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Meet Alaxis Aesthetic Doctors
Dr. Donald Ng
MBBS (Singapore)
Diploma of Dermatology (University of Cardiff, Wales, UK)
Dr Donald Ng is an experienced physician with great interest in cosmetic medicine and surgery. He is energetic and constantly looking for better and more effective treatments with a network of doctors. He believes in seeking as much training as possible in cosmetic surgery and medicine as he knows clearly that the standard in this field is very high and there are many more experienced doctors then himself. To this endeavor, he has taken formal fellowship training and board examinations in cosmetic medicine and surgery. In addition, he has traveled all over Asia and trained with many experts in the field. He counts himself fortunate to have many mentors in Japan, Korea, Taiwan, and Thailand as these countries represent uniquely asian cosmetic surgery which is distinctly different. He has also trained in liposuction in Belgium, Italy and Canada. He is not a plastic surgeon by training and works under the supervision of his associate, Dr Sundarason. He does not train in any reconstructive work, only cosmetic work.
Motto: The journey to medical mastery is a never ending road. One needs to take it with a humble and receptive mind and heart. In the company of true masters, one cannot but be greatly humbled.
SMC Registered Qualification:
MBBS (Singapore)
Diploma of Dermatology (University of Cardiff, Wales, UK)
For a detailed CV of Dr Donald Ng kindly email email@example.com to request.
Trained with the following experts:
- Dr Zurek Longin – Cosmetic Surgeon, Cosmetic Surgicenter of Sydney, Australia
- Prof Niwat Polnikorn – Dermatologist, Kasemrad Aesthetics Center, Bangkok
- Prof Song – Plastic Surgeon, Shanghai East Hospital, China
- Prof Cuong – Cosmetic Surgeon, Saigon Hospital of Cosmetic Surgery, Vietnam
- Dr Steven Mulholland – Plastic Surgeon, SpaMedica, Toronto, Canada
- Dr Jimmy Chuang – Plastic Surgeon, Wish Plastic Surgery, Taipei, Taiwan
- Dr Park Young Jin – Plastic Surgeon, K Samsung Plastic Surgery, Seoul, Korea
- Dr Kevin Cheng – Plastic Surgeon, Macau
- Prof Nikolay Serdev – Plastic Surgeon, Bulgaria
- Dr Kunihiko Nohira – Plastic Surgeon, Soshundo Plastic Surgery, Sapporo, Japan
- Dr Keizo Fukuta – Plastic Surgeon, Verite Cosmetic Surgery, Ginza, Tokyo, Japan
- Dr Toshitsugu Hirohi – Plastic Surgeon, Ritz Cosmetic Surgery, Tokyo, Japan
- Dr Kenichiro Imagawa – Plastic Surgeon, Yokobi Hair Transplant, Yokohama, Japan
- Dr Tsutomu Mizuno – Cosmetic Surgeon, Anesis Total Beauty Clinic , Nagoya, Japan
- Dr Yukio Shirakabe – Plastic Surgeon, Sapho Clinic, Roppongi , Tokyo, Japan
- Dr Kao Chuan-Hsiang – Facial Plastic Surgeon, Tri-service General Hospital, Taiwan
- Dr Damkerng Pathomvanich – Hair Tranplant Surgeon, Bangkok, Thailand
- Dr Kwi-Ho Park – Facial Cosmetic Surgeon, Cohen Rhinoplasty, Seoul, Korea
- Dr Kotaro Yoshimura – Plastic Surgeon, Professor in Tokyo Medical University, Japan.
Appointed as Clinical Advisor in Cosmetic Surgery to Kasemrad Aesthetic Center, Kasemrad Hospital, Bangkok, Thailand
Appointed as International Clinical Trainer in Cosmetic Surgery to Chiva-Som International Health Resort, Niranlada Clinic.
Speaker History:
- 1st and 2nd Advance Aesthetic Workshop in Bangkok organized by Prof Niwat Polnikorn , Clinical Prof of Dermatology (Jun 2009, Jan 2010).
- 53rd World Congress of the International Academy of Cosmetic Surgery in Seoul (Nov 2009) organized by the Korean Academy of Cosemetic Surgery : Threadlift for Face
- IMCAS Singapore Jul 2011 : Bodytite RF assisted Liposuction and TiteFx non-invasive fat killer.
- Taiwan Academy of Facial Plastic and Reconstructive Surgery Congress Oct 7-9 2011 Taipei. Lower face sliming procedures.
- Cutting Edge SICAAAMS 2012 Geneva Apr 2012: Lower Face Slimming, Melasma Treatment, Combination Liposuction, Periorbital Rejuvenation with Fillers, HA for Cheek Augmentation.
- The 100th Congress of Japan Society of Aesthetic Surgery, Tokyo May 2012 : Lower Face Slimming.
- IMCAS Shanghai July 2012 : Fat Transfer for Breast Augmentation, Lower Face Slimming, Melasma Treatment with Laser
- The 8th Annual Scientific Congress of Ho Chi Minh Society of Plastic & Aesthetic Surgery. August 2012 : Lower Face Contouring
- 13th World Congress of Asian Pacific Academy of Cosmetic Surgery: Shenzhen China, Oct 2012: Fat Transfer to Breast, Facial Slimming.
- Cosmetex 2013 Melbourne Apr 2013 : Fat Transfer to Breast, Facial Contouring with RF Masseter Muscle Ablation, Facial Contouring in the Asian Patient.
- 14th APACS Oct 2013 : World Congress on "Comprehensive Facial Enhancement: How the Masters Do It" : Treatment of Melasma. Facial Sliming.
- 43rd PSCS annual convention Sep 2014 : Fat transfer to Breast, Facial Contouring with RF masseter muscle ablation, Treatment of Melasma.
- American Board Hair Restoration Exams 2014, Held in KL, Malaysia. Participated as Examiner for oral Examination.
Dr. R Sundarason (Associate Plastic Surgeon)
Accredited Specialist Plastic Surgeon (Ministry of Health) M.B.B.S. (Melb.), F.R.C.S. (England), F.R.A.C.S. (Plastic Surgery) (Aus.), F.A.M.S. (Plastic Surgery)
QUALIFICATION/PROFESSIONAL: Bachelor of Medicine, Bachelor of Surgery (Melbourne) Fellow of Royal College of Surgeons (England) Fellow of Royal College of Surgeons (Glasgow) Fellow of Royal Australasian College of Surgeons (Plastic Surgery) Fellow of Academy of Medicine Singapore (Plastic Surgery)
Dr. R. Sundarason received his training both in surgery and plastic surgery. He held a post of Senior Lecturer in General Surgery at the Department of Surgery in the University of Singapore.He was appointed part time visiting consultant in plastic surgery by the Ministry of Health until 1992. Dr. R. Sundarason was also appointed to the Board of Examiners of the Royal Australasian College of Surgeons.Dr. R. Sundarason is accredited as a specialist plastic surgery by Ministry Of Health. He was one of the founding members of the Singapore Association of Plastic Surgeon, and the Singapore Society of Cosmetic (Aesthetic Surgery.) Dr. R. Sundarason is very experienced and highly qualified. He also treated both acute and post burnt deformity cases including Plastic and Reconstructive Surgery of Leprosy patients. His graduate and postgraduate trainings were completed in Australia, United Kingdom and the USA. He is a Fellow of 3 Royal Collages of Surgeons (England, Glasgow and Australia) and the Fellow of Academy of Medicine (Singapore) in plastic surgery. He was also awarded the Fellowship of the American College of Surgeons (F.A.C.S.). Dr Sundarason has extensive experience in cosmetic and hair restoration surgeries.
We adhere to the standards set by MOH closely and all cosmetic surgical procedures are performed in accordance to its standards. Dr Sundarason performs all cosmetic surgeries that requires a consultant plastic surgeon.


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Coach Karen is a 4 time Boston-qualified runner who has over 30 years of middle and long distance experience at the high school, collegiate, post-collegiate and club levels (with the Atlanta Track Club competitive team, Phiddipides Running Team and Rain Country Runners).
While still passionate about running in local 5K, 10K or half-marathons, Karen has spent the past few years passing her love of running on to her very active grade-school boys who seem to want to run everywhere!
She has participated in running camps and clinics throughout her career and is currently completing her coaching certification through FitTour to help runners of all ages and abilities realize their fitness goals.

subset: commoncrawl


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[Plugin: Comprehensive Google Map Plugin] Would you consider custom markers with letters?
Great plugin, Alexander! Really enjoy it's flexibility.
I've got a marker question for you…
Would you consider adding custom markers with letters ("A", "B", …)? This would be useful so that I can assign each unique address a different [letter] marker? (I have a need to list corresponding locations on the side of the map.)
I know this is very similar to what Google does when multiple addresses are returned from a search. Each unique address on the left side of the Google map is assigned a letter that corresponds to a location on the map with the same letter.
I could place my own lettered markers into the
/comprehensive-google-map-plugin/assets/css/images/markers folder, but I thought others might want the benefit of having these markers also.
For a marker example, here is a sprite I believe Google is using for it's lettered markers:
Thanks for such great work!
- The topic '[Plugin: Comprehensive Google Map Plugin] Would you consider custom markers with letters?' is closed to new replies.


