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11 Mission Vs. Vision Statement Examples In Government & Healthcare
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Organizations with clear mission and vision statements as part of a defined strategy are more successful. So, it shouldn't be a choice of mission statement versus vision statement—these are two pieces of the same puzzle you'll fit together when creating your strategic plan. Mission and vision have different, but important, parts to play for your organization. In this article, we'll show you some of the best examples we've seen of the interplay between mission and vision in the local government, healthcare, and finance industries.
This Post: 11 Mission Vs. Vision Statement Examples In Government & Healthcare
It shouldn't be a choice of mission statement versus vision statement—these are two pieces of the same puzzle you'll fit together when creating your strategic plan. Click To Tweet
Vision Vs. Mission Statement: Definitions
Two of our previous articles focused on mission and vision statements individually. The mission is focused on the purpose of your organization and your competitive advantage. The vision is focused on how you want your organization to develop in the future. To recap further:
Mission statements define why your business exists and why it's important, including the problems you aim to solve with your product or service. The mission explains an organization's plan for the present based on what it wants to achieve. Some are long, some are short, some are measurable, others aren't—mission statements vary by industry but all should be unique and realistic.
Vision statements declare where your organization wants to be in the future, framed in an aspirational, forward-thinking manner. Visions should be creative, ambitious, and have a logical connection to the mission. This isn't a road map (that's what your strategy is for), but rather the destination based on your mission. Vision statements help organizations make strategic decisions, aligning effort with goals.
How can you tell the difference? Mission statements are more externally focused, telling the world why the company exists. Vision statements can have both an external and internal focus, guiding employee efforts and strategic decision making, as well as broadcasting your company's grandest ambitions. In some cases, organizations use their own, slightly different definitions of mission and vision, but these statements will always have the company's vision of a better future and language around what differentiates it from competitors. This is a critical first step to defining your strategy.
An Alternative Set Of Terms: Objective, Advantage & Scope
Another way of talking about the concepts of mission and vision is the Objective, Advantage, and Scope framework.
- The objective of the organization is essentially its mission—why the organization exists.
- The advantage your organization offers is what sets you apart. It conveys your value proposition—how you do things differently or better than others—and is very similar to the ideas expressed in a vision statement.
- The scope defines the sphere in which you're operating. It defines what you will and will not do to achieve your objective. For example, you might focus on a particular segment of the population, a particular geographic location, or a particular way of serving your customers. Scope is often incorporated into vision statements.
These terms are less commonly used than "mission and vision statements,"but they can provide value as you try to articulate the uniqueness of your organization.
Qualities Of Mission & Vision Statements
While they are distinctly different, both mission and vision should:
- Inform strategic planning
- Inspire while still being achievable
- Highlight what's unique about your organization
- Align with company values
As far as timing, both mission and vision are the first steps in defining your strategy. You can develop them in tandem or lead with your mission statement. We recommend the latter. Why? It's a common pitfall to create a vision statement that's too lofty and fluffy. If you start with the mission, you'll have a more realistic anchor point for your ideal future state.
Below, we've rounded up some mission vs. vision statement examples to provide inspiration. But if you're ready to start crafting your own vision statement, take a look at our step-by-step process for guidance.
11 Mission Vs. Vision Statement Examples In Government, Healthcare & Finance
Local Government
Hampton County, SC
- Vision: "As one of the most progressive, small counties in the state, Hampton County seeks to uphold its…
- Vibrant Economy…
- Rural Quality of Life…
- And Sense of Community Pride…"
- Mission: "The mission of Hampton County Government is to provide quality public services in a timely and competent manner, and to work with the cooperation of the community and other local government units to create a vibrant and healthy physical, social and economic environment. Services shall be provided in a fair, respectful and professional manner consistent with available human, natural and economic resources."
RED : Everything You Need to Know About Yellowtail Fish
We condensed the vision statement, but you can see a clear tie with the three principles outlined in the mission. Notice the focus of the mission is to show what Hampton County's government provides, whereas the vision shows what a thriving county aspires to. You can also see the language isn't from a template—it's tailored for a small rural county already proud of what it has accomplished, that wants to emphasize its continual focus on cooperation, efficiency, and service to its citizens.
City of Toronto, Canada
- Vision: Toronto's vision is four-pronged:
- Toronto is a caring city.
- Toronto is a clean, green and sustainable city.
- Toronto is a dynamic city.
- Toronto invests in quality of life.
- Mission: To serve a great city and its people
Toronto's vision statement includes four perspectives, all of which most people would consider core tenets of a "great city"—the descriptor used in the mission statement. At a time when urban areas are facing a multitude of challenges, Toronto seems to be using this clear delineation to guide its strategic priorities for the years ahead.
Toronto's vision statements are inspirational yet achievable; they also clearly reflect a desire to be a place that is a source of pride for Torontonians.
Tri-County Health Department (TCHD)
- Vision: "Optimal health across the lifespan for the populations we serve."
- Mission: "Promote, protect and improve the lifelong health of individuals and communities in Adams, Arapahoe and Douglas Counties through the effective use of data, evidence-based prevention strategies, leadership, advocacy, partnerships, and the promotion of health equity."
TCHD is the largest local health department in Colorado and has clearly put time into differentiating its mission vs. vision statements. Words like "optimal" and "lifespan" make it clear that TCHD's vision is broad and aspirational—the organization aims for the best over decades. The mission then reiterates "lifelong," listing the focus of TCHD's activities in conjunction with results it wants to achieve. You can see how the vision and mission align, and easily imagine how the mission statement could translate into goals in a strategic plan.
Houston, TX Department Of Health And Human Services
- Vision: "Self-sufficient families in safe and healthy communities."
- Mission: "The mission of the Houston Department of Health and Human Services is to work in partnership with the community to promote and protect the health and social well-being of Houstonians."
This is one of the more interesting mission versus vision statement examples because it originates from a city department and is more specific than the overarching vision and mission of the city itself. This is not uncommon as it allows larger cities to narrow the focus of mission and visions within specific arenas.
In the case of Houston, the vision is very brief, but the mention of self-sufficiency is unique. This last phrase hints that the department is focusing its efforts on programs and services that foster citizens' independence in attaining safe and healthy outcomes. The mission centers on partnering with the community—deliberate wording that suggests cooperation and goodwill.
Kansas City, MO
- Vision: "Our local government will be nationally known for its transformative efforts that make Kansas City the diverse and sustainable community of choice for people to live, work, and play as a result of its safety; vibrant neighborhoods; business, educational, and cultural opportunities; connectedness; and vitality."
- Mission: "The mission of the City of Kansas City, Missouri is to deliver quality, customer-focused municipal services with an emphasis on public safety, neighborhood livability, job creation, responsible planning for economic growth, infrastructure improvements, transportation systems, public health, and the environment. In pursuit of this mission, we use data to make decisions and measure progress based on economic, social, and environmental factors."
