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'Hardy Boys,' 'Dr. Who' and other vintage shows lead new DVD releases of television programs this week
"Family Matters: The Complete Third Season" (Warner, 1991-92, three discs, $29.98, 25 episodes). This sitcom about a middle-class black family living in the Chicago suburbs continues to shift its focus to nerdy supporting character Urkel (Jaleel White) with head of the family Carl Winslow (Reginald VelJohnson) becoming his straight man, making the show more cartoon-like.
"Tyler Perry's Madea Gets a Job: The Play" (Lionsgate/Blu-ray, 2013, $24.99, featurette). Perry's drag character is ordered by a judge to perform 20 hours of community service at a retirement home, which is, of course, completely upended by her antics. Broad comedy performed before a live audience by Perry and his cast of regulars. (Also on DVD, $19.98)
"Attenborough's Life Stories" (BBC, 2013, $24.98, three episodes). David Attenborough has been chronicling natural history in books, audio programs and television shows for some 60 years. This documentary trilogy of 50-minute programs includes "Life on Camera," "Understanding the Natural World" and "Our Fragile Planet." (Also on Blu-ray, $29.98)
"Bible Stories From the Old Testament" (Lionsgate/History, 2013, three discs, $19.98, nine episodes"Bible Stories From the New Testament" (Lionsgate/History, 2013, three discs, $19.98, nine episodes). These sets collect A&E and History Channel hourlong documentaries on the title subjects, including episodes of "Mysteries of the Bible" and "Biography." The Creation, Adam and Eve, Sodom and Gomorrah, Noah and the Flood, Herod, Mary, the life of Jesus and many more biblical subjects are covered, though none in any great depth.
"Nurse Jackie: Season Four" (Lionsgate, 2012, three discs, $39.98, 10 episodes, audio commentaries, featurettes, bloopers). The title drug-addled nurse (Edie Falco) in a New York City hospital returns, going to rehab and watching co-workers suffer for helping her cover up her addiction. (Also on Blu-ray, $39.97)
"Storage Wars: Volume 4" (A&E, 2012, two discs, $19.98, 16 episodes, deleted scene). More episodes of the reality series about treasure hunters bidding at storage auctions and hoping to find valuable treasures.
"Duck Dodgers: Dark Side of the Duck" (Warner, 2003, two discs, $19.97, 13 episodes, original "Duck Dodgers" theatrical cartoon). Daffy Duck's space-traveling alter ego had a three-season run on the Cartoon Network. These episodes comprise the complete first season.
"The Completely Mental Misadventures of Ed Grimley: The Complete Series" (Warner Archive, 1988, $24.95, 13 episodes). Martin Short's cartoonish character from "SCTV" and "Saturday Night Live" was a literal cartoon for this single-season, Saturday-morning show. In addition to Short, voices are provided by Jonathan Winters, and Short's SCTV cohorts Joe Flaherty, Catherine O'Hara and Andrea Martin. (Available at www.WarnerArchive.com)
"Dora the Explorer: Dora's Butterfly Ball" (Nickelodeon/Paramount, 2013, $14.99, three episodes; sticker sheet). Episodes from the current season of the animated series, including one about Father's Day.
"SlugTerra: Return of the Shane Gang, Volume 1" (Shout! Kids, 2012, $14.97, five episodes, new introduction, three bonus episodes). First DVD release of the animated series that initially aired on the Disney XD cable channel has Eli Shane training an army of magical slugs to take on evil Dr. Blakk.
"Chuggington: Safari Adventures" (Anchor Bay, 2013, $16.98, six episodes, bonus "Badge Quest" episode, featurettes, activity sheets; collectible toy Mtambo train). British animated show with anthropomorphic trains. (Also available without the toy train, $14.98)
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Internist, Rheumatologist
1620 Nectarine St
Fernandina Beach, FL 32034 Phone number & directions
Fernandina Beach, FL 32034 Phone number & directions
License & Identifications
A medical license is required for a doctor to practice medicine in a particular state. Requirements vary by state but most require, at a minimum, post-graduate training in the doctor's specific specialty. An NPI number is a national identifier unique to an individual health care provider and is managed by the Center for Medicare & Medicaid Services (CMS).
Learn more about Dr. Charles Christian:
View the full profile to get information on:
- Qualifications
- What's important to you
View full profile
Patient Reviews
Patient comments (1)
The overall average patient rating of Dr. Charles Christian is Good.
|Patient Ratings
|Ease of Appointment
|Courteous Staff
|Accurate Diagnosis
|Bedside Manner
|Spends Time with Me
|Follows Up After Visit
Specialties & Research
Dr. Charles Christian, MD is board certified in Internal Medicine and Rheumatology.
- He has special expertise in 3 areas:
- Hip Osteoarthritis
- Knee Osteoarthritis
- Rheumatic Diseases
Education & Awards
Dr. Christian graduated from Case Western Reserve University , Cleveland.
Home > Find an Internist > FL > Fernandina Beach Internists > Dr. Charles Christian, MD
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- Additional Information
- Dr. Charles Leigh Christian MD is a male Internist and practices in Internal Medicine and Rheumatology.
- Where does Dr. Charles L. Christian practice?
- Dr. Charles Christian has 2 office locations.
Dr. Charles Christian practices Internal Medicine and Rheumatology near Fernandina Beach, FL.
See Fernandina Beach Internists and Fernandina Beach Rheumatologists.

subset: commoncrawl


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I have custom control - using Win Forms, that contains four TextBoxes, all have property to turn them off or on - I just setting visible parameter on them. I would like to change size and position of the custom control - for example, when I turn off first textbox, I would like to change position of all 3 componets below him, to get them higher. Of course, I would like to work it with every TextBox - every TextBoxes, below TextBox I am changing position, should change position.
I cant achieve it with changing of Position of TextBox in its own property - I can ask TextBox on top of me, if its property is set to on or of, but it dont works, because I dont know order of setting property in the application.
I can change position of TextBox below me - in the property of Top textbox, but I can do that with only one TextBox below, I dont know and cant find out, if two TextBoxes below are not off and fourth TextBox should be on position of second.
I cant change it by using some variable - when I change it, other TextBoxes dont care about it and they have set their position before.
So do you have any idea how could I achieve it?


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Great food the best carne asada fries I've had
Machaca burrito for breakfast is the way to go.
I have eaten all kinds of items and everything is good
They don't have green salsa - ever. And they charge $.60 for substitutions (read: not extra, just a substitution).
Best breakfast burrito with hashbrowns, ham, bacon, egg and cheese! Yum!
Best Chicken tacos I've ever had in my LIFE!!! Get 'em!!
Carne asada fries are the best!!
Grab lots of carrots! Yummo!!!
My favorite is their carnitas burrito.
Best beans in town
Rolled tacos are to dieeee
Come here and never leave.
Buy the carne asada fries and get 2 flower tortillas. Then make 2 burritos out of the fries.


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A better education system is a cause worth supporting
The E-Accountability Foundation announces the
'A for Accountability' Award
to those who are willing to whistleblow unjust, misleading, or false actions and claims of the politico-educational complex in order to bring about educational reform in favor of children of all races, intellectual ability and economic status. They ask questions that need to be asked, such as "where is the money?" and "Why does it have to be this way?" and they never give up. These people have withstood adversity and have held those who seem not to believe in honesty, integrity and compassion accountable for their actions. The winners of our "A" work to expose wrong-doing not for themselves, but for others - total strangers - for the "Greater Good"of the community and, by their actions, exemplify courage and self-less passion. They are parent advocates. We salute you.
Winners of the "A":
Johnnie Mae Allen
Dee Alpert
Francesco Alexander Portelos
Harris Lirtzman
Hipolito Colon
Jim Calantjis
Larry Fisher
The Giraffe Project and Giraffe Heroes' Program
Jimmy Kilpatrick and George Scott
Zach Kopplin
Matthew LaClair
Wangari Maathai
Erich Martel
Steve Orel, in memoriam, Interversity, and The World of Opportunity
Marla Ruzicka, in Memoriam
Nancy Swan
Bob Witanek
Peyton Wolcott
More Details
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The E-Accountability Foundation announces the
'F for Fraud' Award
Fraud comes in many shapes and sizes, and we often do not recognize it or the trickster until the consequences of the deceit begin to hurt us or those we love, know, or hear about in the media. Fraudulent actions are always costly, however, and we must remain vigilant in the global effort to stop these practices whenever and wherever they occur. We celebrate the exposure of those people who greedily go after money or glory rather than work hard for a fair wage in a moral and ethical manner by giving them an "F".