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Our little, coastal "Snow Show" - although a rarity, lasted about 24 hours and produced about 2" of snow. It was just enough to make everything beautiful and pristine for a day - and before it started melting. This is the front of our home a few hours after it really started snowing. As soon as the first flake fell, Cole wanted to go out and play. :-) Here he is sporting his new snow hat. Our back deck...excuse the dead palms. BIIIILLLLL! The dead palms are still out there! ... READ MORE
(UPDATE!)I Am Very Excited to Announce a New Weekly Event…
WHAT'S ON YOUR WALL WEDNESDAY UPDATE! I apologize I was not clear, What's On Your Wednesday will begin next Wednesday, so I can get the word out. See you then! Whenever I enter a home for the first time, for some reason I am drawn to a homeowner's display of art and family pictures, or whatever they happen to have displayed. To me, the art on someones wall is an echo of the homeowner's personality and taste. So what do you have on your walls? Please join me every Wednesday and share ... READ MORE
My Bermuda Journey…
I started a post about our cruise to Bermuda several days ago, but never completed it when we all got sick. I finished it tonight, but when it posted it showed up several posts down below. (?) hmmmmm.... Anyhoo - here is the link. Please join me for a "warm" journey thru Bermuda. http://gritsglamour.blogspot.com/2009/01/sending-you-warm-thoughts.html No Bermuda shorts required! ... READ MORE
Before And After…Unsightly Kitchen Soffits
Sometimes - Less IS More! Welcome to my very first Met Monday post sponsored by Susan @ Between Naps on the Porch. You can visit her blog, not only for some wonderful decorating ideas, but also to see all the bloggers who are following Met Monday. ( http://betweennapsontheporch.blogspot.com/ ) What is Met Monday? A showcase of Before and After Projects. Who doesn't love Before and After pics?! So let's get going... A few posts ago, I shared one of my on-going kitchen projects - dealing with ... READ MORE
The Making of a Mantle
(this design article was taken from my website, The Everyday Decorator. http://www.the-everyday-decorator.com ) Homeowners often agonize over finding what they consider the perfect print, or the perfect vase, or the perfect mirror. Most of the time, from my experience, its not always finding the "perfect objects of display" - rather displaying the objects in a perfect way that better suits the space. It would be so much easier if I could list a set of rules for which to strictly follow. ... READ MORE
My newest eBay purchased arrived today!
So it really is true! Good things do come in small packages. In the middle of showing homes to clients, running home to eat chicken soup because I still feel like poo-poo, taking some Sudafed Cold (non drowsy my patootie!)...a small package arrived. I knew what it was instantly. I was sooooo excited. AND....I had the perfect spot for it.I have this minuscule plate stand. There it sat....all alone...with nothing to display of its own. I mean - what does one put on such a small plate stand? ... READ MORE
My Nobe is All Stopped Up!
Hey guys! I'll try not to breathe on anyone, and cover my mouth when.....I......sn-....sneeze! Achoo! We are all sick with the flu - so it will be a few days before I post. My kitchen projects are on hold. I am bummed because I was making such progress. Anyway - look for pics next week. If you are one of the three winners, please email me at [email protected] and give me your mailing address. I am excited to see my newest follower....Angelo Surmelis from HGTV's Rate My ... READ MORE
Sending You Warm Thoughts….
Last May, Bill and I decided on the spur of the moment to take a cruise. My busy season in real estate was just around the corner, and I needed something to rejuvenate me before the "crazy" began. We joined a website a few years ago which sends notices of deals on last minute cruises. We had come close several times to going on several cruises, but something always popped at the last minute. This time - all the stars were aligned. We were able to get a 7-day Bermuda cruise leaving from ... READ MORE
And the Winner's Are:
Cole just pulled three lucky names from the basket. He wanted to pull more...so I'm sorry I don't have another gift. LOL Congrats to the following winners: Southern Living "All Recipes" Recipe Book: Cami @ Creating Myself Pair of Red Southern Living Wall Shelves Alisa @ Carolina Panache Single Southern Living Wall Shelf Mishelle @ The Crowned Casa ( I promise the promised bribe had NOTHING to do with it!) :-) I will be notifying each of you, but if you see this before I ... READ MORE
Hurry, Hurry While There's Still Time…
(see a few posts below for more details on give-aways) Last chance to get your name into the pots for my give-aways!! Tomorrow afternoon, Price Waterhouse will select three names....just kidding. Cole will draw three names from a hat. He is accepting bribes of animal crackers and/or frozen fruit pops. :-) If you have not already done so, add your name to the list and specify a certain item (or both!) you would like to be considered for. Good luck......! Happy Winning.....Barb ... READ MORE
Are You a "House" Whisperer?
When Do You Know When Something DOESN'T Work ? This was a question asked on a message board recently. Do you know when something isn't right? My answer was very simply - yes. And when its not right, it just won't do. I know you have heard of the Horse Whisperer ....I am the House Whisperer. LOL My home speaks to me and tells me when something doesn't "feel" right. When the House ain't happy - Barb's not happy...or something like that. Case in point my current project...or maybe I should ... READ MORE
I've Been Tagged!
Tag - Your It! My friend KD @ Southern Whimsey (http://www.southernwhimsey.blogspot.com/) tagged me for a game of Picture Tag. Sounds like fun, so I'll play along. The object of picture tag is to:1) Choose the 4th folder where you store your pictures on your computer.2) Select the 4th picture in the folder.3) Explain the picture.4) Tag 4 people to do the same.No Cheating! (cropping, editing, etc) It! So here goes: File #1: Amanda File #2: Amanda and Blake File #3: ... READ MORE
New Year's Poll Results…
Why am I not surprised? "Keeping My Sanity" seem to outrank any other goal for 2009! :-) You can see the results of this poll above. Thanks to everyone who participated...it was fun! Happy Sanity Keeping...Barb ... READ MORE
Pay It Forward Friday…
PAY IT FORWARD FRIDAY! I have had such a wonderful time creating this blog and meeting and talking with so many great people - be they other bloggers or those who simply enjoy visiting blogs. I am grateful to each of you for all of your support in my first few months of blogging. That said, I would like to hold a give away. I am calling it my Pay It Forward Friday! I am giving away two different items. The first item is a pair of Southern Living @ Home Wall Sconces. They are red metal ... READ MORE
Michelangelo's David American Tour…
I will never forget my excitement whenever we were traveling to various European cities, and had plans to see some famous works of art. For example, on our first trip to Paris we went straight to the Louvre to see Mona Lisa. Very disappointing by the way, but that's another post! :-) On our tour through Italy, I was equally excited to be able to see David. Now David - he was every bit wonderful as I imagined he would be - in all his "splendor." *wink* I received this email today - and ... READ MORE
Oils and Vinegars and Oh My!
It's nearing midnight here in the South, so you know what that means? Barb is in one of her "moods" to decorate and re-design. As part of my on-going "In-Home Shopping" excursion, and in conjunction with my After-Holiday Clean-Up, I decided to try a few new things on my kitchen countertops. Because I love to cook, I like to keep my oils and vinegars and such at close hand. They sit right next to my cooktop. I have olive oil, canola oil, red wine vinegar, balsamic vinegar, a pepper mill, ... READ MORE
Another New Award….and Some GREAT NEWS!!!
Wow! Another great award! This one comes from Kathy @ EmptyNester. http://mbkatc230-emptynester.blogspot.com/ Have you been to Kathy's website? No? Then, you really must. She nominated myself and nine other special blog friends to receive this award, and I must say I am honored to be placed amongst this special group. The purpose of this award is to give it to someone who is showing gratitude or a good attitude ("if life hands you lemons, make lemonade") within their life or blog. If you are ... READ MORE
A Gift of Time…
Welcome to My Very First Tablescape Tuesday...sponsored by Susan @ Between Naps on the Porch. Visit her blog to get a full list of all Tablescape Tuesday participants. http://betweennapsontheporch.blogspot.com My tablescape actually began yesterday morning. No, I didn't start pulling out dishes and such...the day that lead up to my tablescape started yesterday morning. It was my first day back at the office after the Holidays. I'm sure lots of you shared the "ugh!" moment with me at the ... READ MORE
What a Wonderful Gift From My Husband!
My husband gave me an awesome gift tonight - absolutely the most thoughtful thing he's given me in a while, and it came directly from the heart. What is it you say? Well, I will share it all tomorrow. I am participating in Susan's Tablescape Tuesday @ Between Naps on the Porch. http://betweennapsontheporch.blogspot.com/ It's my very first tablescape, and I am very excited to join along. So what does my husband's gift and Tablescape Tuesday have in common? More than you know! LOL Until ... READ MORE
A Very Special Award…
Kathy at Empty Nester ( mbkatc230-emptynester.blogspot.com/ ) has given me this very special award. It means a lot because of its significance. "Blogs who receive this award are 'exceedingly charming' say its authors.This blog invests and believes in the PROXIMITY-nearness in space, time and relationships. These kind bloggers aim to find and be friends. They are not interested in prizes or self-aggrandizement. Our hope is that when the ribbons of these prizes are cut, even more friendships are ... READ MORE


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It is included on the Kyoukai no Kanata Original Soundtrack "Beyond the Melodies".
In the anime series, the song was supposed to be the trump card against a particular fruit-type yomu whose weakness is the presence of cute girls and dance and song. According to Hiroomi Nase, it was the same song used in the most recent encounter with this yomu, and was successful in taking the yomu's attention enough for someone to eliminate the creature.
The song's lyrics contain inspirational messages related to friendship and facing the hardships of life, accompanied with a combination of guitars, electronic music and percussion common to Japanese pop music.
Romaji LyricsEdit
Tenohira ni tsukanda yakusoku wa
Sora no kanata ni
Kimi to tomoni nagashita namida
Kiseki wa guuzen janai doryoku no keshou de
Motto mae e susumou akiramenai kokoro
Wasurenai yo egao donna tsurai michi mo
Kujike sonna hitori no yoru wa
Nakama wa kakegae no nai bokura no seishun
Zutto mae he susumou hikari no hate made mo
Itsumo kimi ga soba ni iru
Motto mae e susumou akiramenai kokoro
Wasurenai yo egao donna tsurai michi mo
Tenohira ni tsukanda yakusoku wa
English Translation*Edit
We hold in our hands a promise
A treasure that won't ever disappear
The tears we shed together
Miracles don't happen by chance
Let's keep on pushing forward
I won't forget to smile
Let's keep on pushing forward
I won't forget to smile
*English translation incomplete from lines 21 to 33