Both are longer statements, but each serves its distinct purpose without being repetitive. The vision statement describes Kansas City's ideal world and uses ambitious phrasing like "nationally known" and "transformative." The mission statement echoes the elements emphasized in the vision, particularly the customer focus, but also includes how the city will achieve its mission, with a focus on data-based decisions and measurable progress. This is a classic example of the lofty, future-focused vision and practical, present-day mission that helps to direct strategic decision making.
Mayo Clinic
- Vision: "Mayo Clinic will provide an unparalleled experience as the most trusted partner for health care."
- Mission: "To inspire hope and contribute to health and well-being by providing the best care to every patient through integrated clinical practice, education and research."
Mayo Clinic is internationally renowned for its quality of care, which is mirrored in its wording of these statements. The mission strikes a nice balance of aspirational and practical, with phrases like "hope" and "best care" grounded by the mentions of the clinic's three operational focuses that will help it achieve its aims. The vision manages to address both patient experience and clinic reputation, while still being high-reaching and forward-thinking.
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Alliance Health
- Vision: Our vision is to be a leader in transforming the delivery of whole person care in North Carolina's public sector.
- Mission: Our mission is to improve the health and well-being of the people we serve by ensuring highly effective, community-based support and care.
Alliance Health is a managed care organization that partners with more than 2,000 private healthcare providers to help people recover from mental illness and substance use disorders. Its vision is powerful in that it references becoming a "leader" and talks about "transforming" the delivery of care; it's a statement that's likely to inspire employees and help them see the value of their work. The vision also uses the phrase "whole person care" to highlight what's unique about the organization. Finally, it clearly states the scope of terms of a specific geographic region—North Carolina.
St. Jude Children's Research Hospital
- Vision: "To accelerate progress against catastrophic disease at a global level."
- Mission: "The mission of St. Jude Children's Research Hospital is to advance cures, and means of prevention, for pediatric catastrophic diseases through research and treatment. Consistent with the vision of our founder Danny Thomas, no child is denied treatment based on race, religion or a family's ability to pay."
St. Jude's mission statement is well-crafted for many reasons. It's action-oriented and weaves in a mention of the organization's core value based on the founder's vision. That value has a direct tie to how the hospital will achieve its mission. The vision is inspiring and ambitious, but doesn't promise something it can't deliver—the focus is on "progress" versus eradication. Both statements use the word "catastrophic," linking the mission and vision and clarifying the focus of the organization.
Fox Chase Cancer Center
- Vision: "To be a regional, national, and international leader in oncology nursing care and services."
- Mission: "The mission of Fox Chase Cancer Center is to prevail over cancer by marshaling hearts and minds in bold scientific discovery, pioneering prevention, and compassionate care."
Fox Chase is a good mission vs. vision statement example because it uses different language in each, without confusing the two. The mission is creatively written, using phrases like "prevail" and "marshaling hearts and minds" that infuse energy and ambition into the statement. It also outlines the three areas it will focus on. The vision statement is briefer and centered on leadership, making it clear that this is its "destination."
Financial Sector
Fidelity Bank
- Vision: We will be known as the most caring, trusted, and respected source of financial guidance and solutions in the communities we serve.
- Mission: Like smart friends, we care deeply about your well-being and we will use our hearts and our heads to deliver the clarity and confidence you need to get where you want to be. (A more succinct version that's also stated on their site: To help families, individuals, and businesses in our communities get where they want to be.)
In this case, the mission and vision fit nicely hand-in-hand. Together, they show how the character of the institution plays into what it hopes to achieve: Fidelity's desire to be a caring bank will carve out its reputation as the most trustworthy source of financial guidance in the places where it operates. Both statements also put great emphasis on personal relationships, using phrases like "friends," "families," and "communities."
Eastern Caribbean Central Bank (ECCB)
- Vision: To be a model institution delivering exceptional service and influential policy advice to support the development of a thriving currency union.
- Mission: Advancing the good of the people of the currency union by maintaining monetary and financial stability and promoting growth and development.
Founded in part to help facilitate the growth and development of the Eastern Caribbean States, ECCB's purpose (mission) is to advance the financial capabilities of a large group of people, which is markedly different from Fidelity's mission. Its vision is specific in that it clearly sets a priority (everything employees do should be in service of creating a "thriving currency union") and tells how to get there (through providing "exceptional service" and "influential policy advice").
It's important to note that mission and vision statements are usually paired with core values, which help employees and leadership alike align their work with the standards and desired outcomes of the organization. Once you have your mission, vision, and values solidified, it's critical to weave all three into the operations of your organization. And remember, don't let these statements sit and gather dust. Incorporate them into company communications and review them regularly to make sure you're on track and keeping everyone engaged.
ClearPoint can help you organize and manage these foundational elements of your strategy as you build out goals and measure progress. Contact us to learn more.
Source: https://bloghong.com
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Interviewee: Nazanin Azari – Account Manager / Author: Francine Finkler
For non-interpreters, the common assumption is that to become an interpreter; the only necessity is to be fluent in two languages and to be sensible about the nuances of the target and source languages. After all, interpreters are simply being asked to convey the meaning of a set of words in a different language. However, other factors such as the client and their unique needs, the use of appropriate vocabulary, pronouns, and many other considerations are ignored if you think of interpreters as machines that merely change one word for another.
To better understand the importance of broadening your skills as an interpreter, I interviewed one of our Account Managers at MCIS. Nazanin Azari is going to talk about her experience taking the course "Interpreting for LGBTQIA Newcomers." In her interview, she explains the benefits of participating in the workshop. She will highlight the importance of keeping up with the rapid developments in the profession, as well as her perspective on the importance of participating in the training program as an internal staff.
If you're interested in the course, it is available ONLINE for FREE!
How was the LGBTQIA training valuable to you as a member of MCIS?
The most important reason that I took the training was to build and develop a relationship with the other students. Over the past few years, I have established a connection with interpreters that are working in the same language combinations of Farsi<>English. I have also built a telephone and e-mail based relationship with many other resources when I helped in coordinating and the dispatch of our language aid and professional interpreters. This training provided me with the opportunity to convene and join our valued resources in a different and more friendly forum. I would also like to encourage other MCIS internal staff to utilize these workshops as an opportunity to meet and form a bond with our valuable freelance interpreters and translators.
How do you believe the training can be beneficial to language professionals?
Participating in continuing competency programs is a great tool to understand the developments of language demands of different social, legal and medical sectors. The need for interpretation is highly related to the continuing changes and requirements of the vulnerable individuals that are in need of language solutions. To provide the best services, it is necessary to adapt to each situation. Moreover, even if you are not a member of the LGBTQIA community, as an interpreter, you get to be their active voice for the duration of the time you are offering your professional assistance. It is essential to be able to address LGBTIA members and their needs respectfully.
We must build trust with people that might be afraid of judgement. Learning the proper pronouns to be able to address the client respectfully is a great icebreaker. By making a professional approach, you will help the client to feel safe and to be able to express themselves freely.