Winners of the 2004 F For Fraud Award:
The Belmont Learning Center (Los Angeles)
Teen Boot Camps (International)
The Drug/Insurance/Government Testing and Screening Coalition
Welcome to parentadvocates.org
and e-accountability
We celebrate the alliance of the Internet with the awareness that for too many years people elected to publicly-funded positions have promoted policies that do not serve the public interest. We challenge those who believe they can continue to violate the public trust to stop and listen to the buzz of millions of people visiting websites, emailing each other, blogging and chatting online about what is going on behind closed doors. We promise to hold you responsible for your actions. We call this process "e-accountability".
Housing Homeless Youth Poses Challenge for Mayor de Blasio
Government Lies, Corruption and Mismanagement
| Posted 3/28/2015 at 11:26 AM
During the mayoral campaign, Mayor Bill de Blasio said he would see to it that all homeless youths had beds. But after advocates for the homeless filed a federal lawsuit accusing the city of illegally denying young people shelter, just before he took office, the mayor has surprised them, they said, by fighting the case. "It's incredibly disheartening," said Kimberly Forte, a supervising attorney with the Legal Aid Society, which filed the lawsuit in the waning days of Mayor Michael R. Bloomberg's tenure in 2013. "We're not seeing a new way of thinking about young people when it comes to homelessness from this administration."
Success Stories: Positive Outcomes
| Posted 3/27/2015 at 9:26 AM
Founded in 1948, Learning Ally helps K-12, college and graduate students, veterans and lifelong learners – all of whom read and learn differently due to blindness, visual impairment, dyslexia, or other learning disabilities. Through its support programs and audiobooks, Learning Ally enables families and teachers to help students thrive and succeed. The organization provides support to parents and students through events, webinars, personal consultations and other tools; and integrated learning management systems and professional development for teachers.
Kids Can Publish Has Joined StoryMonsters Ink
Success Stories: Positive Outcomes
| Posted 3/21/2015 at 9:34 AM
Getting published in a national magazine just got a lot easier for kids! Children in kindergarten through 12th grade who love to write—and teachers who would like their students to embrace the written word—are now able to submit their writing samples to Kids Can Publish to be considered for publication in the nationally circulated StoryMonsters Ink magazine. The magazine recently joined together with Kids Can Publish, a division of Five Star Publications, Inc., to help broaden its audience.
Tom Schweich, Missouri Auditor and Gubernatorial Candidate, Committs Suicide
Current Events
| Posted 3/14/2015 at 12:56 PM
There are questions surrounding the suicide of Missouri Auditor and GOP gubernatorial candidate Tom Schweich that we are never going to be able to answer. Foremost is this: Why did Schweich, a husband, father and highly accomplished public servant, become so agitated over a suspicion that a state Republican Party leader might be falsely telling people that he was Jewish? Schweich's associates say that, in the days leading up to Feb. 26, when he shot himself in his Clayton, Mo., home, he became increasingly distraught about his belief that he was the target of an anti-Semitic whispering campaign.
Math Fans Celebrate Once-in-a-Century Pi Day March 14, '15
Current Events
| Posted 3/14/2015 at 12:06 PM
It's around this time every year that math nerds unite to celebrate National Pi Day, a day honoring the mathematical constant pi, or 3.14. March 14 (or 3/14) is officially Pi Day because the date represents the first three digits of the number used to represent the ratio of the circumference of a circle to its diameter. But this year, Pi Day is taking it up a notch. On 3.14 at 9:26:53 a.m., the date and time will correspond to the first 10 digits of pi: 3.141592653. ...And one more cool thing about Pi Day: It also happens to be the birthday of Albert Einstein.
The Story of Allison Bates: How a CPS Teacher Went From 'Excellent' to 'Do-Not-Hire' in One Year
Government Lies, Corruption and Mismanagement
| Posted 2/18/2015 at 9:11 AM
The Chicago Public School System resembles the island in William Golding's "Lord of the Flies": give the administrators some power, and they will misuse it.
A Revitalized Teacher Union Movement: Reflections From The Field
Government Lies, Corruption and Mismanagement
| Posted 2/15/2015 at 12:19 AM
On the one hand, we need to fight to improve our public schools by organizing our members and allies to speak out against a variety of problems, including poorly rolled-out initiatives; large class sizes; lack of music, art, physical education, counselors, and librarians; restrictive curriculum mandates; and rogue principals. On the other hand, speaking out can play into the hands of the privatizers as they seek to expand privately run charters in what is already the nation's largest publicly funded private school voucher program.
New York Parent To Mrs. Obama: Save Our Children!
Government Lies, Corruption and Mismanagement
| Posted 2/7/2015 at 9:55 AM
Your husband, our president, is surrounded by policymakers who are not listening. It is high time that mothers evaluate what is best for our children! I am writing you an open letter because you receive much mail and my letter could be tossed aside by one of your aides. I am also interested in consensus and feel that this is a time when the more voices that are heard, the better our correction course may be!
Whistleblower Protection Act Wins with an Exclamation Mark
Success Stories: Positive Outcomes
| Posted 1/26/2015 at 7:01 PM
Today, after an 8 1/2 year legal ordeal, federal air marshal whistleblower and GAP client Robert MacLean won a Supreme Court decision affirming that his disclosures were covered by the Whistleblower Protection Act (WPA). MacLean publicly warned in 2003 that the Department of Homeland Security (DHS) planned to pull federal air marshals, sworn to protect the public, from commercial aircrafts targeted for an ambitious overseas terrorist attack. The key legal issue was whether the law's statutory free speech rights can be canceled by agency secrecy regulations.
What Happens When Kids Don't Have Internet at Home?
Stories & Grievances
| Posted 1/21/2015 at 12:19 AM
As part of an effort to bridge the so-called digital divide—the gap between rich and poor when it comes to access to technology—the Kent School District has for six years given every student a laptop, beginning in seventh grade. But some of these students don't need to carry the bags home—because they can't get online there. It's a problem that districts are increasingly facing as they turn to technology to revolutionize their teaching.
Special Education
Parentadvocates' Resources For Special Education Advocacy
An Introduction to Developing the IEP: FAQs on the Process of Obtaining the Best Individualized Education Program For Your Child
A Day in the Life of a Parent of a Child With Autism
Separating Fact From Fiction in Special Education Reform
General Education
Booker T. Washington Middle School 54, Grievance Brings Retaliation
Education Policy Becomes a Matter of National Security
New York City Teacher Theodore "Teddy" Smith and the Perfect Storm of Injustice
Bloomberg's Secret Government is Unknown Even to Him
Parents of Audrey, a Child With Special Needs, Sue Illinois School District 7 and Two Law Firms For Denial of FAPE
Personal Injury Statutes of Limitations: A Legal Guide
Parentadvocates.org Files Amicus Curiae Brief in Support of Jacob Winkelman v Parma City School District
Can We Tape? And, a Legal Website Directory
Without a Prayer For Relief: The NY State Supreme Court is Bought By Guide One Insurance Company and a Church, Madison Avenue Presbyterian
Editorial: The New York City Department of Education is a Sham and Mike Bloomberg is the Flim-Flam Man
How The New York City "Gotcha Squad" Gets Tenured Teachers Declared "Incompetent", and Placed in a Rubber Room by Betsy Combier
RICO in the New York State Unified Court System: How the Courts Steal Your Property, Your Children, and Try To Destroy Your Life...And How You Can Stop Them
Rules Regulations & Laws
Just Asking
What Do You Think?
Current Events
Stories & Grievances
Website Problems
© 2003 The E-Accountability Foundation


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quick linksCURRENT ISSUE PREVIOUS ISSUES NEWS INDUSTRY NEWS COLUMNISTS BROWSE EDITORIAL TOPICS WEBINARS
resourcesABOUT MT AUTHOR GUIDELINES CLASSIFIEDS EDITORIAL CALENDAR MEDIA GUIDE MASSAGE MART SCHOOLS & EDUCATION FEEDBACK
Older Patients, Stroke Risk and Manipulation
The first population-based study in the United States to evaluate stroke risk following spinal manipulation – and the first involving older adults – suggests that "[c]hiropractic cervical spine manipulation is unlikely to cause stroke in patients aged 66 to 99 years with neck pain.
Viewpoints: Massage Reduces Nonspecific Shoulder Pain, Improves Function
While seemingly universal, pain and stiffness in the shoulders can be a significant cause of disability. Often a pain that does not go away on its own, shoulder complaints tend to linger, sometimes for 12 months or longer.
How We Can Help the Injured Brain
The majority of patients with mild traumatic brain injuries recover within seven to 10 days. If concussion signs and symptoms continue beyond seven days, the diagnosis changes from acute concussion to post-concussion syndrome.
Pain Is Only a Piece of the Puzzle
More often than not, when a patient presents to the office, it is for a pain complaint: headache, neck pain, low back pain, sciatica, carpal tunnel, etc.