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111 Gibson Avenue, Rutland, Vermont
$119,900Contract Pending
$119,900Contract Pending
111 Gibson Avenue, Rutland, Vermont 05701 - Beautiful Victorian home lovingly restored!! Meticulously maintained with many original details - stained glass windows, detailed woodwork & wood flooring! Wonderful spacious floor plan with charming wrap-around porch - perfect for relaxing after a hard day! This home is being used as a single family home, but can be easily returned to a two-family home with income potential! The home features Stainless Steel Appliances, New Snow Pans installed in 2016 on the Roof, New Relined Chimney, New Porch Roof, Slate Roof Inspected and Repaired Yearly, New Gutters, New Hot Water Tank (fall of 2016), Brand New Oil Tank to be installed July 2017, Freshly Painted Interiors, Replaced all old wiring, Brand New Garden Time Shed and superb garden area!! This home really does have it all!!
View Virtual Tour
Listing Courtesy of RE/MAX Four Seasons


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Words containing beidenfle
Found 1 words containing beidenfle. Browse our Scrabble Word Finder, Words With Friends cheat dictionary, and WordHub word solver to find words that contain beidenfle. Or use our Unscramble word solver to find your best possible play! Related: Words that start with beidenfle
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I'm looking for opinions on whether to run both rear beadlocks or just one. Mostly I'm just woods riding on varied terrain with roots and rocks and am of average ability and weigh about 200 pounds. Maybe it is just me but changing the rear by hand with both locks is a real b***h! If you use 1, what do you put in the hole?
Beadlocks on WR 400
When I buy a tire from a dealer I have them install it for free or I don't buy it there.Most dealers will if you remove the wheel. It sounds like the terain your riding requires low tire presure and the 400 has lots of power
to cause the tire to slip and flats may ocure at the valve stem.
I had a post on this a while back. There were a lot of opinions. If you do a user name search for jchantzwr400f in the wr forum, you will see a post for rim locks. It was earlier this summer, so go back a couple of months and you'll see it.
It depends on how hard you ride if you need the 2. If your on the gas all the time and your only using 1 you have a greater chance you could spin the tire. If your just casually trail riding you should be fine with one.
There are little rubber plugs to use in the hole but I'm not sure where to get them. I know hardware stores sell items like that.
Somebody wanna offer up a theory as to why the YZ only has 1 rimlock and the WR has 2?
That was one of my questions. In fact, the DRZ's only have one. Don't know about XR's or KTM's.
Run two!
I've always drilled an extra hole on any bike I've had. If you get a flat, you are more likely to make it back to the truck with two. Helps to better balance the tire as well.
And buy two nice tire irons and learn to change the tire yourself!
Don't pay that dealer to do something that simple.
It is a rewarding task!
Been doing it since I was 14!
Matter of necessity!
Somebody wanna offer up a theory as to why the YZ only has 1 rimlock and the WR has 2?
Because the trip to the truck off the track is much shorter than if you are way out in the khack?
That is my guess.
Somebody wanna offer up a theory as to why the YZ only has 1 rimlock and the WR has 2?
Prob'ly because you have a better chance of riding the WR on a flat tire...
Here's my theory, a YZ has a 19" wheel, and "supposedly" an 18" wheel with a taller sidewall will have more traction, so the tire would slip easier than on a 19" wheel. I also say I have to agree with the whole "balance" thing too. I ice race all winter, and have had tires slip with only one beadlock (01 KX250), BUT there is an awful lot of traction in those 1000 screws in that back tire!!
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UW Bothell held its first Business Diversity Program Small Business Fair on Sept. 5. The University encourages local businesses — especially those owned by women, minorities and veterans — to serve as vendors for its office supplies, catering, awards, flowers, photography and other goods and services.
The School of Interdisciplinary Arts & Sciences internship course BIS 495 Worlds of Work allows students to apply their skills in a professional environment where they can look at a possible career. Meet some of the students in the summer class.
UW Bothell graduate Cruz Garibay, who grew up in an immigrant family in Yakima and was the 2015 UW Homecoming King, won a 2018 Fulbright grant to Brazil. The fifth UW Bothell recipient of a U.S. student Fulbright award, Garibay will work as an English teaching assistant at a university.
Research led by Jesse Zaneveld, an assistant professor of biological sciences at UW Bothell, found that the bacterial communities that live inside each of our guts react very differently from person to person under stress. The researchers published a perspective piece in Nature Microbiology outlining the microbial version of the Anna Karenina principle.
Butch de Castro, associate professor in the UW Bothell School of Nursing and Health Studies, is moving to the UW School of Nursing in Seattle as the school's associate dean for diversity, equity and inclusion.
Office of Advancement and External Relations:
19128 112th Ave NE, Suite 102
Bothell, WA 98011
Laura Mansfield, Director of Digital Communications
425-352-3642 (desk)
After hours: 425-324-1100
Our communications team works to uncover and tell UW Bothell's best stories that support our mission and brand.
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text:
First Published on www.independent.co.uk on November 21st 2016.
MPs call for legalisation of cannabis amid warning UK is falling behind in its drug policies
Cannabis is the most widely used illegal drug in the UK, with 6.7 per cent of adults aged 16 to 59 using it in the past year
The UK should follow in California's footsteps and legalise cannabis, according to a new report into domestic drugs policy.
If cannabis were made legal and regulated by the Government, taxation from sales and savings on criminal justice costs could net the Treasury up to £1bn, claimed the report, which has been backed by MPs from all the major political parties.
Cannabis is the most widely used illegal drug in the UK, with 6.7 per cent of adults aged between 16 to 59 using it in the past year, according to the Home Office.
The report from liberal think-tank the Adam Smith Institute and Volteface accused the Government of failing in its current cannabis policies, which it called an "embarrassment".
"The Government strategy is based around three main pillars: reducing demand, restricting supply and building recovery. All three are failing," it said.
Instead, it proposes a regulated form of legalisation which it claims would ensure quality and purity and reduce crime.
Former deputy prime minister Nick Clegg and former health minister Norman Lamb joined Labour and Tory figures to argue that the UK should follow the lead of the United States, where recreational cannabis use is already legal in a number of states including Washington and Colorado.
Four further states, including California, voted to legalise marijuana in this month's elections, while Germany is preparing to legalise cannabis for medical purposes and Canada makes plans for all-out decriminalisation.
The Netherlands effectively decriminalised cannabis decades ago while Portugal legalised it in 2001.
Mr Clegg said UK ministers should start planning for the possibility of a legal market for the drug so as not to fall behind other nations.
"British politicians need to open their eyes to what is happening in the rest of the world," he said. "Cannabis prohibition is being swept away on a tide of popular opinion and replaced with responsible legal regulation.
"Now is the time for ministers to start writing the rules for this legal market, including age limits and health warnings, so that we can finally take back control from the criminal gangs."
Mr Clegg also wrote a letter to the British Medical Journal last week in which he claimed cannabis is "much safer" than many other medicines in use and said continuing to criminalise it was "absurd".
The new report referred to the "last major campaign to legalise cannabis in this country" over six months by the Independent on Sunday in 1997, which culminated in a protest in central London attended by tens of thousands.
However, it said the march organised by the newspaper was a "high-water mark of the campaign rather than a stepping stone in the stream of progress towards regulation".
The report argued that Britain's "dark ages" drugs policy has failed to stop drugs being manufactured and used, and to stop associated crime, corruption and killing.
It called for "root and branch" reform to legalise and regulate cannabis to ensure it meets acceptable standards, and to remove the market for criminal gangs.


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text:
In response to an attack, a character has two tools to prevent damage: a reflexive action or the free dodge.
Reflexive Actions
Reflexive actions can be used for a variety of defensive actions, and are not limited to those listed. A full treatment on actions in combat is available which delineates guidelines for use, but a reflexive action may be used creatively so long as the player wishing to do so has their narrator's approval. None the less, some common themes among reflexive action defense include:
By rolling fast-twitch musculature minus manipulation against the attacker's attack roll in a roll-off and winning, a successful block puts a limb or held object between the damage source and the target. If the blocker uses a weapon or shield to block, no damage is dealt on a successful block, though the object in question could potentially be destroyed if something like a drill-hammer is the source of danger. Blocking can not be used to negate bullet damage.
By rolling motor skills minus manipulation against the attacker's attack roll and winning, a successful parry negates attack damage using a graceful deflection with one's weapon. Parrying can not be used to negate bullet damage, but can be used to knock a gun's barrel aside if within 5 feet of the shooter. Parrying a bladed weapon with one's hand causes one damage to the hand.
The easiest way to avoid damage from an attack is to not be there when the attack lands. By rolling fast-twitch musculature for a leap or motor skills for a graceful turn against the attacker's attack roll in a roll-off and winning, damage is negated. Dodging can not be used to negate bullet damage unless the character has fourth-tier reflex training or is within 5 feet of the shooter. Also keep in mind that a dodge requires movement, and so a character cannot attempt a dodge without specifying where he will end up. If cornered, a dodge is not possible without first removing impediments to movement.
Apply two armor, but must be used as a reflexive action to do so. This reflexive action may be used in response to seeing an enemy draw a gun, though it may not be used in response to a drawn gun being swung to face the character without fourth-tier reflex training except at range of 5 feet or less from the shooter. Holding a shield at head level will also prevent firearms from dealing triple damage from shots to the head. Once positioned, the armor from the shield applies to all attacks originating from the 90 degree arc in front of the shield, with or without further reflexive actions to reposition the shield, unless an action is taken to negate the shield's effect by pushing it aside or otherwise removing it from the line of sight to the target.
Free Dodge
Each round each player has one free dodge in addition to their one reflexive action. The free dodge may be used either as another dodge against a second source of damage than the reflexive action, or to re-roll the dodge check once to see if you can roll out of the way or fall prone. The free dodge is a move of desperation, and leaves the opponent without any further means of defense.
When the reflexive action and free dodge have been exhausted, or should a character choose to not defend against a given attack, a success on the attack roll by the attacker deals damage. This represents a solid hit, and is likely to leave a mark. When in a brawl with many opponents, it is often important to prioritize which targets most need a parry or dodge and saving defenses when necessary.