It is also essential to build your level of professionalism to be able to remain unbiased and impartial towards the client's story and experience of life. Especially if you can not relate to the client's knowledge or even if you share a common and bothersome experience. Developing a relationship and sharing personal information can affect the results of your work; therefore, the training is a great learning experience to avoid such a crucial mistake.
Finally, participation in the training program will facilitate the comprehension of derogatory words, which enables us to avoid using those terms in favour of the relevant terminology related to LGTBAI* people. All interpreters must be cautious that some expressions and words can be incredibly offensive to the public. Adapting your vocabulary to the needs of your clients will affect the results and then the level of acceptance from the client towards you. Your professionalism combined with your updated knowledge of the language developments and appropriate terms will promote more just results.

subset: commoncrawl


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These are strange times, there's no question about that. With the coronavirus pandemic wreaking havoc throughout the sports world — and the real world — our lives have been filled with uncertainty for the past month.
Fans everywhere have gone weeks without being able to watch their favorite teams, and the void in our lives has made us all realize how much we take the crazy, wonderful world of sports for granted. But we're here to help.
In honor of these strange times, we've dug through the archives to bring you seven of the strangest stats and facts in sports history. Hopefully, they can keep you entertained and occupied while we wait for normality, or something like it, to return. Here they are in no particular order:
Statistically, Tom Brady has a higher percentage of making the Super Bowl than Steph Curry does of making a three-pointer
You're given $10,000 and you have to bet on one: Tom Brady making the Super Bowl or Steph Curry making a three-pointer. It seems like it'd be an obvious choice; Tom Brady might be one of the greatest quarterbacks of all-time but making a single three-pointer is a lot easier than making it all the way to the Super Bowl over the course of an entire NFL season. However, in his 19 years as an NFL starter, Brady has made the Super Bowl on nine occasions, which is about 47 percent of the time. Curry has attempted 5739 three-pointers in his career and made 2495 of them, giving him roughly a 43 percent success rate. That means, statistically, it's been more likely for Brady to make the Super Bowl than for Curry to make a three-pointer over the course of their careers. We'll see if Brady can keep that stat alive with his new team next season.
In 1919, an MLB pitcher earned a complete-game win despite being struck by lightning
Ray Caldwell was an MLB pitcher in the early 20th century for the Yankees, Red Sox and Indians. He had a decent career, finishing with a 133-120 record after 11 years in the league, but he's best known for his performance in a game in August 1919. Caldwell was on the mound for the Indians against the Philadelphia Athletics and had the team up 2-1 in the top of the ninth inning. Suddenly, a lightning bolt struck the field.
"The bolts flashed here and there, causing much excitement," Harry P. Edwards wrote in The Sporting News just over a century ago. "There was a blinding flash that seemed to set the diamond on fire and Caldwell was knocked flat from the shock of it."
According to Edwards, Caldwell "lay stretched out in the pitcher's box," and his teammates feared "he may have been killed." However, he picked himself up from the ground moments later to the amazement of fans and teammates. After presumably rubbing some dirt on it, he proceeded to get the final out to seal the win.
Michael Phelps holds the record for the longest golf putt ever made on camera
Michael Phelps is an athlete with plenty of amazing records to his name, but you probably never knew this was one of them. The legendary swimmer is also an avid golfer, even playing in the Pro-Am at the Phoenix Open the past couple of years, but his highlight in the sport came back in 2012 Alfred Dunhill Links Championship in Scotland. Facing 53 yards to the hole, Phelps pulled out his putter and proceeded to absolutely drain the shot. The 159-foot bomb still holds the record for the longest made putt ever caught on camera.
Larry Fitzgerald has more tackles than drops in his career
Larry Fitzgerald has been the model of consistency throughout his 16-year NFL career, but this stat takes it to superhuman levels. Fitzgerald only has 29 total drops to his name, which is 11 less than his 40 total tackles. It's impressive not only because it shows how reliable he's been as a wideout, but also because 40 tackles is a pretty darn high number for a receiver. Wideouts are often out of the play when a turnover happens, so it shows Fitz's determination and never-quit attitude.
Former NBA player Eric Money played — and scored — for two teams in the same game
In November 1978, the Nets and the 76ers squared off in an early-season NBA matchup. Things started to get weird when a Nets player received his second technical foul, resulting in an ejection. He kicked a chair while walking off the court, which led referee Richie Powers to T him up again. Nets coach Kevin Loughery, who had one tech already, furiously protested, and Powers slapped HIM with two additional technicals.
Because receiving three technical fouls in a game is supposed to be impossible, the Nets convinced NBA Commissioner Larry O'Brien to let them replay the second half of the game at a later date. However, between the first game in November and the second game in March, guard Eric Money was traded from the Nets to the 76ers. He ended up officially playing the first half for New Jersey and the second half for Philadelphia. It resulted in one of the strangest stories, and box scores, in NBA history.
If you took away all of Wayne Gretzky's goals, he'd still be the NHL's all-time points leader
This is more amazing than it is strange. Gretzky is the all-time NHL leader in points (2,857) and goals (894), both impressive stats on their own, but if you took away all of Gretzky's goals he would still be the all-time points leader. Even if "The Great One" had never scored a single NHL goal, his 1,963 assists would put him 62 points ahead of Jaromir Jagr (1,921) in second place.
More men have walked on the moon than have scored an earned run against Mariano Rivera in the playoffs
Mariano Rivera's legendary career for the Yankees has its fair share of impressive stats, but this one stands out above the rest. In 140 innings of postseason baseball, Rivera only allowed 11 earned runs. That figure is less than the 12 people who have walked on the moon. It is literally more common for a human being to have stepped foot on the moon than it is for them to have scored against Rivera in the playoffs. When he retired from the MLB in 2013, Rivera ensured that this stat will live on forever.
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More Projects
Public Realm
Urban Design
The delivered landscape provides the focus for this new, all-through academy made up of a nursery, a three-form-entry primary school; a six-form-entry secondary school; and a sixth form of up to 300 pupils. It successfully gels the old school buildings with the exciting contemporary architectural insertions.
The internal courtyards convey the quality of a University Quad, whilst the outer spaces combine a natural and ecological perimeter with zones of recreation, animation and fun with outdoor gyms, incidental basketball courts and table tennis tables woven through.
Client: Bouygues
Scope + Status: Design and technical delivery
Consultants: Pollard Thomas Edwards Architects


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Janice Bryant Howroyd in 1978 turned a $900 dollar loan into a billion-dollar business — and you may be surprised how she got there.
Janice Bryant Howroyd is the founder and CEO of ActOne Group, a global enterprise that provides employment, workforce management, and procurement solutions. She is the first Black woman to own a billion-dollar business and has a net worth of $390 million.