A Well-Kept Secret: 5 Element Acupuncture, Part II
Supervising acupuncture interns at a TCM college, it has always struck me how funny it is to hear the clinic manager tell the patients that the Five Element clinic specializes in treating emotions, as if patients with physical pain have no emotions!
Striking a Blow to the Medical Monopoly
The U.S. Supreme Court has issued a landmark ruling in North Carolina State Board of Dental Examiners v Federal Trade Commission.
Treating Beyond Pain
More often than not, when a patient presents to the office, it is for a pain complaint. Headache, neck pain, low back pain, sciatica, carpal tunnel... The pain is often the focus of the patient's mindset, and they don't often have any thought of what comes after the pain.
Recreational Cannabis Use and TCM
Many people are drawn to cannabis for its effects physically, mentally and emotionally. Medically, cannabis has some legitimate uses, however the scope of this article is limited to the recreational use of cannabis.
Managing Tibialis Posterior Tendon Injuries
The tibialis posterior is the deepest, strongest and most central muscle of the leg, with fibers originating from the tibia, fibula and interosseous membrane.
Treating GERD and Incontinence: Focus on Trigger Points
Gastroesophageal reflux disease (GERD) is defined as the regurgitation of stomach acid in the esophagus. Previously, it was thought that GERD was caused by a hiatal hernia, but recent trials suggest the cause is an inability of the hiatal sphincter to contract normally.
There Really is No Room for Sexism
Recently, Matteo* (a transgender male) approached me during a break in an advanced shiatsu class in Berlin where he was one of two men in a group of 20 women. "Pamela. Don't forget to remind the translator to include male endings."
Synergy Doesn't Happen in Silos: Acupuncture in Hospitals and Other Healthcare Settings
As acupuncture and traditional East Asian medicine continue to intersect and integrate with biomedical approaches, the conversation about integration expands and becomes richer.
Converting More Patients to Your Practice
In 2013 and 2014, the theme was "the money is in the list." This meant that if you had a big email list, you were really making some "cha-ching." Unfortunately, having thousands of emails doesn't equate to thousands of dollars in profit.
The Way We Are Designed: A Conversation with Gil Hedley, PhD
I was first introduced to the work of Gil Hedley by Tom DiFerdinando. He gifted me Gil's DVD series.
TCM Congress in Rothenburg is Largest in Western World
In the medieval town of Rothenburg, deep set within the Bavarian countryside in Southern Germany, the TCM Kongress Rothenburg each year draws around 1.200 participants from more than 40 different countries to attend the biggest TCM conference in the Western world.
What Do You Know About Physician Compare?
Physician Compare is a website that allows consumers to search for and obtain information about physicians and other health care professionals who provide Medicare services.
News in Brief
ACA Exec. Vice President Out, Acting EVP In; F4CP Executive Director Retires; New ED Named.
Keep Seniors Safe: Age-Proofing the Home
I want to give Dr. Claudia Anrig kudos for her Dec. 1, 2014 column, which highlighted safety issues youngsters might encounter in the home.
God and the Chiropractor
My wife went to church last Wednesday night and brought home a CD of the pastor's message. As she handed it to me, she said, "You should listen to this; you'll like it." Our family regularly goes to church and our faith plays a major role in our lives.
An Excerpt from TCM Case Studies: Pediatrics
This excerpt is reprinted with permission from Jamie Wu. TCM Case Studies: Pediatrics was released in 2014 by People's Medical Publishing House.
The Dietary Supplement Research Dilemma
I do not care what the truth is, one way or another; I just want to know it. And when it comes to dietary supplements, the truth can be hard to find for a number of reasons.
Will You Be an Amplifer or a Mute?
These times are changing, and changing quickly. There have been many challenges to this profession throughout the past few years. The challenge is to talk, then talk and talk some more about this medicine.
Joint Supplements for Athletes (Part 2)
A fairly recent discovery in nutrition supplemental medicine has proven to be a breakthrough in maintaining athletic joint health. Research suggests a combination of undenatured type-II collagen and tetrahydro-iso-alpha acids helps revitalize joint function and performance in athletes.
The Need for a New Medical Model: A Challenge for Biopsychosocial and Ecopsychologica Medicine
Chinese medicine speaks of alignment between humans, heaven and earth. It is a complex view with a focus upon relationship. These are comprehensive ideas with no specific terms in contemporary medical practice.
September, 2004, Vol. 04, Issue 09
Spa Letters
By Steve Capellini, LMT
Author's note: The Spa Letters column features news, personality profiles, trends and plenty of professional possibilities for LMTs in the spa industry. The style is epistolary, meaning the articles are letters to a fictional massage therapist friend of the author.
Dear Lou,
So, the roller coaster ride isn't over yet, just because your new day spa is open, right? In fact, a new ride has just begun.The ups and downs of the long planning stages are over, and you find yourself behind the wheel of a new enterprise with all the unforeseeable problems and challenges that are sure to arise. You're encountering one of those problems right now, in the very first month of operation. Though it's not a pretty one, it's all too typical.
After months of training and coaching, trusting and coddling, and friendship and teamwork, your lead esthetician, Marie, has shown her gratitude by not showing up for work one morning - quitting without giving notice. Isn't it amazing what goes through the minds of some people? What could she have been thinking to justify such an unfair move? Now, she's taking her newfound knowledge with her to a spa that is going to open across town in a few months - a new business, no doubt, inspired by the splash your Spa House made. The competition is going to be fierce, especially with the "insider's" knowledge that Marie will take to the other spa.
"Life is not fair!" You think, and perhaps you're right. But, as we say in the spa industry, Lou, it's no use crying over spilled milk and honey- exfoliating paste. There are a few proactive steps you can take at this interesting juncture.
Job Security - In Reverse
Usually when people talk about job security they mean the employee, not the employer. As a spa owner and employer, you are looking for the same kind of security from your employees as they're looking for from you. How can you trust them when you give them your best and they still abandon ship - like Marie? How can you know they'll be there for you in your time of need? Is there really any such thing as job security in the spa industry?
Spa employees know that their security is somewhat tenuous at times. There are a couple of spas that are unionized but, by and large, the majority is not, and employees have little recourse in instances of dismissal. Perhaps that is part of the reason why so many of them are ready to leave even a good job at the first sign of something better on the horizon. How can you get employees to be loyal?
There are some techniques that work well for some spa owners, such as offering money for continuing education, contingent upon the employee staying in the position for a certain amount of time; increasing pay scales over time often work; and giving people responsibility and a clear path toward career advancement helps, too. However (and this may be just my own overly nihilistic opinion), in the end, employees are out for themselves; they will do what they will do. It is better to go into your role as boss realizing this. It may be a mistake to put too much stock in your friendship with your employees. Doing so will often lead to disaster (emotional, as well as business-wise).
This is not to say you shouldn't be friendly with your staff - that is desirable. Remember though, that part of the relationship, no matter how friendly it gets, is also based on economics. Employees need you for the money you give them. You need them for the money they bring through the clientele. Friendships that exist within that dynamic are sometimes fragile things. That said, now what do you do? You're short an esthetician. In fact, you're short your only esthetician. You've spent months training her and thousands of dollars on the products she was going to use. Ahhhggg!
Of course, you've got to begin the search for a new esthetician. You might offer some of those incentives I mentioned earlier to your new recruit. Tell her you're going to increase her pay over time, and offer money for continuing education after a year of service. Tell her she'll make a higher commission rate as a "senior employee." And treat her like a professional first, a friend second.
In the meantime, before you find this person, you can take some immediate steps that will help you fill the gaps in your appointment book and make sure that you'll never ever again have to explain your complete lack of facial services to clients.
Face Treatment Options
You have a secret weapon on your staff when it comes to facial services. I'm talking about your massage therapists. You haven't been thinking about them offering esthetic-type treatments, but as I told you before, there is no reason to confine their scope-of-practice to the body alone. The face, in fact, is a part of the body, right? There are many face-specific services that massage therapists can perform to the satisfaction of your guests, the great support to your bottom line, and all without greatly angering the folks at the cosmetology board.
For example, I teach an Ayurvedic face massage treatment to students in my spa workshops, and they love it. The treatment consists of an application of herbs that are left to dry and then exfoliated off with the fingers. A hot towel is applied, and then herbal floral water followed by a pressure-point massage with essential oils from India. The treatment is finished up with a short massage using an emollient cream. It takes just half an hour, and it does not include extractions, diagnoses, or any of the specialties of our esthetician colleagues; however, clients report a healthy glow to their skin afterwards, induced, no doubt, by the balancing herbs and oils, plus increased circulation from the massage.