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Listing information
- Beds: 4
- Total Rooms: 14
- Parking Type Description: Inside Entry 3 cars
- Total Square Footage: 3,136 ($133.93 per sq. foot)
- Lot Area: 0.52
- County: Lake
- Style: Colonial
- Baths: 4 (Full: 3 3/4: 0 1/2: 1 Other: 0)
- Stories: 2
- New Construction: No
- Above Ground Sq. Feet: 3,136
- Estimated Annual Taxes: $7,374
- School District: Riverside Lsd
- Year Built: 1989
- Status: Sale Pending
- Fireplaces: 1
- Foreclosure: No
- Below Ground Sq. Feet: 1,144
- Association Fee: $250
- Subdivision: Hunt Club
Amenities Golf Available
Appliances Equipment Audio System, Central Vacuum, CO Detector, Countertop Range, Dishwasher, Garbage Disposal, Humidifier, Microwave, Oven, Refrigerator, Security System, Smoke Detector, Sump Pump
Available Financing Cash, Conventional, FHA/VA
Basement Detail Finished, Walk-Out
Basement Y/N Yes
Cooling Type Central Air
Driveway Paved
Dwelling Type Detached
Exterior Other
Exterior Features Deck, Patio, Sprinkler/Irrigation
Fixer Upper No
Garage Features Access from Unit, Attached, Door Opener, Electric, Water Available
Heating Fuel Gas
Heating Type Forced Air
HOA Fee Includes Association Insurance, Reserve Fund
HOA Y/N Yes
House Faces East
Irregular Lot Y/N Yes
Lot Description Cul de Sac, Golf Course Frontage, Wooded/Treed
Maint Fee Y/N No
Other Full Baths Lower - 1, Full Baths Main - 1, Full Baths Upper - 1, Half Baths Lower - 0, Half Baths Main - 1, Half Baths Upper - 0
Possession Negotiable
Property Subtype Single Family
Restrictions None
Roof Asphalt/Fiberglass
View Description Golf Course, Wooded
Warranty Y/N Yes
Water Sewer Public Sewer, Public Water
Bedroom Second
Bedroom Second
Loft Second
Bedroom Lower
Media Room - Family/Media Room Lower
Office Lower
Other Lower
Dining Room First
Foyer First
Great Room First
Kitchen with Eating Space - Eat-in Kitchen First
Laundry Room First
Library - Library/Study First
Master Bedroom First
Listing Courtesy of Keller Williams Grt Cleve NE


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Southern Sky Optometry Grand Opening
Name: Southern Sky Optometry Grand Opening
Date: June 10, 2017
Time: 11:30 AM - 12:30 PM CDT
Event Description:
Today we are celebrating Southern Sky Optometry GRAND OPENING! Southern Sky Optometry offers professional eye care for the community. They have partnered with Sam's Club and opened their office inside Sam's Club. Southern Sky Optometry is going to have their ribbon cutting Saturday,June 10th! There will be door prizes, food trucks, vendors and much more. The ribbon cutting will take place at 12 o clock, so bring your friends and family! See everyone there!
The Greater Keller Chamber of Commerce | 420 Johnson Road | Suite 301 | Keller, Texas 76248 P: 817- 431-2169 | F: 817- 431-3789 copyright 2010 Keller Chamber of Commerce


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Commercial Property Agent in Melbourne
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Are you tired of giving away your hard earned money in rents, then it's time to invest in a property now! Pride World City project offers spacious and well ventilated apartments in Charholi, Pune having all sort of ultramodern amenities. These 1, 2 and 3 BHK flats are priced at 30.66 Lac to 69.89 L. Search for the properties in Pune with the best of specifications within your budget at 99acres.com.