For every success story, there are moments that lead up to that journey which often times are filled with setbacks. However, it is in those setbacks that we find and learn value within ourselves. It is during that season, where our path to the journey of success begins. In the case of Janice Bryant Howroyd, she has a remarkable story to tell.
In Janice's case, she saw value in people — not things. She noticed many employers viewed employees as simply a business asset and nothing more. In an interview with NBCNews.com she said the following:
"I met with several employment companies, and they had posters on the wall and dollar signs by each applicant's name, it made me feel like a commodity and not a person."
Rather than accepting the status quo, she decided to make the industry better and implement huge improvements over hiring strategies. She ensured that employees felt inspired and encouraged during the work placement process. "If the employees are placed well and are happy, the companies they work for will do well," she says.
Janice has built her company into a billion-dollar business with more than 17,000 clients and 2,600 employees in 22 countries.
Janice explains her full story and her residual success in the new book Acting Up. For the 9-5 employee that wants to start their own business or the entrepreneur who is ready to give up on their start-up, her book gives faith and encouragement to keep pressing forward. She shares key touchstones in her book such as life-long education, asking the right questions, and never compromise. Had Janice not followed these rules, she wouldn't be the woman she is today, nor would she have carved a blazing trail for more Black women entrepreneurs to own billion-dollar businesses.
If Janice did it, so can you.


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A Washington Post story today showed liberals lied about polygamy. They are pushing it.
In June 2015, five liberals on the Supreme Court commanded that homosexual marriage be the law of the land -- both the will of the people and the Constitution be damned.
In dissent, Chief Justice John Roberts asked, "Just who do you think we are?"
He also said, "It is not about whether, in my judgment, the institution of marriage should be changed to include same-sex couples. It is instead about whether, in our democratic republic, that decision should rest with the people acting through their elected representatives, or with five lawyers who happen to hold commissions authorizing them to resolve legal disputes under the law."
The same argument can and should be made about abortion. Let the states decide because America is a collection of 50 states and not a nation divided into 50 compartments.
After the court usurped the authority of all 50 state legislatures and Congress to legalize homosexual marriage, opponents said this will lead to the legalization of bestiality, incest and polygamy.
The press clucked its collective tongue at us.
Politico declared, "No, Polygamy Isn't the Next Gay Marriage. Group marriage is the past—not the future—of matrimony."
The author was Jonathan Rauch, a senior fellow at the Brookings Institution and author of Gay Marriage: Why It Is Good for Gays, Good for Straights, and Good for America.
If homosexual marriage were good for America, then states long ago would have recognized it one by one.
In bypassing legislatures, homosexual marriage is terrible for America because it undermines the authority of the states.
He wrote, "I am a gay marriage advocate. So why do I spend so much of my time arguing about polygamy? Opposing the legalization of plural marriage should not be my burden, because gay marriage and polygamy are opposites, not equivalents. By allowing high-status men to hoard wives at the expense of lower-status men, polygamy withdraws the opportunity to marry from people who now have it; same-sex marriage, by contrast, extends the opportunity to marry to people who now lack it. One of these things, as they say on Sesame Street, is not like the other.
"Yet this non sequitur just won't go away: 'Once we stop limiting marriage to male-plus-female, we'll have to stop limiting it at all! Why only two? Why not three or four? Why not marriage to your brother? Or your dog? Or a toaster?' If there's a bloody shirt to wave in the gay-marriage debate, this is it."
Others made similar arguments. Cathy Young, a contributing editor at Reason magazine, assured us in Time magazine that, "Polygamy Is Not Next."
Oddly, she made the case for polygamy, writing, "In a free society, the private sexual choices of adults should not be criminalized. But they are not automatically entitled to cultural approval or societal support systems."
By "not automatically entitled" simply means wait a few years for the Supreme Court to make it the law of the land.
7 summers later, the push is on to make polygamy the law of the land, likely through Ketanji Brown Jackson, the pedophile friendly justice-in-waiting, and the next liberal majority.
Liberals are good at marketing so they are relabeling polygamy as polyamorous marriage. They change the semantics every few years. Homosexual marriage became gay marriage which became same-sex marriage. It remains an oxymoron because marriage is between a man, a woman and God.
The Washington Post got a nice Drudge link today for its advertorial for polygamy, "How polyamorous people are marking commitment to multiple partners."
By definition, people in the polyamorous crowd are committed only to their own lusts.
Nevertheless, the story said, "In legal terms, polyamorous people are unable to marry all their of partners: It is illegal throughout the United States to marry more than one person at a time. Somerville, Mass., is thought to be the first U.S. city to legally recognize polyamorous domestic partnerships, which it started doing in 2020."
More than a century after Utah banned polygamy in order to be admitted to the union, polygamy is back. TLC began softening the field a few years back with a reality TV series on a polygamist and his wives.
Thus far, LGBT has not added P for polygamy to its alphabet. B for bestiality and I for incest also await addition.
The undermining of American values and the destruction of the American family continue. The assaults never end and come from all directions.
The counterattack begins next month when justices officially repeal Roe v. Wade, likely without John Roberts. What the court giveth without constitutional authority a later court taketh away. In the meantime, 63 million babies have been killed in the womb.


text:
23 September 2016
YWCA Canberra is committed to fostering the growth and development of young women leaders. Each year, our Board of Directors conducts the Board Traineeship Program, which provides women with the opportunity to gain experience in governance, finance and strategic management without the legal and financial responsibilities of being a Board Director.
Here, we speak to Gina Zheng, who is about to complete her Board Traineeship, about her experience over the past 12 months.
Gina is currently in her third year of a Bachelor of Law (Honours) and Development Studies at the ANU. She is also currently the ACT Director of Oaktree and previously sat on the NSW Youth Advisory Council from 2013-15.
How would you describe the Board Traineeship Program to someone who was hearing about it for the first time?
The Board Traineeship Program provides a unique opportunity for young women to gain insight into best-practice governance and strategic operations of a not-for-profit organisation. It is a chance to learn through first-hand experience, and build leadership through conversations and communications.
What compelled you to apply for the Program?
Firstly, YWCA Canberra is a reputable organisation and is a leader in the not-for-profit sector for gender equality through service delivery and advocacy. I saw the program as a great opportunity to become deeper involved with the organisation.
Secondly, as the ACT Director of Oaktree, I have gained thorough hands-on day-to-day management of a not-for-profit, developing skills in people and project management. I applied for the program because I was incredibly keen on gaining insights into broader, higher-level strategic management and governance of not-for-profits.
What were a couple of your personal highlights of the Program?
I had a fantastic experience working with Caitlin Sandercock, the Vice-President of the Board, who was always willing to answer my many questions and was able to provide broader contextual understanding of Board matters.
It was also fantastic to be provided the opportunity to attend sponsored Australian Institute of Company Directors Workshops.
How has the Board Traineeship Program contributed to your development as a young woman leader?
The Program has deepened my understanding of the strategic analysis and considerations of not-for-profits. More tangibly, I have gained an incredible amount of insight into the financial operations of an organisation as well as best-practice governance procedures.