Clients also report feeling relaxed and cared for in that particular way that facials can make you feel. An esthetic treatment is different than a massage treatment. They both have their advantages, and these hybrid "face massage" treatments sometimes offer the best of both worlds. The Ayurvedic treatment can be emulated with a wide range of other products and techniques. Some of the more popular these days are Balinese, Indonesian and Thai. Getting your entire massage staff trained on these modalities is a great way to offer guests something different and assure that you won't be left without a facial service to offer if something like this ever happens again.
Of course, it's always a good idea to be aware of the laws in your state regarding face treatments. You definitely do not want to break any of them; in a few states, it is actually quite difficult to do even simple treatments, like the one I described above, if there is any chance it might impinge on cosmetologists' practice. Just be careful and do the right thing. I'm sure once your business starts to really rock, you'll have more than one esthetician on staff, and perhaps this problem will never arise again, but it's always good to be prepared, right? And don't forget about the new retail possibilities you'll be creating by adding these spa face massage treatments to your menu!
Well, Lou, I hope your second month in business is a little less traumatic than your first, and that you and your partner, Barbara, get the operation running smoothly after these initial hiccups. Always know that in the spa business there are usually more surprises than certainties. This makes for some great rewards, as well as challenges. I know you're up to them all!
Talk to you soon,
Steve Capellini, LMT
Click here for previous articles by Steve Capellini, LMT.
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text: All of my what? Flag By: wondersquid (More by author: Most viewed, Newest, Most Thumbs) previous next random - Fav + Fav Comment Also I need sauce..... For science Recommend tagsx +795 Views: 28880 Favorited: 74 Submitted: 07/21/2014 Share On Facebook Remove from favorites Add to favorites Unsubscribe from wondersquid Subscribe to wondersquid Seems you're using adblock :( This site costs a lot of money and time to run. It's also in danger of shutting down since everyone uses adblock and ads are used to pay the bills. You can donate here or just post some quality content.


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Why not welcome the flirtiest season of the year (hi, spring) by switching to an intoxicating new fragrance? Like one of these.
Style Stakeout: Long, Light Summer Dresses
Before she nailed her signature style, a fresh-faced Katy hit up a party with long, natural hair and gold eyeshadow.
Katy accessorized her shag haircut with a big flower and sported flushed cheeks with berry lips at a birthday bash for gossip blogger Perez Hilton.


text:
Below: A natural view of Glovetopia when it's not inundated in skepticism
Commandeer your own Glovetopia glove and tour around the forest. You can tour your glove as dangerously as you like, so long as it's not at more than 30 dragons per day, you don't suspend traffic, and you don't destroy cast members.
Please don't try Zombie Mode
© copyright 2011–2015
A Pants Aflame production


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"I'm a big fan of the outdoors and I'm looking for a group of people who enjoy more involved adventures."
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eBook ISBN: B00TKXN6KO
[ Contemporary Romance ]
Rowdy Pierce-warrior. Sara Stewart-artist. Macho mercenary rescues posh jewelry designer. Lives collide in a rescue in the desert. Sara falls for her body-to-die-for hero. Rowdy is fascinated by the luscious woman. He doesn't expect to lose his heart. Will their lust turn to something deeper back in the real world?
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American Masters TV Show Watchlist
Photos | Cast & Crew | Episode Guide | Video Clips
An acclaimed series of biographies of artists, writers, filmmakers, musicians and Hollywood stars. Profiles of Alexander Calder, Paul Simon, Charlie Chaplin and music producer John Hammond won Peabody Awards, while films about Edward R. Murrow, Lucille Bal (more…)
Rating: TV-PG
Season 18, Episode 2 James Brown: Soul Survivor
First Aired: October 29, 2003
James Brown (1933-2006), the "Godfather of Soul," is featured in this crisp 2003 profile, which also includes comments from Little Richard, Chuck D, Wyclef Jean, Dan Aykroyd and Rev. Al Sharpton, who says that Brown "changed the whole cultural paradigm of black America." Producer-director Jonathan Marre follows Brown from Augusta, Ga., in 1938, when he danced for nickels and dimes at age 5 in his aunt's "house of ill repute," back to Augusta.
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Page 1
Reductively-responsive siRNA-conjugated hydrogel
nanoparticles for gene silencing
Stuart S. Dunn†, Shaomin Tian†,‡,§, Steven Blake△, Jin Wang◇, Ashley L. Galloway#,
Andrew Murphy#, Patrick D. Pohlhaus#, Jason P. Rolland@, Mary E. Napier†,‡,§,^, and
Joseph M. DeSimone*,†,‡,§,×,¥,¶,°,≡,∥
†Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599
‡Carolina Center of Cancer Nanotechnology Excellence, University of North Carolina, Chapel Hill,
§Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599
△Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA
◇Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030
#Liquidia Technologies, Research Triangle Park, NC 22709
@Diagnostics For All, Cambridge, MA 02139
^Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599
×Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599
¥Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599
¶Institute for Advanced Materials, University of North Carolina, Chapel Hill, NC 27599
°Institute for Nanomedicine, University of North Carolina, Chapel Hill, NC 27599
≡Department of Chemical and Biomolecular Engineering, North Carolina State University,
Raleigh, NC 27695
∥Sloan-Kettering Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, New
York, NY 10021
A critical need still remains for effective delivery of RNA interference (RNAi) therapeutics to
target tissues and cells. Self-assembled lipid- and polymer-based systems have been most
extensively explored for transfection with small interfering RNA (siRNA) in liver and cancer
therapies. Safety and compatibility of materials implemented in delivery systems must be ensured
to maximize therapeutic indices. Hydrogel nanoparticles of defined dimensions and compositions,
*Corresponding email@example.com.
ASSOCIATED CONTENT Supporting Information. Compositions of PRINT particles and additional Zetasizer measurements in
Tables S1-S4. Viability of HeLa cells dosed with PEGylated hydrogels and pro-siRNA hydrogels containing different siRNA cargos,
transfection of HeLa cells with different siRNAs using Lipofectamine, gel electrophoresis of siRNA loaded into and released from
different hydrogels, and analysis of siRNAs by HPLC in Figures S1-S9. Completed author list for abbreviated references. This
material is available free of charge via the Internet at http://pubs.acs.org.
Notes The authors declare competing financial interest. Joseph DeSimone is a founder, member of the board of directors, and
maintains a financial interest in Liquidia Technologies. Liquidia was founded in 2004 to commercialize PRINT technology and other
discoveries of Professor Joseph DeSimone and colleagues at the University of North Carolina, Chapel Hill.
NIH Public Access
Author Manuscript
J Am Chem Soc. Author manuscript; available in PMC 2013 May 02.
Published in final edited form as:
J Am Chem Soc. 2012 May 2; 134(17): 7423–7430. doi:10.1021/ja300174v.
NIH-PA Author Manuscript
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prepared via a particle molding process that is a unique off-shoot of soft lithography known as
PRINT (Particle Replication in Non-wetting Templates), were explored in these studies as
delivery vectors. Initially, siRNA was encapsulated in particles through electrostatic association
and physical entrapment. Dose-dependent gene silencing was elicited by PEGylated hydrogels at
low siRNA doses without cytotoxicity. To prevent disassociation of cargo from particles after
systemic administration or during post-fabrication processing for surface functionalization, a
polymerizable siRNA pro-drug conjugate with a degradable, disulfide linkage was prepared.
Triggered release of siRNA from the prodrug hydrogels was observed under a reducing
environment while cargo retention and integrity were maintained under physiological conditions.
Gene silencing efficiency and cytocompatibility were optimized by screening the amine content of
the particles. When appropriate control siRNA cargos were loaded into hydrogels, gene
knockdown was only encountered for hydrogels containing releasable, target-specific siRNAs,
accompanied by minimal cell death. Further investigation into shape, size, and surface decoration
of siRNA-conjugated hydrogels should enable efficacious targeted in vivo RNAi therapies.
PRINT; soft lithography; siRNA; hydrogel; nanoparticle; gene silencing
Gene silencing via RNA interference (RNAi)1,2 has demonstrated great potential for the
treatment of diseases3,4 by halting the production of target proteins. The major challenge in
realizing the potential of RNAi therapies resides in delivering small interfering RNA
(siRNA) effectively to the cytoplasm of a target cell. With a highly negatively charged
backbone and a molecular weight of ca. 13 kDa, siRNA is unable to effectively cross cell
membranes without assistance. Additionally, siRNA is susceptible to degradation by
ubiquitous RNases in serum. A suitable carrier is required to enhance stability and facilitate
delivery to the cytoplasm of cells. Exemplar carriers include oligonucleotide conjugates5-14,
polyplexes8,15-18, and lipoplexes19-22. After systemic administration, the siRNA carrier
encounters several biological hurdles en route to the target tissue and cell such as clearance
by the reticuloendothelium system, protein fouling, and size requirements to reach particular
tissues. Designing delivery vehicles with surface decorations including stealthing (e.g.
polyethylene glycol, PEG23) and targeting (e.g. peptide13) ligands may promote prolonged
circulation and passive delivery to tissues of interest followed by actively targeting cell
surface receptors for internalization by desired cells.