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Obesity in the Early Childhood Years
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Obesity in the Early Childhood Years
Publisher: National Academies Press 2016
Number of pages: 115
Among the many troubling aspects of the rising prevalence of obesity in the United States and elsewhere in recent years, the growth of early childhood overweight and obesity stands out. This book explores what is known about effective and innovative interventions to counter obesity in young children.
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Long-term lentiviral transduction of human mesenchymal stem cells (hMSCs) greatly enhances the usefulness of these cells. However, such transduction currently requires the use of polybrene, which severely inhibits hMSC proliferation. In contrast, protamine sulfate at 100 μg/ml doubled transduction efficiencies without affecting proliferation or differentiation potential. Expression levels improved 2.2-fold with the addition of a woodchuck hepatitis post-transcriptional regulatory element. Further improvements in transduction efficiencies could be obtained by a modest increase in viral concentrations through increased viral titers or decreased transduction volumes without changing multiplicity of infection, by transducing over multiple days, or by culturing the cells in fibroblast growth factor-2. Centrifugation improved expression but had no effect on efficiency. Transgene expression was stable over 6 weeks in vitro and in vivo. Donor-to-donor and intradonor variability were observed in primary passage through passage 2 cultures, but not at passage 3. These results provide a better optimized approach for expanded use of hMSCs through genetic manipulation.
Mesenchymal stem cells (MSCs) are multipotent adult stem cells that can differentiate into adipocytes, chondrocytes, and osteoblasts [1, 2]. This ability to serve as cell progenitors for different mesenchymal lineages has been used in regenerative medicine to structurally repair injured tissues and restore their lost functions, such as in bone [3] and cartilage repair [4]. In addition, these cells have been found to exert trophic and other local effects that allow them to modulate the immune system, protect surrounding cells from apoptosis, and establish a regenerative microenvironment. The translational potential of these capabilities ranges from treating autoimmune diseases, such as multiple sclerosis [5] and rheumatoid arthritis [6], and other immune disorders such as graft versus host disease [7] to treating ischemic events such as stroke [8], inflammatory disorders such as asthma [9], and other instances of tissue damage such as acute lung injury [10].
The usefulness of MSCs can be greatly expanded through genetic modification of the cells that provide new functions or improve on existing ones. For example, when MSCs were genetically modified to overexpress stromal cell-derived factor-1 [11] or Akt [12] and delivered to mice after a myocardial infarction, the cardiac function significantly improved. MSCs have been genetically modified and used as targeted anticancer drug carriers. For example, when they were modified to secrete interferon-β, survival of mice implanted with either glioma [13] or melanoma [14] significantly increased. MSC homing itself could be improved, as Kumar and Ponnazhagan demonstrated by increasing the expression of α4 integrin [15]. They could also be used to treat genetic diseases for which there currently is no cure [16]. For example, in osteogenesis imperfecta (OI), a disease in which osteoblasts cannot produce either functional or adequate levels of type I collagen [17], MSCs from the patient could be isolated, genetically corrected to produce sufficient amounts of functional collagen, and then re-engrafted into the patient [18]. Finally, genetic modification of MSCs can be a useful tool to track and study normal MSC functions. For example, MSCs have been labeled with lacZ [19] or green fluorescent protein (GFP) [20] in order to study their trafficking behavior.
There are a number of short-term transfection methods available for MSCs. These include viral methods, such as treatment with adenovirus [14, 15, 21], as well as nonviral methods [22] such as liposomes [23] or electroporation [24]. In contrast, methods for long-term/stable genetic modification of MSCs are significantly more limited. One method is to use transgenic mice [25]. However, there are serious limitations with this method, including its restriction to mouse MSCs, which can have different properties from other species, such as human MSCs [26], and the necessity of generating new transgenic mice for every protein under study.
An alternative method is to use lentiviruses to deliver the engineered DNA construct. The current standard protocol for efficient lentiviral transduction of MSCs involves the addition of polybrene during transduction. Unfortunately, as we recently published, polybrene severely inhibits MSC proliferation, even with the addition of fibroblast growth factor-2 (FGF-2), a potent mitogen [27], and without polybrene, transduction efficiency is low [20, 27]. In addition to the increased cost of transducing MSCs, the inhibition of MSC proliferation is problematic for any therapy that requires sufficient cells to repopulate a host, such as the treatment of OI described above. Furthermore, we have some initial indications that polybrene might also affect human MSC (hMSC) differentiation potential in addition to proliferation. For instance, in the study of chondrogenesis in aggregate (pellet) cultures, we observed that pellets made with polybrene-treated hMSCs were smaller and exhibited less cartilage extracellular matrix than untreated controls (unpublished observations). Thus, there is a necessity for an alternative hMSC transduction method that preserves the cells' proliferation and differentiation potential.
In order to overcome these technical limitations, we comprehensively and systematically addressed various factors that affect viral transduction, including the virus, the cells, and the protocol used, in order to evaluate their impact on both efficiency and transgene expression and their effects on proliferation and differentiation. We found for the first time that the use of high concentrations of protamine sulfate is a viable alternative to polybrene that preserves hMSC proliferation and differentiation capabilities while significantly improving lentiviral transduction. In conjunction with protamine sulfate, efficiency was further improved by modest increases in viral concentrations without the need for ultracentrifugation, via increased viral titers or decreased transduction volumes, or by transducing over multiple days. Expression levels could be enhanced with centrifugation or with the addition of the woodchuck hepatitis post-transcriptional regulatory element (WPRE) at the end of the transgene. Furthermore, we investigated for the first time the possibility of transducing hMSCs grown in the presence of FGF-2.
Human MSCs were isolated as described previously [28, 29]. An aspirate from the bone marrow was obtained from the posterior superior iliac crest of consenting adult donors (institutional review board-approved protocol from University Hospitals, Cleveland, OH). The aspirate was washed with Dulbecco's modified Eagle's medium, low glucose (DMEM-LG) (Sigma-Aldrich, St. Louis, MO, http://www.sigmaaldrich.com) supplemented with 10% fetal bovine serum (FBS), screened to support hMSC proliferation and differentiation (hMSC medium) [30]. The cells were centrifuged, resuspended in 5 ml of medium, and placed on top of 35 ml of 65% (vol/vol) Percoll gradient. After 15 minutes of centrifugation at 460g, the top 25% of the gradient was transferred to a new tube and washed again. The cells were then seeded at 1.8 × 105 per cm2 and cultured for 2 weeks before passage. The hMSCs were cultured in 10% FBS DMEM-LG with medium changes every 3–4 days. For maintenance cultures, cells were passaged weekly with 0.25% trypsin/4 mM EDTA (Gibco, Grand Island, NY, http://www.invitrogen.com) and seeded at 3–4.5 × 103 per cm2 in 100-mm tissue culture dishes or T175 flasks.
The bicistronic reporter gene constructs pLV-MND-LR (LR) and pLV-MND-LR-WPRE (LR-WPRE) were created by modifying the fl-mrfp-ttk triple fusion reporter vector [31] (gift from Dr. Zhenghong Lee, Case Western Reserve University) containing firefly luciferase (fl), monomeric red fluorescent protein (mrfp), and herpes simplex thymidine kinase (ttk) driven by a modified myeloproliferative sarcoma virus promoter (MND). First, the herpes simplex thymidine kinase (ttk) domain was removed from the triple fusion construct with BamHI to create the pLV-MND-LR plasmid. For the pLV-MND-LR-WPRE construct, WPRE was obtained from the pLV-MND-P140K-2A-GFP plasmid (gift from Dr. Stanton Gerson, Case Western Reserve University) with BamHI and then inserted into the bicistronic vector after the transgenes. Both plasmids were sent for sequencing to ensure the correct insertion and that the nucleotides were in-frame.
To produce the second-generation replication-incompetent lentivirus, near confluent 293T cells were transfected with the reporter plasmid, pCMVΔR8.91 (packaging plasmid), and pMD.G (vesicular stomatitis virus protein G envelope plasmid) at a ratio of 3:3:1 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) in Opti-MEM culture medium (Gibco). Opti-MEM was replaced with fresh hMSC medium after 12 hours, and viral supernatant was collected after 36 hours and again after another 24 hours at half the volume. The supernatant was sterile filtered, aliquoted, and frozen at −80°C until use. Viral titers were determined with 293T cells by measuring the monomeric red fluorescent protein (mRFP) and ranged from 2.8 × 106 to 7.1 × 106 infectious units (IU)/ml for pLV-MND-LR and from 4.9 × 106 to 15.5 × 106 IU/ml for pLV-MND-LR-WPRE. Viruses were thawed immediately before titer and cell transduction.
Transductions were done in six-well plates. Unless otherwise specified, hMSCs were seeded at 1 × 105 cells per well in triplicate at a final volume of 1 ml per well with a multiplicity of infection (MOI) of 5. Protamine sulfate (catalog no. P4020-1G; Sigma-Aldrich) from a 5 mg/ml stock solution (in DMEM-LG, sterile filtered) was added to obtain the desired final concentration. Cells were transduced for 24 hours before being replaced with 1.5 ml per well. Cells cultured from the initial marrow aspirate and then trypsinized and transduced were termed P0 (passage 0). Experiments were repeated with at least two donors.
In the FGF-2 experiments, either hMSCs were cultured in the presence of recombinant human (rh) FGF-2 (final concentration, 10 ng/ml; Peprotech, Rocky Hill, NJ, http://www.peprotech.com) from primary passage or rhFGF-2 was added at the time of transduction and subsequent medium changes. For the centrifugation and the different transduction volume experiments, hMSCs were first seeded at 1.5 ml per well for 24 hours prior to transduction. In the low-volume experiment, the cells were initially transduced at 0.5 ml per well for 8 hours. Subsequently another 0.5 ml (hMSC medium plus protamine sulfate) per well was added for 16 hours before medium change. Volumes for other size dishes are listed in supplemental online Table 1. In the centrifugation experiments, hMSCs were centrifuged for 1 hour in an Allegra 6KR centrifuge (Beckman Coulter, Fullerton, CA, http://www.beckmancoulter.com) with a GH-3.8 rotor and Micro Plus Carriers at 2,000 rpm (730g) and 1.5 ml per well. The centrifuge was run to bring the temperature up to ~32°C before the experiment.
Three days after the last round of transduction, the efficiency was measured by detecting the mRFP on a BD LSR II (BD Biosciences, San Diego, CA, http://www.bdbiosciences.com) (excitation: 561, emission: 610/20 band pass filter with a 600LP in front) as shown in supplemental online Figure 1. The positive cells were gated using a 525 nm versus 610 nm scatter plot in order to account for the autofluorescence from hMSCs. Expression levels were calculated from the geometric mean of the gated population. Figures show the results of triplicate wells from one representative experiment.
Trypsinized cells were washed, and then resuspended at 2 × 104 cells per milliliter. For the protamine sulfate experiment, the cell suspension was additionally supplemented with protamine sulfate to obtain final concentrations of 0, 50, 100, and 200 μg/ml. The cells were seeded into 96-well plates at 1 × 103 cells per 50 μl per well in triplicate and cultured at 37°C, 5% CO2. After 24 hours and 7 days, the medium was removed and the plate was placed in the −80°C freezer until the day of analysis. The CyQUANT assay (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) was then performed on the wells according to manufacturer instructions.
Human MSCs were transduced at P0 and cultured for an additional 4 days after the start of transduction. The cells were then sorted on the iCyt Reflection flow cytometer (excitation: 561 nm, emission: 615/30 band pass filter) with a 100-μm tip. The 30% brightest and dimmest cells were collected and cultured for an additional week before the start of the differentiation assays. The assays were performed as described previously [29].
To assess the chondrogenic potential of the hMSCs, 2.5 × 105 cells were resuspended in chondrogenic medium (1% ITS+, 10−7 M dexamethasone, 1 mM sodium pyruvate, 37.5 μg/ml ascorbic acid-2 phosphate, and 10 ng/ml transforming growth factor-β1; Peprotech) and seeded into V-bottomed polypropylene 96-well plates. The plates were centrifuged at 500g for 5 minutes and cultured at 37°C for 21 days with medium changes every 2–3 days. The pellets were then fixed in 10% formalin, embedded, sectioned, and stained with toluidine blue.
The adipogenic assay was performed by seeding 2.0 × 105 cells per well into six-well plates in adipogenic medium (DMEM-HG with 1 μM indomethacin, 500 μM 3-isobutyl-1-methylxanthine, 10−6 M dexamethasone, and 10 μg/ml insulin). Medium was changed twice a week. After 21 days, the cells were fixed with 4% formaldehyde. The cells were permeabilized with 0.01% digitonin for 20 minutes and blocked with 1% bovine serum albumin. The cells were then stained with mouse monoclonal anti-adipophilin antibody (catalog no. 610102; 1:10; Progen Biotechnik, Heidelberg, Germany, http://www.progen.de) followed by a fluorescein-conjugated goat anti-mouse secondary antibody (1:1,000; Cappel/MP Biomedicals, Solon, OH, http://www.mpbio.com).
To test whether the transduced hMSCs could be detected in vivo and whether they can differentiate into osteoblasts, the cells were loaded into porous calcium phosphate ceramic cubes coated with fibronectin and implanted subcutaneously on the dorsal surface of CB17 SCID mice. (Animal experiments were approved by the Institutional Animal Care and Use Committee of Case Western Reserve University, Cleveland, OH.) After 6 weeks, the mice were x-rayed, the bioluminescence was measured, and the cubes were harvested. Bioluminescent imaging was done with the Xenogen IVIS Imaging 200 Series system (Caliper Life Sciences, Hopkinton, MA, http://www.caliperls.com). The mice were injected with 200 μl of 12.5 mg/ml luciferin substrate (Biosynth, Itasca, IL, http://www.biosynth.com) intraperitoneally, and a 10-second exposure was taken 5 minutes later. The harvested ceramics were fixed, decalcified, paraffin-embedded, sectioned, and stained with Mallory-Heidenhain. Sections were semiquantitatively analyzed as described previously with a ceramic cube score based on the percentage of positive pores [32].
Unless otherwise stated, significance was assessed by analysis of variance (ANOVA) followed by a Tukey's multiple comparison test. For Figure 2F, a one-sample t test was used to test the null hypothesis that the mean was 1.0. For Figure 3A, a two-way ANOVA showed significant difference between cells frozen and thawed and cells in continuous culture. A Bonferroni post test showed significant difference between the conditions in P1 and P3 (p < .001). For Figure 3B, a repeated measures linear mixed model using Tukey-Kramer adjustment for multiple comparisons was used. When multiple comparisons were used, adjusted p values are reported.
To investigate whether protamine sulfate is a viable alternative for polybrene, hMSCs were transduced at various concentrations of protamine sulfate with a modified dual reporter gene (LR) that codes for a fusion protein containing both luciferase (Luc) and mRFP functional domains [31]. The transduction efficiency was determined by analyzing the fluorescence with flow cytometry (Fig. 1A). An example of the flow cytometry analysis is shown in supplemental online Figure 1. Protamine sulfate concentrations below 8 μg/ml showed no effect on transduction efficiency. However, increasing the protamine sulfate concentration to 50 μg/ml resulted in a 2.4-fold increase in efficiency from 15.7% (at 0 μg/ml) to 37.6%. Further increase in protamine sulfate concentration resulted in a slight but not statistically significant increase in efficiency, although cell detachment became pronounced at concentrations above 100 μg/ml. Hence subsequent experiments were done at 100 μg/ml unless otherwise specified. Unlike polybrene, there was no statistical difference in cell proliferation between untreated cells and cells treated with protamine sulfate at any concentration tested for an extended period of time (7 days) (Fig. 1B). Similarly, trypan blue staining during cell passage showed an identical percentage of dead cells (between 0% and 1%) as controls. This was confirmed with a 4′,6-diamidino-2-phenylindole live/dead assay (supplemental online Fig. 2).
In addition to increasing transduction efficiency, labeling of hMSCs can be enhanced by increasing transgene expression. In an attempt to increase expression of the transgene, a WPRE was added at the end of the transgene in the vector construct. The addition of WPRE did not increase transduction efficiency (Fig. 1C), but it significantly increased the expression level of the transgene by 2.2-fold (Fig. 1D). This was confirmed by Western blot analysis, which showed a 2.4-fold increase in expression (supplemental online Fig. 3). Consequently, the remaining experiments were done with the LR-WPRE vector.
To investigate whether there is a concentration dependence with viral transduction, hMSCs were transduced with increasing MOI while maintaining a consistent protamine sulfate concentration (100 μg/ml). This resulted in a sharp initial rise in transduction efficiency (up to 20 MOI) followed by smaller increments in increased efficiency with further increasing amounts of lentivirus (Fig. 1E). The high viral titers also had no significant effect on cell proliferation (supplemental online Fig. 4). Therefore, the remaining experiments were performed with an MOI of 5, which showed a transduction efficiency of approximately 50%, allowing additional opportunities to investigate further improvements in efficiency.
In addition to changing the ratio of virus to cell (i.e., MOI) in solution, viral concentration can also be affected by changing the transduction volume. To investigate whether viral concentration or the absolute amount of viral particles is more important in determining transduction efficiencies, the same amount of virus (MOI = 5) was added to different volumes of transduction medium (Fig. 1F). Halving the transduction volume to 0.5 ml per well increased transduction efficiency from 50% to 68% (Fig. 1F), similar to the transduction efficiency of 61% when the MOI is doubled to 10 (Fig. 1E). In contrast, changing the seeding density and adjusting the viral particles to maintain a consistent MOI had no effect on efficiency (supplemental online Fig. 5).
To investigate the role that the cells play in the transduction process, we transduced hMSCs over the course of 2 days (MOI = 2.5 × 2 days) or 3 days (MOI = 1.67 × 3 days) while keeping the volume and overall total amount of virus particles constant (MOI = 5). A 2-day consecutive transduction increased transduction efficiency by 40%. Three days of transduction resulted in an additional smaller, although statistically significant, increase of 7% from the 2-day transduction (Fig. 2A). The increased transduction efficiency also correlated with higher expression levels (Fig. 2B), since a 2-day transduction increased expression by 22%, and a 3-day transduction by another 44%. There was no difference in cell proliferation as compared with untreated cells (supplemental online Fig. 6A), and there was no difference in cell proliferation of transduced cells cultured in 60- and 100-mm dishes, and T175 and T500 flasks at up to seven passages (supplemental online Fig. 6B, 6C).
Another method commonly used to increase transduction efficiency is centrifugation after the addition of virus to the medium [33]. Unlike the case with static cultures, centrifugation caused the cells to detach at much lower concentrations of protamine sulfate even without the addition of virus. Major cell detachment was observed at concentrations greater than 30 μg/ml. Importantly, centrifugation alone (without the addition of protamine sulfate) did not improve transduction efficiency. However, 10–30 μg/ml protamine sulfate combined with centrifugation was sufficient to increase efficiency to the same degree as 100 μg/ml under static conditions (Fig. 2C). Whereas centrifugation did not have an effect on transduction efficiency above what can be achieved under static conditions, it did improve the expression level by approximately 36% (Fig. 2D). Centrifuging at lower than 1,500 rpm (411g) had no effect (data not shown).
Although a key characteristic of the lentivirus is its ability to transduce nondividing cells, it transduces dividing cells more efficiently [34, 35]. This led us to hypothesize that treatment of hMSCs with FGF-2, a known mitogen [36], could have a positive impact on transduction efficiency. To determine whether the timing of the addition of FGF-2 had an effect, hMSCs were either cultured in FGF-2 at least 1 week prior to transduction or FGF-2 was added at the time of transduction. In both cases, FGF-2 significantly increased transduction efficiency (Fig. 2E). On average, the efficiency increased from 61% to 70% when FGF-2 was added at the time of transduction, and it further increased to 79% with cells cultured in FGF-2 prior to transduction. Cells treated with FGF-2 before transduction also showed a significant increase in transgene expression, ranging from a 9% to a 155% increase (mean, 49%) (Fig. 2F).
Storage of hMSCs in liquid N2 is commonly practiced to preserve the cells for later use. Freeze-thaw of the cells was shown to not negatively impact the transduction efficiency of the cells at any passage (Fig. 3A). We did notice a slight but significant increase in transduction efficiency with cells that were previously frozen in P1 and P3. We have also observed that thawed cells can exhibit increased proliferation (unpublished data), which could explain this increase in transduction efficiency.