What's your top piece of advice for the next round of Board Trainees?
Go in with an open-mind and eagerness to learn, and don't ever shy away from asking questions! Take as much advantage of all the opportunities and resources available to you, because the Board Traineeship Program is a unique opportunity that will unlikely come around again.
Applications for the Board Traineeship Program are currently open, and will close on Friday 21 October. Download the application form here.
Tags: board trainee, board traineeships, Leadership, Women's leadership, YWCA Canberra Board
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text:
- Research article
- Open Access
- Published:
Recombinant human SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human intervertebral disc nucleus pulposus cells
Arthritis Research & Therapy volume 17, Article number: 253 (2015)
2481 Accesses
59 Citations
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Nutrient deprivation is a likely contributor to intervertebral disc (IVD) degeneration. Silent mating type information regulator 2 homolog 1 (SIRT1) protects cells against limited nutrition by modulation of apoptosis and autophagy. However, little evidence exists regarding the extent to which SIRT1 affects IVD cells. Therefore, we conducted an in vitro study using human IVD nucleus pulposus (NP) cells.
Thirty-two IVD specimens were obtained from patients who underwent surgical intervention and were categorized based on Pfirrmann IVD degeneration grades. Cells were isolated from the NP and cultured in the presence of recombinant human SIRT1 (rhSIRT1) under different serum conditions, including 10 % (v/v) fetal bovine serum (FBS) as normal nutrition (N) and 1 % (v/v) FBS as low nutrition (LN). 3-Methyladenine (3-MA) was used to inhibit autophagy. Autophagic activity was assessed by measuring the absorbance of monodansylcadaverine and immunostaining and Western blotting for light chain 3 and p62/SQSTM1. Apoptosis and pathway analyses were performed by flow cytometry and Western blotting.
Cells cultured under LN conditions decreased in number and exhibited enhanced autophagy compared with the N condition. Medium supplementation with rhSIRT1 inhibited this decrease in cell number and induced an additional increase in autophagic activity (P < 0.05), whereas the combined use of rhSIRT1 and 3-MA resulted in drastic decreases in cell number and autophagy (P < 0.05). The incidence of apoptotic cell death increased under the LN condition, which was decreased by rhSIRT1 (P < 0.05) but increased further by a combination of rhSIRT1 and 3-MA (P < 0.05). Under LN conditions, NP cells showed a decrease in antiapoptotic Bcl-2 and an increase in proapoptotic Bax, cleaved caspase 3, and cleaved caspase 9, indicating apoptosis induction via the mitochondrial pathway. These changes were suppressed by rhSIRT1 but elevated further by rhSIRT1 with 3-MA, suggesting an effect of rhSIRT1-induced autophagy on apoptosis inhibition. Furthermore, the observed autophagy and apoptosis were more remarkable in cells from IVDs of Pfirrmann grade IV than in those from IVDs of Pfirrmann grade II.
SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human IVD NP cells, suggesting that rhSIRT1 may be a potent treatment agent for human degenerative IVD disease.
Lower back pain affects up to 85 % of people at some point during their lives and causes severe incapacity that increases medical expenses and affects the workforce [1]. In the United States, lower back pain results in annual health care and related costs of approximately $100 billion [2]. Although the cause of lower back pain is considered to be an interdependent multifactorial process, intervertebral disc (IVD) degeneration is one of the major pathological processes implicated [3–5].
The IVD is structurally an avascular tissue that includes an inner nucleus pulposus (NP) surrounded by an outer annulus fibrosus (AF), which is sandwiched between the cartilaginous endplates of contiguous vertebral bodies. The nutritional supply to NP cells depends on diffusion, primarily through the endplates, which is reduced by aging- and smoking-induced sclerosis of the subchondral bone [6]. A loss of nutritional supply ultimately leads to apoptotic cell death in the IVD [7, 8]. In particular, apoptosis of NP cells is considered to be the first sign of IVD degeneration [9]. Therefore, treatments targeting cell apoptosis need to be developed to inhibit or prevent degenerative IVD disease.
Silent mating type information regulator 2 homolog 1 (SIRT1), a mammalian homolog of silent information regulator 2, is a nicotinamide-dependent class 3 histone deacetylase that is essential for cell survival and increases the longevity of mammalian cells under calorie restriction [10]. In response to cellular energy loss, SIRT1 regulates multiple cellular processes, including the cell cycle, metabolism, and apoptosis [11, 12]. Previously, we hypothesized that SIRT1 might play an important role in homeostasis of IVD tissues, especially during the early phase of IVD degeneration. We subsequently revealed for the first time the expression of SIRT1 in human NP cells, which markedly increases in IVDs during early degeneration stages, thereby promoting extracellular matrix (ECM) metabolism and cell proliferation [13]. More recently, it has been shown that SIRT1 prevents human chondrocyte apoptosis, suppresses catabolic changes in gene expression, and accelerates osteoarthritis in conditional SIRT1-deficient mice [14–16]. SIRT1 has been considered to inhibit apoptosis by deacetylation of nuclear proteins such as p53 and nuclear factor κB (NF-κB) [17, 18]. Thus, it is conceivable that SIRT1 is a key regulator of mammalian musculoskeletal cell survival through modulation of apoptosis.
SIRT1 is also known to regulate the autophagy–lysosome pathway [19]. Autophagy-related genes Atg5, Atg7, and Atg8, which regulate autophagy, are deacetylated by SIRT1 in a nicotinamide-dependent manner, and this deacetylation is accelerated by limited nutrition [19]. Autophagy is an important cell survival mechanism activated by calorie restriction in which cells digest and recycle their own damaged proteins and organelles [12]. It has been revealed that autophagy is associated with certain degenerative diseases such as osteoarthritis [20], Parkinson's disease [21], and Alzheimer's disease [22]. Autophagy and apoptosis are closely associated in the pathological processes of diverse human diseases [19].
On the basis of the antiapoptotic and proautophagic roles of SIRT1, we hypothesized that SIRT1 might play a key role in the homeostasis of IVD cells under nutrient deprivation at the early phase of IVD degeneration. Furthermore, exogenous administration of recombinant human silent mating type information regulator 2 homolog 1 (rhSIRT1) protein into an IVD might contribute to cell survival by inhibition of apoptosis though autophagy induction. The purpose of this study was to elucidate the effects of exogenous SIRT1 on autophagy and apoptosis under limited nutrition in human IVD NP cells.
Ethics statement
The collection and use of human IVD specimens were approved by the ethics committee of Kobe University Hospital. All samples were obtained in accordance with the World Medical Association Declaration of Helsinki Ethical Principles for Medical Research Involving Human Subjects. This study was approved by the institutional review board of Kobe University Graduate School of Medicine. All patients provided written informed consent.
