagy inhibitor 3-methyladenine (3-MA; Selleck, Houston, TX, USA) or agonist rapamycin (Selleck) before the LIPUS stimulation. During the LIPUS stimulation and the autophagy agonist and inhibitor treatment, the medium were changed every 3 days. When the medium were changed, the autophagy agonist and inhibitor were re-added to the medium.
Western blotting
Protein was extracted from the cells using a total protein extraction kit (KeyGEN), and equal amounts of protein (20–25 μg) per sample were loaded into sodium-dodecyl sulfate polyacrylamide (SDS-PA) gels and resolved by electrophoresis. After blotting the proteins onto nitrocellulose membranes, the latter were blocked with skim milk for 2 h at room temperature and incubated overnight with anti-Beclin1 (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-LC3 (1:1500; Novus Biological, Littleton, Colorado, USA), and anti-β-actin (1:1000; Cell Signaling Technology) antibodies at 4 °C. The following day, the blots were washed thrice with Tween-20 in PBS and incubated with peroxidase-conjugated goat anti-mouse secondary antibody (1:5000; Santa Cruz, Dallas, TX, USA) at 37 °C for 2 h. After the final three washes, the membranes were developed by exposure to chemiluminescence reagents (ECL kit; KeyGEN).
Electron microscopy
Harvested cells were washed in ice-cold PBS, fixed with 2% glutaraldehyde (Sigma-Aldrich, St. Louis, MO, USA), and washed twice with PBS. Cells were post-fixed with 1% osmium tetroxide (Sigma-Aldrich), dehydrated, and treated with propylene oxide (Sigma-Aldrich) before being embedded in epoxy resin (Sigma-Aldrich). The blocks were cut into thin sections, stained with lead citrate (Sigma-Aldrich), and observed under the electron microscope (JEM-1011, JEOL, Akishima, Tokyo, Japan).
The MSCs were seeded onto slides and cultured for 10 days. And then the MSCs were fixed with 4% paraformaldehyde for 30 min on ice, washed twice with PBS, and incubated with 3% H2O2-methanol solution at room temperature for 10 min. Micromass pellets were washed twice with PBS, fixed for 24 h in 10% formalin, embedded in paraffin, and cut into 5-μm thick sections. The latter were deparaffinized, rehydrated, and then washed with PBS. After a 5-min incubation with 0.5% Triton X-100 (KeyGEN), the cells/sections were blocked with 10% goat serum in PBS for 30 min and incubated overnight with anti-LC3B antibody (1:200; Novus Biological) at 4 °C. The slides were washed thrice with the blocking solution, incubated with fluorochrome-conjugated secondary antibody, and counterstained with diamidine phenylindole (DAPI; Molecular Probes, Waltham, MA, USA). The LC3 punctae were observed and counted under a confocal microscope (Dmi 6000-B, Leica, Brunswick, Germany) [25, 26].
Immunocytochemistry (ICC)
The pellets were washed twice with PBS, fixed for 24 h in 10% formalin, embedded in paraffin, and cut into 5-μm thick sections. The pellet sections were incubated with 3% H2O2 in methanol at room temperature for 10 min to quench the endogenous peroxidase, washed thrice with PBS, and blocked with goat serum at room temperature for 20 min. The sections were then incubated overnight with anti-COL2 antibody (1:200; Acris, Herford, NRW, Germany) at 4 °C, washed thrice with PBS, and incubated with horseradish peroxidase (HRP)-conjugated anti rabbit secondary antibody (50 μl; Santa Cruz) at 37 °C for 30 min. After washing thrice with PBS, 3,3-diaminobenzidine (DAB) was added for color development, and the sections were counterstained with hematoxylin (KeyGEN). Three slides were observed per condition, and positively stained cells were counted in three randomly selected areas per slide.
Toluidine blue staining
To determine the presence of glycosaminoglycans, the pellet sections were washed thrice with PBS, fixed with 4% paraformaldehyde at room temperature for 20 min, and washed again. The slides were then stained with toluidine blue for 30 min, washed with PBS, and observed under an inverted microscope.
Quantitative real-time (qRT)-PCR
Primer sequences for qRT-PCR
Primer sequences
Statistical analysis
All data were expressed as mean ± standard deviation (SD) of three independent experiments and analyzed using SPSS 23.0 software (IBM Corp, Armonk, NY, USA). Multiple groups were compared by single-factor analysis of variance (ANOVA) and two groups by pair-wise Student's t test. P values < 0.05 were considered statistically significant.