Viral transduction of hMSCs was also slightly better at P1 and P2 compared with P0 and P3 (55% and 52% vs. 48% and 38%, respectively), although it was not significant due to the high variability at P1 and P2, for which the transduction efficiencies ranged from 39% to 89% (Fig. 3B). For cells transduced at P0, one donor had an efficiency of 80%, but the remaining four donors clustered tightly around 40%. There can also be high intradonor variability as one donor had a transduction efficiency of 39% and 75% at P1 and P2, respectively, whereas another had an efficiency of 89% and 42% at the same passages. Such donor-to-donor variations are normal observations with hMSCs [37]. Donor age (27–61 years old) and gender had no significant effect.
In order to test whether transduction and the insertion of the transgene affects the differentiation potential of hMSCs, the brightest and dimmest 30% of the cells based on their fluorescent intensity were sorted, and chondrogenic, adipogenic, and osteogenic assays were performed. In the chondrogenic pellet assay, both low and high expressing cells showed cartilage formation similar to that of untransduced cells. Furthermore, cells expanded in the presence of FGF-2 formed larger pellets and had more matrix (Fig. 4).
In the adipogenic assay, lipid formation was observed in all conditions. To confirm that the lipid formation was not isolated to untransduced cells, the MSCs were stained for adipophilin. Colocalization of red fluorescence with adipophilin staining indicated lipid formation in lentiviral transduced hMSCs (Fig. 5).
To test osteogenic potential, transduced MSCs were loaded into porous calcium phosphate ceramic scaffolds that were implanted subcutaneously on the dorsal surface of immunocompromised mice. After 6 weeks in vivo, bioluminescent imaging showed strong transgene expression (Fig. 6A). Concurrently, the same cells cultured in vitro confirmed that fluorescent expression remained consistent for at least seven passages (about 7 weeks) (Fig. 6B). Histologic analysis of the implanted ceramic cubes showed that bone formation was similar in high and low sort and control cells. There was no statistical difference in the ceramic cube scores between the transduced and the control cells in both FGF-2 and non-FGF-2-treated cells. Interestingly, cubes loaded with FGF-2-treated cells had more bone formation (Fig. 7).
Currently, the addition of polybrene during transduction is required to obtain efficient lentiviral transduction with hMSCs. Unfortunately, we have found that polybrene dramatically impacts hMSC proliferation and possibly their differentiation capabilities as well [27]. In our attempts to devise an alternative method to preserve hMSC proliferation and differentiation, improve hMSC transduction, and enhance transgene expression, we first established a baseline for comparison with 100 μg/ml protamine sulfate (as a polybrene alternative), the inclusion of WPRE at the end of the transgene, and an MOI of 5. Next we investigated variables affecting the virus, the cells, and the protocol. Those variables included viral concentration, number of rounds of transduction, centrifugation, the effect of FGF-2, freeze/thaw cycle, passage number, donor variability, and seeding density. All these methods had an impact on both transduction efficiency and transgene expression.
Protamine sulfate is a Food and Drug Administration-approved, relatively inexpensive polycation most commonly used to neutralize heparin overdose. It is believed to improve transduction by neutralizing the negative charge between the cell surface and the viral particles [38]. It was first suggested as an alternative to polybrene by Cornetta and Anderson in 1989 [39]. In that report, 5–10 μg/ml protamine sulfate resulted in the highest transduction efficiencies with the retrovirus, with either increasing toxicity or decreased transduction efficiency at higher concentrations. However, in addition to using a different viral vector, the cells used were rapidly dividing NIH 3T3 cells and another cell line, which typically experience greater transduction efficiencies than primary cells [39]. When these authors investigated transduction of primary cultures at 10 μg/ml, they were unable to obtain efficiencies above 1% without coculture with the retroviral producer cells that provided continuous high viral titers. Investigators have since used protamine sulfate for labeling of MSCs with superparamagnetic iron oxide for cell tracking by magnetic resonance imaging [40] and attempted to use for transduction with lentivirus [41, 42]. As it has always been used at concentrations below 8 μg/ml, which we found to have no effect on hMSC lentiviral transduction, their efforts have had limited success. As a consequence, protamine sulfate is rarely used for hMSC transduction. In contrast, when the concentration was raised to 50 μg/ml, transduction efficiencies increased 2.4-fold, although concentrations above 100 μg/ml affect the ability of hMSCs to bind to the surface of the plate during seeding or cause the cells to detach from the dish. The improvement was great enough that in some experiments at an MOI of 5, protamine sulfate alone was sufficient to increase transduction efficiency to as high as 88%. Thus ultracentrifugation is not required to concentrate the viral supernatant in our studies. Furthermore, unlike polybrene, protamine sulfate did not affect the cells' ability to divide, thus allowing lower amounts of virus to be used for clinical or experimental purposes, potentially decreasing the initial number of MSCs required for therapy, and thereby enhancing the utility for therapeutic applications in which MSCs serve as regenerative cells. Although not as drastic, we also found that polybrene affected the proliferation of human umbilical vein endothelial cells, whereas protamine sulfate does not (data not shown). Thus, these results are potentially applicable to other cell types.
In addition to improving transduction efficiencies, we sought to improve expression levels with the addition of WPRE at the end of the gene construct, which had been shown to improve transgene expression of the viral vector [43, 44]. Although constructs with WPRE have been used previously with hMSCs [45–47], the degree of enhancement on lentiviral transduction of hMSCs was unknown because such vectors have not previously been directly compared with constructs not containing this element.
The addition of the WPRE resulted in two benefits to our system. First, whereas the transduction efficiency did not increase with WPRE, the expression level of the transgene increased 2.2-fold. This confirmed previous results with 293T cells, for which the addition of the WPRE did not increase the percentage of transduced cells, affect the copy number of the transgenes, or affect the frequency of transcription, but instead increased the expression of the vector, possibly through stabilization of the transcript in the nucleus [43]. Second, we observed that the addition of WPRE benefited viral production as viral titers approximately doubled.
It is clear that viral concentration is key to obtaining high transduction efficiency. Increasing viral concentration can occur from either increasing the amount of virus in the transduction solution (increasing MOI) or by decreasing the transduction solution volume while keeping the MOI constant. Both resulted in higher transduction efficiencies. Unfortunately, since hMSCs are not grown in suspension, the volume is limited to the lowest volume possible that does not desiccate the substrate-attached cells.
An alternative method to increase transduction efficiency is the centrifugation of the cells in transduction medium, which is also termed spinoculation. Despite the small size of the virus and the relatively low g force, centrifugation is believed to function by effectively increasing the concentration of viral particles near the cells [33]. However, unlike the case with T cells, centrifugation alone had no effect on improving hMSC transduction and protamine sulfate continues to be required. Interestingly, centrifugation causes extensive cell detachment at protamine sulfate concentrations above 30 μg/ml, even without the addition of virus; this is not observed in static cultures. One possible explanation is that centrifugation can also concentrate the protamine sulfate, despite its small size (~5.1 kDa). Alternatively, it is possible that the stress from centrifugation causes the cells to be more susceptible to deleterious effects of protamine sulfate.
Ultimately spinoculation of hMSCs presents a number of different challenges. A larger volume is needed to provide sufficient medium coverage, which decreases the viral concentration and counteracts any increase in concentration from the spinoculation itself. In the end, this resulted in the observation that transduction efficiencies were not different from those of static cultures, although a lower concentration of protamine sulfate was needed and transgene expression increased by 36%. There are also issues with scalability. It is difficult to expand beyond the six-well plates used in these experiments. Our attempts at spinning T75 flasks have led to regions of cell death on the flask due to desiccation.
Although important, viral concentration is not the only viral factor that affects transduction efficiency. When the total amount of virus was held constant but spread out over multiple days, efficiencies improved despite the lower viral concentration at each day. This suggests that at any given time, there are a limited number of cells that are susceptible to transduction.
In addition to manipulating the virus, it is possible to affect the cell as well. One common treatment used to modify hMSCs is the addition of FGF-2 in culture. Human MSCs are slowly proliferating cells that can lose their differentiation potential with increasing passage [36], thus limiting the number of cells available from each donor. The addition of FGF-2 to cultures has the advantage of increasing the proliferation rate while delaying the loss of chondrogenic potential (senescence) and maintaining immune suppressive capabilities with increasing passage [36, 48]. In addition, FGF-2-treated hMSCs form larger pellets with more extracellular matrix in the chondrogenic pellet assay [4, 36]. Our results show for the first time that FGF-2-treated hMSCs also form more bone matrix in the in vivo ceramic cube assay, thus possibly extending FGF-2 treatment of hMSCs for osteogenic repair as well.
The increased proliferation of hMSCs provides another opportunity to increase transduction efficiency. Although it is true that lentiviruses can transduce nondividing cells [49], lentiviral transduction is superior with rapidly dividing cells [34, 35]. Our results with FGF-2-treated hMSCs are consistent with this. When FGF-2 was added during the time of transduction, the improvement in efficiency and increase in transgene expression was donor-dependent. It is possible that this relates to the length of time it takes for the cells of a particular donor to respond to the mitogen. In contrast, if the cells were cultured in FGF-2 for at least a week before transduction, we saw both increased efficiency and transgene expression.
Human MSCs are known to have donor-to-donor variability [37], and there have been reports of donor-to-donor variability with hMSC transduction [45]. Interestingly we found this variability to be dependent on passage number. hMSCs in primary culture and at passage 3 were fairly consistent, whereas high variability was found at passages 1 and 2. Furthermore, there can also be high intradonor variability. It is possible that this relates to the fact that proliferation rates vary between donors at passages 1 and 2, and that by passage 3 the proliferation rates stabilize.
Our studies indicate that hMSCs can be efficiently transduced using a protamine sulfate concentration of 100 μg/ml at low volumes over 3 days. Furthermore, inclusion of WPRE at the end of the transgene leads to higher viral titers and increased transgene expression. Transduction efficiency and transgene expression can be improved by transducing at passage 1 and by treatment with FGF-2 for 1 week prior to transduction. With the capacity to easily, reliably, and inexpensively transduce hMSCs, it will be possible to efficiently use genetic engineering to add or delete a function, improve or replace an existing one, and gain insight into the biology of these cells through the use of reporter genes. This will significantly impact not only scientific progress with these cells but also their clinical and therapeutic translational potential.
The authors thank Margie Harris for assistance in isolating the hMSCs, R. Michael Sramkoski and Jonida Toska for their assistance with the cell sorting, Yunhui Kim for her assistance with creating the viral construct, Amad Awadallah for his assistance with the histology, Vance Holt III and Dr. Danielle MacKay for their help with the immunohistochemical stain, and Christine Lee for her assistance with the Western blot. We also thank David Carrino for his help reviewing the manuscript and with the Western blot. This research was supported by the L. David and E. Virginia Baldwin Foundation. P.L. was supported in part by National Institutes of Health T32 GM007250.
P.L.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; Y.L.: conception and design, collection and/or assembly of data, data analysis and interpretation; D.P.L.: collection and/or assembly of data, data analysis and interpretation; D.C. and M.S.: data analysis and interpretation; A.I.C.: conception and design, data analysis and interpretation, financial support.
The authors indicate no potential conflicts of interest.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Meet our senior team
We're different to other consultancies. For starters, as an SME ourselves, we've been through all the pains of growth, passing the 100 people mark in 2015. We've got a great track record of supporting over 15,000 entrepreneurs and businesses over the past decade. We've got a great partner network to connect you into. And we are focused on helping generate tangible bottom line results.
Oh, and we've got a fantastic team of people with a wealth of expertise to bring to your business, and much first hand experience of the growth journey as well. Check us out, and the clients and brands we've worked for; we hope we'll be adding your company to the list of clients we serve.
Bev Hurley CBE,
Bev is a serial entrepreneur, having run several successful businesses during her career and bringing a wealth of direct experience to the team. She has led the YTKO Group through five years of 30% year on year growth, including the challenge of doubling the team twice in a matter of weeks, but Bev also knows how to deal with the hard times – market turmoil, client collapse, bad staff, procurement mendacity, financial squeezes and a whole lot more!
Her particular strengths are commercialising innovative new product and services, procurement, business roadmapping, and developing effective growth strategies that encompass markets, money, people, as well as leadership, risk and change. Bev has developed several pioneering and successful new brands within the YTKO Group, and enjoys helping clients get innovation to market.
Bev created and still occasionally delivers GetSet's suite of strategic Growth Programmes, each tailored for a critical stage of the business journey. Over 1000 like-minded experienced CEOs have benefited from connecting with each other through these programmes.
As an experienced angel investor and NXD, Bev also works with clients to create compelling investor propositions and strengthen senior management teams and boards.
Clare McKee,
When businesses want more clients they ask Clare how to get them. Clare started her sales and marketing career in telesales with Birmingham Post & Mail, then moved into field sales to help independent retailers in the city centre compete with the big boys.
She joined Orange as a Regional Sales Manager, winning numerous contracts from local authorities and national brands. Clare also devised the first mobile handset 'quick guide', knowing that no one ever reads the handbook!
Clare then set up an online mobile phone recycling business, winning large recycling contracts from major banks and insurance companies. She sold it to a competitor prior to joining ijento, an enterprise level web analytics/customer experience software company. There she helped global brands such as Tesco, Direct Line and The National Lottery significantly improve online customer experience and marketing ROI.
With a Master's degree in digital marketing, Clare enjoys fixing broken marketing – fast. She's doubled client website conversions in weeks, trained over 500 businesses in every aspect of marketing, and saved tens of thousands of pounds by nailing clients' sales and marketing strategies and priorities.
Ian Gardner,
Ian has more than 35 years experience in business and corporate banking, working for some of the UK's leading banks in credit and sales management roles. A very solid background in credit assessment led to a position as Senior Credit Analyst where Ian gained a comprehensive understanding of the Banks' policies and practices when lending to small businesses. Specialist positions have also included managing regional teams focussing on manufacturing SMEs with turnovers from £1m – £20m and improving bespoke banking propositions for the professional services sector, including solicitors and owner-managed accountancy practices. Ian was also selected to provide independent support to Directors and SME owner-operators as part of a high profile review instigated by the Financial Conduct Authority in 2012.
In more recent years Ian worked at a Community Finance Development Institution across the West Midlands whilst also setting up his own travel business specialising in cruises, winning a Social Media Award in the first year of operation.
Ian's particular expertise at GetSet Midlands is in carrying out financial reviews for clients to ensure they get the best deals from the most appropriate funding source.
Kathy Poole,
With over 25 years in business, operations and account management across the banking, leasing, asset finance and commercial loan industry, Kathy has held senior roles at a major high street bank, one of the UK's leading asset finance brokerages, and a highly successful Community Development Finance Institution in the West Midlands.
Former positions have also included business development manager and ambassador for the Stoke on Trent & Staffordshire Jobs and Growth Fund, a £4M grant fund for growing businesses from a wide variety of industry sectors. In this role Kathy was instrumental in helping drive the full allocation of the fund and securing a second tranche of finance.
A warm communicator with a real understanding of the benefits that making connections and new relationships bring to businesses, Kathy thrives on ensuring that collaborations with our partners bring results. With a wealth of connections across local authorities, Local Enterprise Partnerships and a wide range of private sector organisations, plus our fast-growing GetSet client community of growth-ambitious businesses, Kathy is adept at creating tailored partnership arrangements that deliver results.
Philip Stott,
Phil Stott is an accredited Price 2 Project Director and our specialist in the energy and construction sectors. Phil is a current member of the BEIS Green Construction Board, Energy Managers Association, Green Skills Alliance and BEIS Supply Chain Impact Group. He also contributed to the recent Government Bonfield Review into energy policy in the UK and continues to advise on the implementation of that review through the Industrial Strategy and Cabinet Office balanced scorecard projects.
Phil has worked with a number of leading industry businesses including Carillion, Willmott Dixon, Viridor, Bam Nuttall, Kingfisher Group, Affinity Sutton and the big 6 energy companies. He also has provided consultancy to the Scottish Government, GLA, Northern Ireland Housing Executive and the All Parliamentary Renewable and Sustainable Energy Group.
In 2014 Phil led an ambitious EU Horizon 2020 project to map skills infrastructure and was named UK Coordinator for a number of subsequent construction and energy projects. That year he also won the Green Build Breakthrough Award. Phil is currently heading our public sector projects in the West of England, but his expertise is available to GetSet clients.
Helen Stratton,
An experienced marketer and commercial executive with a strong track record on both the agency and client side, Helen has worked with many national and international brands to develop, manage and implement marketing strategies and campaigns.
Helen has held various senior level marketing positions with consumer and entertainment companies including Britvic, Sara Lee, Wella GB and The Walt Disney Company. She brings considerable experience in strategy development, marketing, new product development and marketing communication.
More recently, she managed and led the launch of the GetSet for Growth programme in Solent, a business, marketing and finance support programme for SME's. The programme has engaged over 5,500 local businesses, enrolled over 750 clients and has helped raise over £3.5m in private investment.
Helen specializes in creating and implementing comprehensive marketing plans utilising new media and/or traditional media tactics as well as defining and developing brand positioning. Her broad experience across a wide variety of industries gives her a clear and complete understanding of all aspects of marketing and commercial executive roles.
She thrives from supporting people and companies in the process of building and transforming businesses through clear strategies and marketing solutions and always placing the customer at the heart of everything. With an MBA focused on strategy and marketing, Helen has helped numerous businesses with their growth strategies, both SME and Enterprise level, working closely with the marketing teams and business owners.
Emma Blake,
With over 15 years of Finance expertise, Emma has extensive Operational and Advisory experience. Emma fast tracked her corporate career from graduate to finance director within the world of Aerospace & Defence at BAE SYSTEMS Plc (think fighter jets, tanks and submarines…), delivering as a qualified Accountant, winning CIMA Young Business Manager of the Year, completing her MBA and becoming one of the industry's youngest finance leaders.
Emma went on to join Deloitte as Director leading their CFO advisory practice, managing teams across New York, Japan, Kuwait and London, and delivering large scale global finance transformation and turnaround programmes.
Emma is passionate about robust processes and structure in business… systematising for success. She transforms teams across Business Performance, Management Reporting, Process, Systems and Controls.
Following her successful corporate career, Emma founded and scaled her own business which retailed to 170 countries worldwide. She successfully sold this in 2015 to a Tech Investment firm. Since then Emma has expanded her investments in the property sector and also shares her expertise as business mentor / non-exec director, advising entrepreneurs on rapid business growth.
Call us on 0800 917 9881 to find out more or contact us below.