Culture of human nucleus pulposus cells
Thirty human NP tissue samples were obtained from consenting patients during surgical procedures (lumbar disc herniation, 13 cases; lumbar spinal stenosis, 10 cases; idiopathic scoliosis, 9 cases). There were 18 males and 14 females with a mean age of 32.4 years (range 11–80 years). Degeneration grades of the IVDs were determined by magnetic resonance imaging and classified according to the Pfirrmann grading system [23]. The degeneration grades were represented by 9 cases of grade II IVD and 23 cases of grade IV IVD. All Pfirrmann grade II IVDs were obtained from young patients with idiopathic scoliosis (Table 1). To clearly compare early degenerative IVDs (Pfirrmann grade II) with advanced degenerative IVDs (Pfirrmann grade IV), moderate degenerative IVDs of Pfirrmann grade III were not analyzed in the present study.
The NP was carefully separated from the AF immediately after surgery. The tissue specimens were centrifuged and then subjected to sequential enzyme digestions at 37 °C with 0.025 % (w/v) collagenase P (Roche Applied Science, Indianapolis, IN, USA) and 0.001 % (w/v) deoxy-ribonuclease 2 (DNase 2) (Sigma-Aldrich, St Louis, MO, USA) for 12 h. The digested NP tissues were placed in complete tissue culture medium consisting of Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 10 % (v/v) heat-inactivated fetal bovine serum (FBS; Sigma-Aldrich), 100 U/ml penicillin, and 100 mg/ml streptomycin. When confluent, the NP cells were released and transferred to 35-mm culture dishes for 3 days of preculture at 37 °C in 20 % (v/v) O2, 5 % (v/v) CO2, and 1000 mg/L glucose. These isolated cells were defined as NP cells.
To induce nutritional stress, NP cells were cultured in DMEM supplemented with 1 % (v/v) FBS, defined as the low nutritional (LN) condition, compared with 10 % (v/v) FBS defined as the normal nutritional (N) condition. In this study, to extrapolate possible clinical applications of SIRT1 to IVDs, exogenous supplementation of rhSIRT1 (Sigma-Aldrich) and not transfection of the SIRT1-encoding gene was employed. To inhibit autophagy, we used 3-methyladenine (3-MA; Santa Cruz Biotechnology, Santa Cruz, CA, USA), a specific inhibitor of the early stages of the autophagic process.
After 3 days of preculture, the NP cells were assigned to one of four culture groups (Table 2) as follows: group N, normal nutritional (N) condition cultured with 10 % (v/v) FBS; group LN, low nutritional (LN) condition cultured with 1 % (v/v) FBS; group LN + SIRT1, low nutritional (LN) condition cultured with 1 % (v/v) FBS and rhSIRT1 (10 μM); and group LN + SIRT1 + 3-MA, low nutritional (LN) condition cultured with 1 % (v/v) FBS, rhSIRT1 (10 μM), and 3-MA (5 mM). Introduction of rhSIRT1 using a protein delivery reagent with culture medium was repeated every 3 days at the same time as media changes.
Delivery efficacy of rhSIRT1
rhSIRT1 was introduced into NP cells using Pro-DeliverIN™ (OZ Biosciences, Marseille, France), a protein delivery reagent, according to the manufacturer's instructions. The delivery efficiency of Pro-DeliverIN™ in NP cells was measured using rhSIRT1 labeled with a HiLyte Fluor™ 555 Labeling Kit-NH2 (Dojindo Molecular Technology, Rockville, MD, USA). A microscopic visual count was performed as described previously to calculate the delivery efficiency of rhSIRT1 [13].
Analysis of total NP cell numbers
NP cells that were approximately 60–70 % confluent were treated with 10 μM rhSIRT1 or 5 mM 3-MA under N or LN conditions in six-well plates at a density of 1.5 × 105 cells/well. Briefly, after 3, 7, and 14 days of initial treatment, images of five random fields were obtained with Motic Images Plus 2.2 software (Shimadzu, Kyoto, Japan). The number of NP cells in each well was then calculated and represented as the percentage of the baseline cell number.
Immunohistochemical staining
After 72 h of treatment, the cells were fixed in 4 % (w/v) paraformaldehyde/phosphate-buffered saline (PBS) for 15 minutes at room temperature, permeabilized, and blocked in 0.2 % (v/v) Triton X-100 in PBS for 30 minutes. To analyze autophagy, the cells were incubated with a primary antibody against human light chain 3 (LC3; Santa Cruz Biotechnology) overnight at 4 °C, followed by an Alexa Fluor 594–conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 30 minutes at room temperature. Images were obtained using a BZ-9000 microscope (Keyence, Osaka, Japan).
Analysis of autophagy activity
Autophagy activity was assessed by using an autophagy staining kit (Cayman Chemical, Ann Arbor, MI, USA). The absorbance of monodansylcadaverine (MDC), which enters multilamellar bodies by both an ion-trapping mechanism and interactions with membrane lipids as a probe to detect autophagic vacuoles, was measured using a microplate reader after 6, 12, 24, 48, and 72 h of treatment. Autophagy activity was calculated as the MDC absorbance normalized to the cell number.
Analysis of apoptosis
Apoptosis was assessed using a MEBCYTO apoptosis kit (MBL International, Nagoya, Japan) and flow cytometry. Early apoptotic cells stained only with annexin V conjugated with fluorescein isothiocyanate (annexin V-FITC), and late apoptotic/necrotic cells stained with propidium iodide (PI) as well as annexin V-FITC. After 3, 7, and 14 days of treatment, treated NP cells were isolated, centrifuged, and then incubated in 100 μl binding buffer containing annexin V-FITC and PI. Apoptotic cells, indicated by positive staining for annexin V-FITC only or annexin V-FITC and PI, were counted by flow cytometry and represented as the percentage of the total cell number.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting
Treated NP cells were lysed on ice for 30 minutes in lysis buffer containing protease and phosphatase inhibitors (Roche Diagnostics, Basel, Switzerland). Total cellular proteins were centrifuged and collected. The separated proteins were transblotted electrically onto a blotting membrane (Amersham Biosciences, Arlington Heights, IL, USA). The membranes were probed with primary antibodies followed by a horseradish peroxidase–conjugated secondary antibody. Proteins were visualized with ECL Plus reagent (Amersham Biosciences) and a Chemilumino analyzer LAS-3000 mini (Fujifilm, Tokyo, Japan). The expression of acetylated and total p53, as well as of acetylated and total NF-κB proteins, was detected to elucidate the deacetylation potential of SIRT1. LC3 and p62 protein/sequestome 1 (p62/SQSTM1) were detected to monitor autophagy. In addition, Bcl-2-associated X protein (Bax), B-cell lymphoma (Bcl-2), caspase 3, and caspase 9 proteins were analyzed to determine the involved pathways of SIRT1 during apoptosis. The intensities of the bands were quantified using ImageJ analysis software, version 1.45 (NIH Image; National Institutes of Health, Bethesda, MD, USA). The antibodies used in this study were as follows: rabbit monoclonal or polyclonal antibodies against acetylated and total p53, acetylated and total NF-κB, LC3, p62/SQSTM1, Bax, Bcl-2, caspases 3 and 9, and cleaved caspases 3 and 9 (Cell Signaling Technology, Danvers, MA, USA); a mouse-monoclonal antibody against α-tubulin (Sigma-Aldrich); and horseradish peroxidase–conjugated anti-rabbit and anti-mouse antibodies (Amersham Biosciences).