Characterization of MSCs
Effects of autophagy agonist and inhibitor on MSCs
The protein expression of autophagy-related gene Beclin1 and LC3 in MSCs was examined by western-blot analysis after rapamycin or 3-MA treatment for 24 h. Beclin1 expression and ratio of LC3II/LC3I in MSCs significantly increased (p < 0.05) after treatment with 1, 5, or 10 μM rapamycin, with the highest at 10 μM rapamycin (p < 0.05). Beclin1 expression and ratio of LC3II/LC3I in MSCs significantly decreased (p < 0.05) after treatment with 1, 5, or 10 μM 3-MA, with the lowest at 10 μM 3-MA (p < 0.05) (Fig. 1b). The autophagosome formation in MSCs was observed by electron microscopy. The morphometric ultrastructural analyses showed that autophagosomes were increased in rapamycin treatment group compared with the control group and 3-MA treatment group in MSCs (Fig. 1c). Immunofluorescence staining also showed that LC3-positive cells were significantly increased (p < 0.05) in rapamycin treatment group compared with the control group and 3-MA treatment group in MSCs (Fig. 1d).
LIPUS inhibits autophagy and promotes chondrogenesis of MSCs
Autophagy inhibits MSC chondrogenesis
LIPUS stimulates the chondrogenic differentiation of MSCs by inhibiting autophagy
Consistent with results presented before, LIPUS stimulation significantly decreased autophagy in the differentiating MSCs compared to the unstimulated cells in the presence of rapamycin (p < 0.05 for all indices; Fig. 4a–c). However, compared to the 3-MA-treated cells, the degree of autophagy was still significantly higher in the rapamycin-treated cells, even after LIPUS stimulation (Fig. 4a–c). In addition, compared to the unstimulated state, LIPUS stimulation also increased the chondrogenic markers in the differentiating MSCs in the presence of rapamycin. However, LIPUS-mediated chondrogenesis was more pronounced following additional autophagy inhibition by 3-MA compared to that after rapamycin treatment (Fig. 5a-d). Taken together, LIPUS can enhance MSC differentiation into chondrocytes by inhibiting autophagy, although it cannot completely block the autophagic pathway.
The aim of this study was to determine the role of autophagy in chondrogenesis of MSCs and whether LIPUS affects chondrogenesis of MSCs via regulation of autophagy. We found that autophagy activation suppressed chondrogenesis of MSCs, but autophagy inhibition promoted chondrogenesis of MSCs. In addition, we demonstrated that LIPUS promoted chondrogenesis of MSCs via autophagy inhibition.
The lack of regenerative ability in chondrocytes and the absence of nerves and vasculature in cartilage severely limits articular cartilage repair after injury. Autologous chondrocyte implantation (ACI) is a promising therapeutic option for severe cartilage defects and has shown satisfactory clinical outcome [27, 28, 29]. However, the outcome of ACI is limited by insufficient number of chondrocytes, the lack of specific niches within the articular cartilage, and individual variability [30, 31, 32]. MSCs can proliferate extensively ex vivo while maintaining their multipotent differentiation abilities, making them an ideal cell type for cell-based repair strategies [33, 34]. In addition, BMSCs are easily isolated and can differentiate into various lineages, including the chondrocytes, under optimal conditions [35].
The chondrogenic differentiation of MSCs is affected by soluble biological factors, cell-cell interactions, and the local microenvironment [36]. Several studies have demonstrated in vitro chondrogenesis of MSCs in pellet culture or on three-dimensional scaffolds in the presence of TGF-β, accompanied by the synthesis of cartilage-specific matrix proteins [37, 38, 39, 40, 41]. Consistent with these reports, we found that the chondrogenesis-related genes COL2, AGG, and SOX9 were upregulated in MSCs cultured in the chondrogenic medium containing TGF-β and other soluble factors.
Autophagy, a cellular degradation process that provides energy and macromolecular building blocks, is essential for cell survival and differentiation [42] and also plays an important role in the differentiation and self-renewal of stem cells [13, 14, 43]. However, the role of autophagy in the chondrogenesis of MSCs is still poorly understood. We found that the autophagy agonist rapamycin significantly decreased the chondrogenic gene signature such as COL2, AGG, and SOX9 in the differentiating MSCs, whereas the autophagy blocker 3-MA promoted chondrogenesis, indicating an inhibitory role of autophagy in the chondrogenic differentiation of MSCs.