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Life Arts
Horoscope for June 2009(Page 1 of 1 pages)
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"Mine!" I growled as she walked out the room.
"What?!" Her mother asked.
"Your daughter she's my mate." I said.
"NO!" She yelled. "YOUR 16 AND SHE IS 10."
Alexia is a 10 year old girl that is doing normal 10 year old things. Except getting a mate already. The weird thing is her dad's best friend and her sisters brother.
Tanner is a 16 years old and haven't found his mate yet. Until he really takes a good look at his little sister's 10 year old best friend. He starts to realize that that is his mate.
D3c3mb3rba3 Jun 23
It's funny because the girl playing Alexis in real life is Madison Beer and everyone makes jokes because she is Dating Jack Gilinsky who is a few years older than her. They make jokes saying she needs to stop skipping school,that she's like 10, and Jack is her babysitter.😂
anime_lover227 Sep 06, 2016
10 to 16 six year age difference is not that big of a deal cause when she's 20 and he's 26 it'll be okay I've seen bigger age differences
mik0608 Apr 08, 2016
Wow 10 year olds just look older and older now days like for real
- Jun 11, 2016
I guess guys really can't trust how girls look, she's 10 and someone about to be charged as a pedophile.
diiamond_infinitii Jun 22, 2016
She don't loom like a 10 year old ......to young for make up honey
lowkeyems Jun 21, 2016
Lmao Madison Beer ? As a 10 year old? This is gonna be interesting af lmfao