Hydrogels and nanogels have been explored as delivery vector candidates for transfection of
cells with siRNA. Effective gene knockdown in kidney, epithelial, ovarian, and hepatoma
cell lines has been achieved by siRNA-associated hydrogels and nanogels24-28. Due to their
robust and tunable mechanical properties, hydrogel micro- or nano-particles may enable
delivery of siRNA to a wide range of tissues in vivo including delivery to circulating cells.
Bottom-up approaches for encapsulating siRNA in nanogels through electrostatic attraction
post-fabrication may result in dynamic association of cargo, uncontrollable cargo release,
and modification of particle surface properties. Particle Replication in Non-wetting
Templates (PRINT®) technology allows for fabrication of hydrogels with control over size,
shape, composition, surface chemistry, and modulus such that delivery properties may be
tuned to particular applications29-36. Encapsulation of siRNA in hydrogels may be
engineered with PRINT technology, which allows for direct physical entrapment or covalent
incorporation of siRNA during particle fabrication.
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Results and Discussion
Transfection of cancer cells with siRNA electrostatically entrapped in hydrogels
Initially, a highly cationic, moderately crosslinked hydrogel composition (Table S1) was
pursued to allow for physical entrapment and electrostatic association of siRNA within
cylindrical (diameter [d] = 200 nm; height [h] = 200 nm) PRINT particles prepared by a
film-split technique. To promote cytocompatibility and dispersibility of highly cationic
hydrogel nanoparticles in aqueous media, amine handles on hydrogels were reacted with
succinimidyl succinate monomethoxy PEG2K (Fig. 1a). After PEGylating the hydrogels, a
concomitant decrease in the ζ-potential was observed (Table S2), resulting in a low surface
charge that would be minimally toxic to cell membranes. Steady release of siRNA from the
hydrogel particles in PBS at 37 °C was observed over time, reaching maximum
concentration around 48 h (Fig. 1b). By gel electrophoresis, siRNA loading was determined
to be about 1.4 wt%, and encapsulation efficiency of siRNA in hydrogels was determined to
be ca. 28% (1.4 wt% final siRNA loading in particles relative to the 5 wt% siRNA charged
into the composition of the pre-particle solution). Scanning electron microscopy (SEM)
analysis of the hydrogel particles demonstrates their cylindrical shape and dimensions (Fig.
To evaluate the transfection potential of particles loaded with siRNA, a stably-transfected,
luciferase-expressing human cervical cancer (HeLa/luc) cell line was utilized for in vitro
studies. Particles were dosed on HeLa cells for 4 h followed by 72 h incubation. Due to the
positive charge of the particles, the PRINT hydrogel particles were readily internalized into
the HeLa cells (Fig. 2a) as determined by flow cytometry. Dose-dependent knockdown of
luciferase expression (Fig. 2b) was observed for HeLa cells incubated with the anti-
luciferase siRNA-charged particles with a half-maximal effective concentration (EC50) of
ca. 6 nM siRNA. Conversely, PRINT hydrogel particles charged with a control siRNA
sequence did not elicit gene knockdown, implying that transfection was sequence-specific
(Fig. 2b). Additionally, both particles were found to be cytocompatible with HeLa cells (Fig.
S1). Confocal microscopy of HeLa cells dosed with PEGylated particles (Fig. 2c,d) shows
the internalization of fluorescent particles and their distribution throughout the cellular
cytoplasm as well as the perinuclear region. Even though the desired cell activity was
achieved with these particles, subsequent efforts to modulate in vivo behavior by
conjugation of targeting ligands to the particle surface resulted in extensive premature
release of siRNA. For example, 50% loss of encapsulated cargo was observed during a
sequence of reactions to add targeting moieties to the particle surface (Fig. S2).
siRNA prodrug for triggered release from hydrogels
In order to combat the premature release issues with the PRINT hydrogel particles described
above, an alternative prodrug strategy was employed which involved covalently conjugating
the siRNA directly to the PRINT hydrogel particles. Acrydite DNA oligonucleotides have
been incorporated into hydrogels for nucleic acid hybridization assays37,38 while a CpG
oligonucleotide methacrylamide has been copolymerized into aciddegradable microparticles
to invoke immune responses39,40. For the triggered release of therapeutic conjugates from
particles or delivery vectors under biologically relevant conditions, acidlabile41-43,
enzymatically degradable44,45, and redox-sensitive linkages46 have been examined.
Glutathione and reducing enzymes are present at high concentrations inside cells relative to
the extracellular space in normal and cancer cell lines47. Considering the serum stability of
disulfide conjugates46,48, cleavage of disulfide linkages in therapeutic conjugates may
selectively occur in the intracellular reducing environment. Disulfide-siRNA conjugates to
polymers and lipids have been previously reported6,7,10-12 as reductively-labile systems.
Dunn et al.
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In this work, siRNA was derivatized with a photopolymerizable acrylate bearing a
degradable disulfide linkage for reversible covalent incorporation into the PRINT hydrogel
nanoparticles (Fig. 3a). In the designed 'pro-siRNA hydrogels', it was envisioned that the
siRNA cargo would be retained in the particle until entry of the particle into the cytoplasm
of a cell, where the disulfide linkage would be cleaved in the reducing environment,
allowing for release and delivery of the siRNA (Fig. 3b). A water-based pre-particle
composition containing the disulfide siRNA pro-drug and a higher content of hygroscopic,
liquid monomers (Table S3) was applied to fabricate cylindrical (d = 200 nm; h = 200 nm)
loosely-crosslinked cationic PRINT hydrogel particles using a film-split technique. SEM
micrographs confirmed the dimensions and shape of cylindrical siRNAcontaining particles
(Fig. 3c).
To study the response of pro-siRNA hydrogels to a reducing environment, non-disulfide
acrylamide siRNA-based hydrogel particles and native siRNA-complexed particles were
prepared as controls (Fig. 3a). Release of native siRNA-NH2 occurred rapidly from these
porous, loosely-crosslinked hydrogels (Fig. S4). Fig. S5 showed that different siRNAs were
loaded into hydrogel particles and remained associated with particles directly after
harvesting. Unconjugated siRNA was found in hydrogels charged with native siRNA or
siRNA macromers due to incomplete conversion; therefore, in the following experiments all
particles were extensively washed with 10x PBS to remove the sol fraction containing free
siRNA. The time-dependent release of the siRNA from the pro-drug PRINT hydrogel
particles was evaluated under physiological and reducing conditions (Fig. 4a). The siRNA
was retained in the hydrogel particles over 48 h at 37 °C in PBS while the siRNA was
quickly released from hydrogels when incubated in a reducing environment (5 mM
glutathione), reaching maximum concentration around 4 h (Fig. 4a).
Moreover, the siRNA conjugate did not even leak out of the hydrogel particles when they
were exposed to high salt concentration buffer (10x PBS) at 37 °C for 4 h (Fig. 4b).
Incubation of the non-disulfide, non-degradable acrylamide control siRNA hydrogel
particles did not result in release of the siRNA under reducing conditions as expected.
Stability of the siRNA covalently conjugated to the PRINT hydrogel particles was tested by
incubation of the particles in serum (10% FBS) as a function of time. Over 48 h, the siRNA
in the pro-drug PRINT hydrogel particles could be protected from degradation by RNases
when incubated in serum while siRNA in the form of the simple macromonomer in the
absence of the particle to protect it was rapidly degraded in serum under the same conditions
(Fig. 4c).
pro-siRNA hydrogels for gene silencing
The PRINT particles were designed to have a positive zeta potential to facilitate cell
internalization and endosomal escape by including an amine monomer (AEM, 2-
aminoethylmethacrylate hydrochloride). It is known that excessive amine content in
hydrogels may disrupt and destroy the plasma membrane, eliciting cell death. Conversely,
an insufficient amine content may not enable efficient cell uptake and endosomal escape for
transfection. To optimize cytocompatibility and gene silencing efficiency of pro-siRNA
hydrogels, the AEM content was varied from 5 to 50 wt% (Table S3). Pro-siRNA hydrogels
were not PEGylated as were siRNA-complexed hydrogels whereby the cytocompatibility of
bare particles would be determined. For systemic delivery of particles in vivo, sheddable
coatings that reveal bare particles are attractive to enable targeting and endosomal escape for
effective gene delivery49,50.
ζ-potentials of cationic hydrogels increased with amine content and the Z-average diameters
(Dz) of the resultant particles ranged from 250 to 350 nm (Table 1). Encapsulation of the
siRNA in the hydrogel PRINT particles reached a roughly constant value once the amine
Dunn et al.