Statistical analysis
Data are expressed as the mean ± standard deviation. Three-way analysis of variance with the Tukey–Kramer post hoc test was used to assess differences in experimental groups. Student's t test was used to evaluate differences between the Pfirrmann grades II and IV groups. P values <0.05 were regarded as statistically significant. Statistical analyses were performed using PASW Statistics 18 (IBM SPSS, Armonk, NY, USA).
Introduction of recombinant human SIRT1 into nucleus pulposus cells under low nutritional conditions maintained deacetylation potential
The delivery efficiency of 10 μM rhSIRT1 using protein delivery reagent was 37.9 % in our previous study [13]. In the Pfirrmann grade II IVD (n = 1), introduction of rhSIRT1 reduced acetylated p53 but did not reduce acetylated NF-κB levels. In Pfirrmann grade IV IVDs (n = 4), introduction of rhSIRT1 reduced both acetylated p53 and acetylated NF-κB levels. This acetylation potential of rhSIRT1 was more prominent in Pfirrmann grade IV IVDs than in the Pfirrmann grade II IVDs. These findings indicate the deacetylation potential of rhSIRT1 under LN conditions (Fig. 1a, b).
Recombinant human SIRT1 inhibited the decrease in nucleus pulposus cell number under low nutritional conditions
In this experiment, morphological changes were not observed in any experimental group (Fig. 2a).
In Pfirrmann grade II IVDs (n = 5), the total number of NP cells in group N showed a time-dependent increase over 14 days (day 14 + 52.2 % vs. day 0). NP cells were treated with each condition in six-well plates at a relatively high density of IVD cells (approximately 50–60 % confluent). NP cells did not double, even after 14 days of culture in 10 % (v/v) serum-supplemented media. This result suggests the very slow cell proliferation of human NP cells or reduced cell division due to the confluence in our cell culture system. The total number of NP cells in group LN + SIRT1 showed a slight increase and was significantly higher than that in group LN at only day 14 (P < 0.05). In addition, the total NP cell number in group LN + SIRT1 + 3-MA was significantly lower than that in group LN + SIRT1 at every time point (P < 0.05) (day 14, group LN: −22 %, group LN + SIRT1: −18 %, group LN + SIRT1 + 3-MA: −32 % vs. Group N) (Fig. 2b). In Pfirrmann grade IV IVDs (n = 5), the number of NP cells in group N showed a time-dependent increase (day 14 + 27.3 % vs. day 0). Cell numbers in group LN were significantly lower than those in group N at every time point (P < 0.01). The number of NP cells in group LN + SIRT1 showed a time-dependent increase with a significant difference compared with that in group LN at days 7 and 14. However, NP cell number in group LN + SIRT1 + 3-MA was significantly lower than that in group LN + SIRT1 at days 7 and 14 (P < 0.05) (day 14, group LN: −22 %, group LN + SIRT1: −8 %, group LN + SIRT1 + 3-MA: −34 % vs. Group N) (Fig. 2b).
Recombinant human SIRT1 accelerated autophagy activity under low nutrition conditions
First, autophagy was quantitatively evaluated by the absorbance of MDC. In Pfirrmann grade II IVDs (n = 5), the normalized autophagy activity of NP cells in group N showed no significant alteration for 72 h. Autophagy activity in groups LN and LN + SIRT1 was significantly higher than that in group N at 24 h (P < 0.01). However, there was no significant difference in normalized autophagy activity between groups LN and LN + SIRT1. Autophagy activity in group LN + SIRT1 + 3-MA was significantly lower than that in group LN + SIRT after 12 h (P < 0.01) (24 h, group LN: +115 %, group LN + SIRT1: +88 %, group LN + SIRT1 + 3-MA: −14 % vs. group N) (Fig. 3a). In Pfirrmann grade IV IVDs (n = 5), the normalized autophagy activity in group N showed no significant alteration for 72 h. Autophagy activity in group LN was higher than that in group N after 24 h. Moreover, autophagy activity of NP cells was significantly stimulated in group LN + SIRT1 compared with group LN after 48 h (P < 0.05). However, autophagy activity in group LN + SIRT1 + 3-MA was significantly lower than that in group LN + SIRT1 after 48 h (P < 0.01) (72 h, group LN: +66 %, group LN + SIRT1: +136 %, group LN + SIRT1 + 3-MA: +21 % vs. group N) (Fig. 3a).
Next, immunofluorescence analysis for LC3 was performed at 72 h after treatment. In Pfirrmann grade II IVDs, immunoreactivity for LC3 was measured in group N, as the basal level, which showed little difference among groups N, LN, and LN + SIRT1. In Pfirrmann grade IV IVDs, LC3 immunoreactivity of group LN was higher than that of group N. Furthermore, LC3 immunoreactivity in group LN + SIRT1 was higher than that in groups N and LN. However, in group LN + SIRT1 + 3-MA, LC3 immunoreactivity was attenuated, together with a decrease in the number of NP cells (Fig. 3b).
Finally, we examined autophagic flux by monitoring LC3 and p62/SQSTM1 at 72 h after treatment. LC3-I expression levels were quite low, making it difficult to measure the ratio of LC3-II/LC3-I. Therefore, we measured the expression level of LC3-II compared with that of tubulin. In the Pfirrmann grade II IVD (n = 1), Western blot analysis revealed the SIRT1 treatment did not affect protein expression. In Pfirrmann grade IV IVDs (n = 3), a significant increase in the amount of LC3-II was detected in group LN + SIRT1 compared with group LN and LN + SIRT1 + 3MA (P < 0.05). In addition, the amount of p62/SQSTM1 in group LN + SIRT1 was significantly decreased compared with the other groups (P < 0.05) (Fig. 3c, d).