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Aug 11 2011
KABOOM! Hamas terrorist killed in 'work accident' at his home in Gaza
A member of Hamas's armed wing, the Izzadin a-Kassam Brigades, was killed by an explosion in his Rafah home in the southern Gaza Strip late Wednesday night, Palestinian news agency Ma'an reported.
J POST The man was killed in an explosion, a suspected "work accident" likely caused while the man was preparing an explosive device. It was under investigation by Hamas security forces, according to the report. The man killed was reportedly Ali Nayef al-Haj, a 20-year-old resident of the central Gaza Strip.


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"It' happened once, get over it! We ain't got shit to talk about!"
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Damn, this is....it's complicated!
Story of Dana Williams & Desmond Green
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🎶🎶🎶I got fake people showing fake love to me🎶🎶🎶(but Dezi's is real?)
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You can tell this is old since Rihanna is holding a blackberry phone...man I remember when I used to be on those things 24/7
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___andreaaaa Nov 25, 2016
If I was a guy and she said that she's pregnant by me, it's no questions asked. Idc if the baby isn't mine. Robyn is my baby mama, that's it
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Oh hellll nah. Girl u dont need to stay for dinner no more, ill cook for u if its that serious


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Kinsa's Hot Jobs and Current Food and Beverage Job Opportunities
Welcome! If you are a Job Seeker, you may Sign Up below to Create Your Free Private Account. Our Job Seeker interface makes it easy for you to submit a new resume to our recruiters, apply to new job postings you see today, receive new job posting alerts, as well as track which jobs you have already applied to.
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I just had a breast reduction four days ago and my surgeon told me I could remove the bandages two days ago... I've noticed that they are two different sizes and I'm not sure if it's swelling or if it's because something might actually be wrong. Is uneven swelling normal?
My breast are very uneven after my breast reduction... Is this something I should be worried about? (Photos)
Doctor Answers 8
Early post op, some advices:
Thank you very much for sharing your concerns with us.
4 days after Breast Reduction, it's not time enough to express a valid opinion of results.
On the other hand, generally to avoid the normal p.o. swelling, I recommend to limit the arms movement for two weeks.
Also it's not advisable to carry weight and take the antiinflamatory painkillers drugs.
Finally, I recommend you always use a therapeutic bra.
Dr. Emmanuel Mallol Cotes.-
Uneven Breasts
Thank you for your pictures. I recognize the difference but this may be due to swelling. Give your breasts time to completely heal which is around 3 months. Best wishes
Uneven breast after a breast reduction
Thank you for the question. As you can see from the other responders is just far too early to tell. It's very common to have variation in size after surgery and sometimes I can fluctuate depending on how do you sleep. If you're concerned call your surgeon. Good luck
You might also like...
Breast reduction
It is too early to tell. If you are concerned about a hematoma, then you need to be seen in person. .
Learn more: https://www.stevenwallachmd.com/breast/breast-reduction/
Give it time, but they do look a little different
You may hear this from every plastic surgeon that replies to your questions, but it is the truth- you are just way too early to judge your results. I know this is frustrating for you, but I have seen major changes through the healing process in both directions- some things that looked perfect at first and ended up looking less than perfect as well as some things that looked less then desirable in the beginning and ended up a homerun. You just have to let time pass and your body heal. Most will say give it at least six months before drawing any conclusion. The good news is that if you do require a revision, there is always a solution. In your particular case, it does appear that some of your incision lengths are different but that may even out with time.
Breast reduction
It's very early yet and yes, swelling can make that kind of difference but this is something you really want to show your own surgeon. If you were symmetrical before the surgery I'm sure they removed The same amount on each side and you should be fine. Little trickier if you were asymmetrical before the surgery.
My breasts are very uneven after my breast reduction...is this something I should be worried about?
Thank you for sharing your question and photographs. I see your area of concern but at only 4 days after surgery, and without an in-person examination, it is difficult to offer definite advice on your long-term results. Some asymmetry in size is not uncommon and as hard as it is, you need to allow more time to pass for your results to mature. Your surgeon should be able to provide you with greater reassurance based on the amount of breast tissue removed from each breast. Hope this helps.
Learn more: Http://www.nelson-center.com/breast-reduction.html
My breast are very uneven after my breast reduction...
Significant, especially asymmetric, swelling can be a sign of a complication. After reviewing your photographs, I do see some asymmetry in some of the photographs, not in others. Overall, the degree of breast asymmetry is not impressive.
Always best to let your plastic surgeon know of your concerns; he/she will be in the best position to advise you and/or provide you with meaningful reassurance.
Best wishes for an outcome that you will be pleased with
Learn more: http://www.poustiplasticsurgery.com/Procedures/Procedure_breastReduction.htm
These answers are for educational purposes and should not be relied upon as a substitute for medical advice you may receive from your physician. If you have a medical emergency, please call 911. These answers do not constitute or initiate a patient/doctor relationship.


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With the business of organized retail becoming more competitive with the entry of several deep-pocketed players—including Reliance Industries Ltd and the Aditya Birla Group—Pantaloon Retail (India) Ltd, part of the Future Group, and which operates the Big Bazaar chain of stores, is pulling out all stops to win consumers, including offering services such as tailoring, laundry, even vacuum-cleaning homes and repairing electronic appliances.
"We will be adding a whole range of services one by one," said Rajan Malhotra, chief executive, Big Bazaar. The services will be offered by a new unit, Future Services.
According to Malhotra, Pantaloon will launch the services in its hypermarkets in Mumbai in the next three weeks and will follow it up with launches in its stores in Bangalore and the National Capital Region. He said the company has already put in place the logistics for such services in Mumbai and is in the process of doing so in other cities.
While organized retail in India is still at a nascent stage, it is growing as people become more affluent and consumption patterns change. That's translating into a business opportunity for organized retailing and attracting players ranging from Bharti Enterprises Ltd to Reliance Industries.
Pantaloon managing director Kishore Biyani believes there is potential even for shoe-shines and doctors in modern malls and stores.
Pantaloon already provides some services—including painting, carpentry and plumbing—in its home improvement stores, Home Town, but it will now have a full-fledged entry into a sector synonymous with millions of small and unorganized service providers. At Home Town, which resembles large western do-it-yourself or DIY stores, the mantra for Indian consumers is "don't do it yourself" because India, with a population in excess of a billion, has a huge pool of affordable labour.


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