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content was greater than or equal to 20 wt% (Fig. S6). The encapsulation efficiency was
determined to be ca. 35% for AEM contents ≥ 20 wt% while when only using 5% AEM, the
encapsulation was lower (ca. 15%). When the pro-drug, disulfide-containing siRNA
hydrogel PRINT particles were dosed onto luciferase-transfected HeLa cells (HeLa/luc) for
5 h followed by 48 h incubation at 37 °C, dosedependent knockdown of the luciferase
expression was observed (Fig. 5a) for hydrogels with amine contents greater than 5 wt%
AEM. Cytocompatibility was maintained at the lower amine contents and dosing
concentrations (Fig. 5b). It appeared that the 30% AEM-containing PRINT hydrogel
particles provided the ideal combination of gene silencing efficiency (EC50 ~ 20 nM siRNA)
and cytocompatibility (even at high dosing concentrations).
To further investigate the in vitro gene knockdown efficacy of the PRINT hydrogel
particles, the 30% AEM-based hydrogel composition was utilized with four different cargos:
(1) native luc siRNA, (2) degradable disulfide luc siRNA, (3) non-degradable, acrylamide
luc siRNA, and (4) degradable disulfide control siRNA. Zetasizer analysis of the hydrogel
PRINT particles indicated that their size and charge were similar (Table S4) and gel
electrophoresis (Figs. S7) allowed for confirmation of the release of the various cargos.
After dosing the particles on cells and incubating for 48 h, cell viability was maintained
above 80% for all of the samples across all dosing concentrations, except for the particles
charged with the free siRNA (Fig. S8). Uptake of all of the hydrogel particles approached
saturation at around 50 μg/mL particle concentration (Fig. 6a). Dose-dependent silencing of
luciferase expression was elicited notably for the pro-drug, disulfide-based siRNA-
containing hydrogel particles while the control particles did not elicit significant gene
knockdown (Fig. 6b).
The transfection efficiency between siRNA-complexed, PEGylated particles and pro-siRNA
hydrogels is comparable, which can be explained by differences in release characteristics.
PEGylated particles release siRNA slowly in PBS while pro-siRNA hydrogels rapidly
release siRNA under intracellular conditions. Confocal microscopy images of HeLa cells
dosed with particles (Figs. 6c-f) illustrates uptake of the particles, which accumulated
largely in the cytoplasm and perinuclear region. Particles appear significantly brighter in
Figs. 6c-f compared to Figs. 2c,d due to the higher fluorescence intensity emitted by
DyLight 488 compared to fluorescein. Given the internalization of cationic particles by
HeLa cells and distribution throughout the cytoplasm and perinuclear area, potential exists
for the encapsulation of other therapeutic nucleic acids in hydrogel nanoparticles to transfect
diseased cells.
siRNA may be physically or electrostatically entrapped within hydrogel nanoparticles to
provide effective gene knockdown. Nevertheless, to retain cargo during particle
functionalization and under physiological conditions, a covalent incorporation approach was
deemed necessary. To our knowledge, this is the first work to polymerize siRNA in
hydrogel particles for controlled intracellular delivery. The siRNA cargo was found to be
adequately protected by the particles and only released upon reaching the reducing
environment of the cytoplasm for effective gene knockdown. Physical dimensions and
surface functionalization of pro-siRNA hydrogel nanoparticles with targeting and stealthing
ligands are currently under investigation to enable in vivo systemic delivery of siRNAs for
the treatment of diseases.
Dunn et al.
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Experimental Section
2,2′-dithiodiethanol, acryloyl chloride, PEG700 diacrylate, disuccinimidyl carbonate (DSC),
2-aminoethyl methacrylate hydrochloride (AEM), 1-hydroxycyclohexyl phenyl ketone
(HCPK), fluorescein O-acrylate, tetraethylene glycol, and Irgacure 2959 were purchased
from Sigma Aldrich. Poly(vinyl alcohol) 75% hydrolyzed MW ≈ 2 kDa was obtained from
Acros Organics. SS-mPEG2K and PEG1K dimethacrylate were from Polysciences, Inc.
mPEG5K acrylate and SCM-PEG2K-biotin were from Creative PEGWorks. Anti-human
CD71 (Transferrin Receptor) biotinylated OKT9 monoclonal antibody was purchased from
eBioscience. UltraAvidin was obtained from Leinco Technologies. Tetraethylene glycol
monoacrylate (HP4A) was synthesized in-house and kindly provided by Dr. Matthew C.
Parrott, Dr. Ashish Pandya, and Mathew Finniss. PRINT molds were graciously supplied by
Liquidia Technologies. siRNAs were purchased as duplexes from Dharmacon, Inc. Sense
sequence of amine-modified and native anti-luciferase siRNA: 5′-N6-
GAUUAUGUCCGGUUAUGUAUU-3′; anti-sense: 5′- P-
UACAUAACCGGACAUAAUCUU-3′. Sense sequence of amine-modified and native
control siRNA: 5′-N6- AUGUAUUGGCCUGUAUUAGUU-3′; anti-sense: 5′-P-
CUAAUACAGGCCAAUACAUU-3′. All other reagents were obtained from Thermo
Fisher Scientific, Inc. in molecular biology grade or RNase-free when available.
Synthesis of siRNA macromers
Degradable disulfide macromer precursor: 2,2′-dithiodiethanol (15 mL, 0.12 mol) was
dissolved in anhydrous DMF (250 mL) in a 500-mL round-bottomed flask containing NEt3
(20.5 mL, 1.2 eq) under a N2 blanket to which acryloyl chloride (11.0 mL, 1.1 eq) was
added dropwise and allowed to react for 8 h. Crude product was extracted into
dichloromethane against 5% LiCl and purified via silica gel chromatography
(EtOAc:hexanes) to provide monoacrylate-substituted 2,2′-dithiodiethanol (63% yield). 2-
((2hydroxyethyl)disulfanyl)ethyl acrylate (10 g, 48 mmol) was dissolved in anhydrous
acetonitrile (100 mL) in a N2-purged 250mL round-bottomed flask, followed by addition of
disuccinimidyl carbonate (14.8 g, 1.2 eq). The reaction proceeded for 8 h and product was
purified by silica gel chromatography (EtOAc:hexanes 4:1) to afford 2-N-
hydroxysuccinimide, 2′acryloyl-dithiodiethanol as a clear, viscous liquid (82% yield). 1H
NMR (600 MHz, CDCl3) δ = 6.46 (dd, J = 17.4 Hz, 1H), δ = 6.2 (dd, J = 10.7 Hz, 6.8 Hz,
1H), δ = 5.90 (dd, J = 10.7 Hz, 1H), δ = 4.59 (t, J = 6.5 Hz, 2H), δ = 4.45 (t, J = 6.5 Hz, 2H),
δ = 3.05–3.00 (m, J = 6.8 Hz, 4H), δ = 2.87 (s, 4H).
Non-degradable siRNA conjugate precursor: N-hydroxyethyl acrylamide (15 mL, 0.14 mol)
was dissolved with DSC (51.9 g, 1.4 eq) in ACN:DMF 4:1 (250 mL) and reacted for 16 h.
Afterward, ACN was removed via rotary evaporation and product was extracted into EtOAc
against 5% LiCl. Product was concentrated and purified by silica gel column
chromatography (EtOAc:hexanes 4:1) to provide 2-(succinimidyl carbonate)ethyl
acrylamide (80% yield) as a fine white solid. 1H NMR (600 MHz, CDCl3) δ = 6.50 (br, 1H,
NH), δ = 6.34 (dd, J = 17.1 Hz, 1H), δ = 6.19 (dd, J = 10.3 Hz, 6.4 Hz, 1H), δ = 5.71 (dd, J
= 10.3 Hz, 1H), δ = 4.46 (t, J = 5.0, 2H), δ = 3.71 (m, J = 5.5 Hz, 2H), δ = 2.87 (s, 4H).
siRNA macromers: siRNA-NH2 (2 mg, 148 nmol, anti-luciferase siRNA or control
sequence) was dissolved in DEPC-treated PBS (200 μL) in a 1.5-mL RNase-free Eppendorf
tube. Separately, 2N-hydroxysuccinimide, 2′-acryloyl-dithiodiethanol (5.2 mg, 100 eq) or 2-
(succinimidyl carbonate)ethyl acrylamide (3.8 mg, 100 eq) was dissolved in RNase-free
DMF (150 μL) and added to the solution of siRNA. The reaction was allowed to proceed for
36 h where additional 100 eq of the acrylate or acrylamide were added to the reaction
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mixture every 12 h. 5 M NH4OAc (50 μL) and EtOH (1.1 mL) were added to the reaction
mixture, which was vortexed for 15 s. The sample was incubated in a -80 °C freezer for 4 h
followed by centrifugation (14 krpm, 4 °C, 20 min) to pellet the siRNA. The supernatant
was decanted and the pellet was washed twice with 70% EtOH (ice-cold) to provide siRNA
prodrug (79% yield). HR-ESI-MS: m/z found for siRNA sense strand [M-H]− = 6832.366;
m/z calc. for disulfide macromer [M-H]− = 7067.676; found [M-H]− = 7067.855; m/z calc.
for siRNA acrylamide macromer [M-H]− = 6974.506; found [M-H]− = 6974.871.