Mechanical stress has also been shown to be an important regulatory factor in MSC differentiation, and mechanical compression such as shear stress induces chondrogenesis in MSCs by upregulating the chondrogenic genes [44, 45]. LIPUS also produces mechanical stress in the form of acoustic waves and enhances the matrix gene expression in mature chondrocytes [17, 19, 46]. Furthermore, our and others' studies have shown that LIPUS augmented TGF-β1-induced chondrogenic differentiation of MSCs [20, 21]. Ebisawa.et al. indicated that pellet culture of MSCs is essential for the induction of chondrocyte differentiation and that TGF both accelerates differentiation and facilitates acquisition of cell machinery to respond to the LIPUS signal [20]. In our previous study, we also indicated that LIPUS promoted TGF-β1-induced chondrogenesis of MSCs, represented by increased expression of COL2, AGG, and SOX9 genes. In addition, as integrins are a type of transmembrane cell surface stress receptor and mechanistic target of the rapamycin (mTOR) plays a key role in autophagy, we found that LIPUS increased the integrin and p-mTOR expression of MSCs. Moreover, the positive effects of LIPUS on chondrogenesis of MSCs were prevented by integrin and mTOR inhibitors. These results suggested us that integrin-mTOR signaling pathway might mediate the LIPUS-induced inhibition of autophagy [21]. In the present study, we found that LIPUS enhanced chondrogenesis by inhibiting autophagy. However, addition of an autophagy agonist (rapamycin) suppressed the pro-chondrogenic effects of LIPUS to some extent, indicating the involvement of other pathways in MSC chondrogenesis. Nevertheless, our findings provide new insights into cell-based articular cartilage regeneration.
In conclusion, autophagy plays a key role in chondrogenesis of MSCs, and LIPUS promotes the chondrogenic differentiation of MSCs via autophagy inhibition. Therefore, we deduce that autophagy plays a key role in the promotional effects of LIPUS on the chondrogenesis of MSCs. Our findings provide the mechanistic basis for cartilage repair using LIPUS and MSCs.
We thank the National Natural Science Foundation of China (No.81501941, 81772437) for financial support.
This work was supported by the National Natural Science Foundation of China (Grant Number 81501941,81772437).
Availability of data and materials
All data generated and/or analyzed during this study are included in this published article.
Authors' contributions
PX and XL conceived and designed the study. TZ and XW performed the experiments. XW wrote the manuscript. QL, KC, and XL reviewed and edited the manuscript. All authors read and approved the final manuscript.
Authors' information
All the authors are researchers at Department of Rehabilitation Medicine, Nanjing First Hospital, Nanjing Medical University, China. Peng Xia and Xueping Li are the leaders of our research group.
Ethics approval and consent to participate
The experimental protocol relating to rats was approved by the Nanjing Medical University Ethics Committee of Nanjing Hospital (20150829).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Grabbed this from Facebook. May we all defend the faith so boldly in the face of such adversity.
----The Courageous Child-Martyr of the Cristero Prayed the Daily Rosary—
Today is the feast day of the martyr, Saint José Luis Sánchez del Río (March 28, 1913 – February 10, 1928). This Saint was a Mexican Cristero who was put to death by government officials because he refused to renounce his Catholic faith. His death was seen as a largely political venture on the part of government officials in their attempt to stamp out dissent and crush religious freedom in the area. He was dubbed "Joselito."
José Luis Sánchez del Río was born on March 28, 1913, in Sahuayo, Michoacán. He attended school in his hometown and later in Guadalajara in Jalisco. When the Cristero War broke out in 1926, his brothers joined the rebel forces, but his mother would not allow him to take part. The rebel general, Prudencio Mendoza, also refused his enlistment. The boy insisted that he wanted the chance to give his life for Jesus Christ and so come to Heaven easily. The general finally relented and allowed José to become the flagbearer of the troop.
The Cristeros nicknamed him Tarcisius, after the early Christian saint, martyred for protecting the Eucharist from desecration.
During heavy fighting on January 25, 1928, Mendoza's horse was killed, and José gave his horse to the general so that the fight could go on. Then he sought cover and fired at the enemy until he ran out of ammunition. The government troops captured the boy and imprisoned him in the sacristy of the local church.
José's martyrdom was witnessed by two childhood friends. It was later reported that José was captured by government forces, who ordered him to renounce his faith in Christ, under threat of death. He refused to apostasize.
To break his resolve, he was made to watch the hanging of another Cristero that they had in custody, but instead José encouraged the man, saying that they would soon meet again in Heaven after death.
In prison, José prayed the Rosary daily and wrote an emotional letter to his mother, saying that he was ready to fulfill the will of God to whom he dedicated himself. His father attempted to raise a ransom to save him, but he was not able to appease the government in time to do so, thus failing to secure the release of his son.
Others recalled the gruesome events that transpired after the government's failure to break José's resolve on the evening of February 10, 1928:
"Consequently they cut the bottom of his feet and obliged him to walk around the town toward the cemetery. They also at times cut him with a machete until he was bleeding from several wounds. He cried and moaned with pain, but he did not give in. At times they stopped him and said, 'If you shout, "Death to Christ the King" we will spare your life'. José would only shout, 'I will never give in. Viva Cristo Rey!'" In front of his parents, he was cruelly executed.
The remains of José Luis Sánchez del Río are enshrined above a side altar in the Church of Saint James the Apostle in Sahuayo, his hometown.