Characterization of siRNA prodrug precursors was carried out on a 600 MHz Bruker NMR
Spectrometer equipped with a Cryoprobe and siRNA macromonomers were analyzed by an
IonSpec Fourier Transform Mass Spectrometer FTMS (20503 Crescent Bay Drive, Lake
Forest, CA 92630) with a nano electrospray ionization source in combination with a
NanoMate (Advion 19 Brown Road, Ithaca, NY 14850) chip based electrospray sample
introduction system and nozzle operated in the negative ion mode as well as reversed phase
high-performance liquid chromatography (Figs. S9).
Fabrication of hydrogels via PRINT process
Pre-particle solutions were prepared with listed compositions at 2.5 wt% in RNase-free
DMF (for physically entrapped siRNA) or DEPC-treated water containing 0.01 % sodium
dodecyl sulfate (for prodrug siRNA, where all remaining steps were conducted in a humidity
room maintained at 70% relative humidity). Specifically, for each composition, solid and
liquid monomers were dissolved in respective pre-particle solution solvent at a total weight
percent of 2.5%. The film-split technique for preparing particles was performed as described
in the following: using a #5 wire wound rod (R.D.S.), 150 μL of pre-particle solution was
cast at 6 ft/min on a sheet of poly(ethylene terephthalate) (PET), followed by brief
evaporation of solvent with heat gun to yield a transparent film (delivery sheet). 200 × 200
nm cylindrical Fluorocur-patterned PRINT molds (Liquidia Technologies) were laminated
against the delivery sheet with moderate pressure (40 psi) and then gently delaminated. The
filled mold was laminated against corona-treated PET and subsequently cured in a UV
chamber (λmax = 365 nm, 90 mW/cm2) for 5 min. After photocuring, the mold was removed
to reveal an array of particles on PET. Particles were harvested off PET with water
mechanically using a cell scraper (1 mL/48 in2). Particles were washed via centrifugation
(15 min, 14 krpm, 4 °C), removal of supernatant, and re-suspension in water. Particle yield
was determined by thermogravimetric analysis (Q5000IR, TA instruments). Pro-siRNA
hydrogels were washed repeatedly with 10x PBS containing 0.05% PVA 2 kDa to remove
the sol fraction.
Hydrogels with electrostatically-entrapped siRNA were PEGylated by reacting particles
with SS-mPEG2K (2 wt eq, weight equivalents relative to particle mass) using pyridine (2 wt
eq) in DMF at 2 mg/mL particle concentration for 6 h at room temperature followed by
washing particles with water. For post-functionalization of particles with ligands, SCM-
PEG2K-biotin (2 wt eq) was reacted with particles in DMF at 2 mg/mL for 2 h along with
pyridine (4 wt eq). NHS-mPEG12 (2 wt eq) was added to quench non-biotinylated amines
with pyridine (4 wt eq) in DMF at 2 mg/mL for 30 min. Particles were centrifuged, isolated
through removal of supernatant, and washed with PBS. UltraAvidin (0.25 wt eq) was
conjugated to biotinylated particles in PBS at 2 mg/mL for 20 min, followed by
centrifugation, washing with PBS, and adding biotinylated OKT9 antibody (20 wt meq) for
another 20 min at 2 mg/mL, after which particles were washed with PBS to remove
unconjugated antibody and re-suspended in PBS at 2 mg/mL.
Particle characterization
Scanning electron microscropy (SEM) enabled imaging of hydrogels that were dispersed on
a glass slide and coated with 2 nm of Au/Pd (Hitachi S-4700). ζ-potential measurements
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were conducted on 20 μg/mL particle dispersions in 1 mM KCl using a Zetasizer Nano ZS
Particle Analyzer (Malvern Instruments Inc.).
Analysis of siRNA by gel electrophoresis
2.5% agarose gel in TBE buffer was prepared with 0.5 μg/mL ethidium bromide. For
studying release of siRNA from hydrogels, aliquots of particle dispersions were centrifuged
(15 min, 14 krpm, 4 °C) for recovery of the supernatant at various time points and frozen.
Similarly, aliquots of siRNA prodrug incubated in 10% FBS at 37 °C were taken at various
time points and frozen for storage. 12 μL of sample (supernatants from particle dispersions,
siRNA solutions, or particle dispersions) was mixed with 3 μL of 6x loading buffer (60%
glycerol, 0.12 M EDTA in DEPC-treated water) and loaded into the gel. 70 V/cm was
applied for 25 min and the gel was then imaged with ImageQuant LAS 4000 (GE). Analysis
of siRNA band intensity was conducted with Image J software for quantification. siRNA
loading was calculated by comparing the maximum amount of siRNA released to the
particle mass. Encapsulation efficiency of siRNA was calculated by comparing wt% of final
siRNA loading to the siRNA charged into the pre-particle composition (5 wt% of particle).
Cell culture
Luciferase-expressing HeLa cell line (HeLa/luc) was from Xenogen. HeLa/luc cells were
maintained in DMEM high glucose supplemented with 10% FBS, 2 mM L-glutamine, 50
units/mL penicillin and 50 μg/mL streptomycin, 1 mM sodium pyruvate and non-essential
amino acids. All media and supplements were from GIBCO except for FBS which was from
Mediatech, Inc.
In vitro cell uptake analysis
HeLa/luc cells were plated in 96-well plates at 10,000/well and incubated overnight at 37°C.
Cells were dosed with fluorescently-tagged particles in OPTI-MEM at 37 °C (5 % CO2) for
4 h or indicated time for cell uptake studies. Fluorescently-tagged particles were prepared by
incorporation of fluorescent monomers (fluorescein O-acrylate for PEGylated particles and
DyLight 488 maleimide for pro-siRNA particles) in the particle composition and
copolymerization of these monomers into particles matrix. After incubation, cells were
washed and detached by trypsinization. After centrifugation, cells were resuspended in a 0.4
% trypan blue (TB) solution in Dulbecco's Phosphate Buffers Saline solution (DPBS) to
quench the fluorescein fluorescence from particles associated to the cell surface. Cells were
then washed and resuspended in DPBS or fixed in 1% paraformaldehyde/DPBS, and
analyzed by CyAn ADP flowcytometer (Dako). Cell uptake was represented as percentage
of cells that were positive in fluorescein fluorescence.
In vitro cytotoxicity and luciferase expression assays
HeLa/luc cells were plated in 96-well plate at 10,000/well and incubated overnight at 37°C.
Cells were dosed with particles or Lipofec-tamine 2000/siRNA mix in OPTI-MEM at 37 °C
(5 % CO2) for 4 or 5 h, then particles were removed, and complete grow medium was added
for another 48 h incubation at 37°C. Cell viability was evaluated with Promega CellTiter
96® AQueous One Solution Cell Proliferation Assay, and luciferase expression level was
evaluated with Promega Bright-Glo™ Luciferase Assay according to manufacturer's
instructions. Light absorption or bioluminescence was measured by a SpectraMax M5 plate
reader (Molecular Devices). The viability or luciferase expression of the cells exposed to
PRINT particles was expressed as a percentage of that of cells grown in the absence of
particles. Half-maximal effective concentration (EC50) of siRNA required to elicit gene
knockdown was determined by applying the dose-dependent gene knockdown data to a
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log(inhibitor) vs. response variable slope non-linear function in GraphPad Prism software
for given siRNA concentrations (calculated from particle concentration and siRNA loading).
Confocal laser scanning microscopy
HeLa/luc cells were plated in 8-well chamber slides (BD) at 10,000/well and incubated
overnight at 37°C. Cells were dosed with green fluorescent particles (fluorescein or DyLight
488 labeled) in OPTI-MEM at 37 °C (5 % CO2) for 4 h. Cells were then washed three times
with PBS, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.5%
saponin for 30 min at room temperature. Cells were stained with 100 nM phalloidin-
AlexaFluor 555 for actin for 1 h at room temperature, and then counterstained with 30 μM
DAPI for 15 min. Images were collected with LSM710 confocal laser scanning microscope
(Carl Zeiss).
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
This work was supported in part by the Carolina Center for Cancer Nanotechnology Excellence (NIH-U54-
CA119343 and NIH-U54CA151652), National Institutes of Health Director's Pioneer Award (1DP1OD006432)
and R01 (R01EB009565), University of North Carolina Cancer Research Fund, the Chancellor's Eminent
Professorship of Chemistry at the University of North Carolina at Chapel Hill, and a sponsored research agreement
with Liquidia Technologies. We thank Dr. J. Christopher Luft, Dr. Warefta Hasan, Jing Xu, and Luke Roode for
helpful discussions throughout this work as well as Dr. Kevin P. Herlihy for particle illustrations in Figures 1 and 2.
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Figure 1.
(a) Reaction scheme for PEGylation of hydrogels with succinimidyl succinate
monomethoxy PEG2K (SS-mPEG2K), (b) time-dependent release of siRNA from particles
incubated at 2 mg/mL and 37 °C in PBS, and (c) Scanning electron micrograph (SEM) of
particles illustrating their 200×200 nm cylindrical dimensions (scale bar = 2 μm).
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Figure 2.
siRNA delivery with PEGylated cationic hydrogels to luciferase-expressing human cervical
cancer (HeLa/luc) cells. (a) Cellular uptake. HeLa/luc cells were dosed with particles for 4 h
followed by trypan blue treatment and flow cytometry analysis. (b) Luciferase expression.
HeLa/luc cells were dosed with particles for 4 h followed by removal of particles and 72 h
incubation in media. Data are representative of at least three independent experiments. The
error bars represent standard deviation from triplicate wells in one experiment. (c) and (d)
Confocal micrographs. HeLa/luc cells were dosed with 50 μg/mL particles containing (c)
luciferase or (d) control siRNA cargos for 4 h. Cellular actin cytoskeleton was stained with
phalloidin (red) and nuclei with DAPI (blue), while particles (green) were labeled with the
fluorescent monomer fluorescein O-acrylate during particle fabrication.
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Figure 3.
(a) Structures of degradable and non-degradable siRNA macromers as well as native siRNA,
(b) Illustration of pro-siRNA hydrogel behavior under physiological and intracellular
conditions, and (c) SEM micrograph of pro-siRNA, 200 × 200 nm cylindrical nanoparticles
(scale bar = 2 μm).
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Figure 4.
Release profiles and stability of siRNA in 30% AEM-based hydrogels. All hydrogels were
washed with 10x PBS buffer to remove the sol fraction containing unconjugated siRNA
before release studies were performed. (a) Time-dependent incubation of pro-siRNA
hydrogels (1 mg/mL) in PBS and under reducing conditions (glutathione, 5 mM) at 37 °C.
(b) Selective release of the disulfide-coupled siRNA prodrug (PD) from hydrogels under
reducing conditions compared to the acrylamide (AA) macromer and native siRNA (NH2).
Hydrogels were incubated in 10x PBS with or without 5 mM glutathione for 4 h at 1 mg/mL
and 37 °C. (c) Retention of siRNA integrity when conjugated to hydrogels after exposure to
10% fetal bovine serum (FBS) over time. Naked siRNA PD macromer was incubated at 36
ug/mL in 10% FBS for given times, proceeded by storage of solution. pro-siRNA hydrogels
were incubated at 1.2 mg/mL in 10% FBS at 37 °C for given times followed by incubation
in 10x PBS (5 mM glutathione) for 4 h at 1.2 mg/mL and 37 °C to release siRNA.
Differences in siRNA migration observed in gels among the standards and samples which
were released from hydrogels incubated in PBS and 10x PBS may arise from the differences
in salt concentrations of sample solutions.
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It seems that Hollywood is starting to catch on to what many viewers already know: Some of the best shows on TV are not on a network; they're streaming over your internet connection. Shows such as House of Cards and Orange Is the New Black from Netflix have been capturing people's imagination for years, although major awards have often eluded them. But in the first months of 2015, one announcement after another pointed toward a sort of streaming content renaissance.
Oct 12, 2007
CrossRef, a non-profit membership association founded and directed by publishers, announced that BSI British Standards has joined as a new CrossRef member. As a member, subject to the creation of an appropriate protocol, BSI will be able to register standards with CrossRef and implement interlinking with other scholarly and scientific publications. This will be achieved through use of the DOI (Digital Object Identifier) System for which CrossRef provides content registration services. CrossRef includes hundreds of publishers and societies, with 28.9 million content items registered to date. BSI British Standards has 27,000 current standards. CrossRef and BSI British Standards will join forces to establish a working group of interested parties, made up of both CrossRef members and outside organizations, to discuss best practice for assigning DOI names to standards.
(www.crossref.org, www.bsigroup.com)


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Introducing the Honey Badger Award for Extracurricular Excellence
Every spring and summer, some college football players trade exerting their energy on the gridiron toward exerting in in various, um, extracurricular activities including DUIs, arrests and other freelance activities.
Since these incidents are so copious, Rant Sports has created the Honey Badger Award for Extracurricular Excellence to be given to the team that participates in the most extra-curricular activity from the day after the National Championship game up to Aug. 1. I know, I know, it's a tough task– but someone has to do it.
We'll be bringing you the weekly rankings based on our own tracking of this extra activity from week to week each Friday afternoon.
Here are the inaugural rankings– things are off to a slow start, but I'm sure they'll pick up soon:
Honey Badger Award for Extracurricular Excellence- Week One
Week One brought us only two off-field shenanigans, one by a player involved with the Kentucky Wildcats and the other from the Florida State Seminoles.
On Tuesday, Seminoles running back James Wilder was arrested for the third time in less than a year, this time for failing to appear in court for a charge of driving with a suspended license. Maybe he was scared to drive to court. News flash, James, there are these magical vehicles called buses that can get you places without you driving. I know, that 21st century technology is mind-blowing!
Kentucky Wildcats offensive lineman T.J. Jones was hit with a with a weapons law violation after reportedly bringing a loaded gun into his former high school's locker room following an altercation. I'll leave this one alone–there's nothing funny at all about guns.
Anyway, the Seminoles and Wildcats are off to a quick lead in the HBAEE award race.
Wish them (less) luck!
Kris Hughes is the College Football Network Manager for Rant Sports. You can follow Kris on Twitter, Google Plus and Facebook
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Marine and Coastal Studies MR1510 The Role of Geospatial Technologies in Coastal and Marine Applications Marine and Coastal Studies • Introduction • Time & Place • Course Content • Reading Materials and Tutorials • Assignments and Assessments • Attendance Marine and Coastal Studies Time & Place • Friday 14.00-16.00 • Lecture Theatre: 105 • Geography Department, St. Marys Marine and Coastal Studies Three components: • Technology • Theory • Applications Marine and Coastal Studies Content • Data and Information as the basis for Knowledge and Understanding • Importance of Access to Data and Information for Studying the Marine and Coastal Environment • Geospatial Data and Information Defined • The Geospatial technologies of remote sensing, GIS, Digital Mapping (and Cartography), GPS, Mobile Data Collection, and the Internet: theory and practice • The Data into Information Pathway – Data Collection – Data Handling and Processing – Data Analysis – Data Visualisation Marine and Coastal Studies Content (details) • An introduction to the fundamentals of airborne and satellite borne remote sensing in coastal and marine environments including an introduction to the sensors and platforms used • An introduction to the fundamentals of digital image processing (DIP) • Example applications of remote sensing to coastal and marine environments e.g. monitoring of sediment movement, change detection, pollution, bathymetry, mangroves, coral reefs, algal blooms, sea surface temperature, oil spills and contingency planning, emergency response… amongst many others • An introduction to desktop and online (Internet- based) Geographical Information Systems (GIS) and Decision Support Systems (DSS): theory and practice Marine and Coastal Studies Content (details) • Integrating disparate data sources for mapping, analysis and modelling • Example applications of GIS to coastal and marine environments • Mobile Field Data collection, Global Positioning Systems (GPS), WAP-enabled mobile phones • Examples of Internet-based Information and Decision Support Systems (DSS) • Data and Information sources for coastal and environmental monitoring, mapping and modelling Marine and Coastal Studies Assignments Three Assignments for Assessment 1. Paper Critique (30%) 2. Module Paper (30%) 3. Group Report & Presentation (40%) 4. Duly Completed Exercises Marine and Coastal Studies Attendance • Compulsory for all elements of the course • Class Attendance Sheet will be taken each week • Medical Certificates (MC) should be filed with Geography office Marine and Coastal Studies Reading Materials •Basic Textbooks on Remote Sensing and Geographical Information Systems (GIS) Online Tutorials •Remote Sensing •Digital Image Processing •GIS •GPS •Online Resources •Coastal and Marine Applications •Mobile Technologies •Online Databases •Online Mapping and Information Systems Marine and Coastal Studies Guest Lectures Fieldtrip/Site Visit Marine and Coastal Studies Contact David R. Green Room G12 Tel. 272324 Email. email@example.com Marine and Coastal Studies Any Questions?
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