Recombinant human SIRT1 attenuated apoptosis under the low nutrition conditions
In Pfirrmann grade II IVDs (n = 5), apoptosis in group N, as detected by flow cytometry, was not altered for 14 days. Apoptosis in group LN + SIRT1 was also unaltered for 14 days. However, the extent of apoptosis in group LN + SIRT1 + 3-MA was significantly increased compared with that of group LN + SIRT1 at days 7 and 14 (P < 0.01) (day 14, group N: 9 %, group LN: 14 %, group LN + SIRT1: 10 %, group LN + SIRT1 + 3-MA: 16 %). In Pfirrmann grade IV IVDs (n = 8), the extent of apoptosis in group LN, LN + SIRT1, and LN + SIRT1 + 3-MA was higher than that in Pfirrmann grade II IVDs. Apoptosis in group N was maintained at the initial level at day 3. In group LN, apoptosis increased in a time-dependent manner compared with group N at every time point (P < 0.01). The apoptotic incidence of group LN + SIRT1 was also increased compared with that of group N at day 7 (P < 0.01), but it was significantly lower than that of group LN at every time point (P < 0.05). Group LN + SIRT1 + 3-MA demonstrated the highest levels of apoptosis among all groups (P < 0.01) (day 14, group N: 9 %, group LN: 24 %, group LN + SIRT1: 14 %, group LN + SIRT1 + 3-MA: 29 %) (Fig. 4a, b). On the basis of our results, basal levels of apoptosis in group N were not so high. This may indicate physiological cell life turnover or programmed cell death. Treatment with rhSIRT1 can suppress limited nutrition-induced non-physiological apoptosis, but it may not affect physiological apoptosis.
Recombinant human SIRT1 modulated the mitochondrial apoptotic pathway under low nutrition conditions
Apoptotic pathway proteins were analyzed in Pfirrmann grade IV IVDs (n = 4) by Western blotting. Higher amounts of both cleaved caspases 3 and 9 were detected in group LN compared with group N. In addition, Bax protein expression was increased and Bcl-2 was attenuated in group LN. However, this enhancement of both cleaved caspases 3 and 9 protein expression in group LN was counteracted in group LN + SIRT1. Moreover, Bax expression was attenuated and Bcl-2 was increased in group LN + SIRT1. This observation shows that rhSIRT1 has an antiapoptotic effect mediated through the mitochondrial pathway under LN conditions. Furthermore, when autophagy was inhibited by 3-MA, rhSIRT1-induced antiapoptosis was attenuated in group LN + SIRT1 + 3-MA (Fig. 5a, b).
In the present study, we demonstrate that reduced nutrition induced apoptosis through the mitochondrial pathway in human IVD NP cells and that rhSIRT1 prevented this apoptosis as well as induced autophagy. Pharmacological inhibition of autophagy resulted in a marked increase in apoptosis, suggesting an antiapoptotic effect of rhSIRT1 through induction of autophagy. Furthermore, these effects of rhSIRT1 on autophagy and apoptosis in human NP cells were more prominent in Pfirrmann grade IV IVDs than in Pfirrmann grade II IVDs.
Published evidence suggests that excessive apoptosis of IVD cells is a potential cause of IVD degeneration [3, 5, 24, 25]. Sudo et al. demonstrated that serum starvation and annular needle puncture models induce apoptosis leading to IVD degeneration, and they also revealed that both Bcl-2 overexpression and caspase 3 knockdown prevent apoptosis and degenerative alterations [26, 27]. We also found that apoptosis of both AF and NP cells is mediated through the mitochondrial pathway in a rat tail static compression model of disc degeneration [28]. In surgically obtained herniated IVDs, Park et al. revealed that NP cell apoptosis is also induced by the mitochondrial pathway [9]. Consequently, prevention of apoptosis in NP cells is considered to be a therapeutic strategy for IVD regeneration. In the present study, reduced serum concentrations induced apoptosis in both early and advanced degenerative IVDs. This proapoptotic effect induced by nutritional stress was more remarkable in advanced degenerative IVDs than in early degenerative IVDs. On the basis of these results, advanced degenerative NP cells are supposed to be more sensitive to nutritional stress than early degenerative NP cells, leading to apoptosis more readily.
Researchers in several studies have reported that prevention of apoptosis by platelet-derived growth factor [29], Bcl-2 [27], caspase 3 small interfering RNA (siRNA) [26], or a caspase inhibitor [30] has positive effects on ECM metabolism in IVD cells. In the current study, introduced rhSITR1 reduced apoptosis under LN conditions in human NP cells from Pfirrmann grades II and IV IVDs. Furthermore, this antiapoptotic effect of rhSIRT1 was attenuated by 3-MA, an inhibitor of autophagy. Therefore, introduced rhSIRT1 prevented apoptosis through autophagy induction. In addition, apoptosis inhibition was more obvious in Pfirrmann grade IV IVDs than in Pfirrmann grade II IVDs. We also found that introduced rhSIRT1 reduced acetylation of p53, decreased proapoptotic Bax expression, apoptotic cleavage of caspases 3 and 9, and increased antiapoptotic Bcl-2 expression in human NP cells. It has been shown that deacetylation of p53 by SIRT1 contributes to inhibition of p53-dependent mitochondrial apoptosis [18, 31]. Thus, introduced rhSIRT1 potentially contributes to NP cell survival by modulation of the mitochondrial apoptotic pathway.
Autophagy is one type of cytoprotective process and ameliorates various types of cell stress. Activation of autophagy improves cell survival, whereas inhibition of autophagy promotes cell death [32]. Autophagy is known to be activated by a variety of stress stimuli, including calorie restriction [33], hypoxia [34], and DNA damage [35]. In addition, SIRT1 serves to activate autophagy under limited nutrition [19]. In the present study, introduced rhSIRT1 activated autophagy in NP cells under nutrient starvation and increased the number of NP cells through suppression of apoptosis that was inhibited by autophagy, which is in line with previous evidence.
Mounting evidence has indicated possible crosstalk between autophagy and apoptosis pathways. The interplay between autophagy and apoptosis may be important throughout development and homeostasis [36, 37]. Inhibition of autophagy by 3-MA or siRNA targeting genes related to autophagy triggers apoptosis in human HeLa cells [33]. Shen et al. reported activation of autophagy in IVDs by interleukin-1β in rat AF cells under serum deprivation. Upon inhibition of autophagy by 3-MA, apoptosis was increased in AF cells. They concluded that autophagy might protect against apoptosis of AF cells as well as against IVD degeneration [38]. The results of the present study demonstrate that autophagy, which was activated by rhSIRT1, inhibited apoptosis, leading to increases in the number of human NP cells. Notably, this effect of autophagy was more prominent in advanced degenerative IVDs. These findings suggest that autophagy plays an important role in the IVD degeneration process.
Distinct apoptotic and autophagic responses to rhSIRT1 in NP cells from Pfirrmann grade IV IVDs, compared with those from Pfirrmann grade II IVDs, may be due to different disease types and degeneration severity. We have previously reported that SIRT1 mRNA expression is higher in Pfirrmann grade III IVDs than in Pfirrmann grades II and IV IVDs [13]. We considered that SIRT1 expression might be induced in the early stage of IVD degeneration when the internal environment of the IVD begins to deteriorate because of disturbances in the nutrient supply and that it then decrease in the advanced stage of IVD degeneration. The role of SIRT1 was altered according to IVD degeneration severity. The results, which show that the sensitivity to rhSIRT1 was different between Pfirrmann grades II and IV IVDs, indicate the safety and effectiveness of rhSIRT1 as a therapeutic agent for the treatment of degenerative IVD disease.
The present study may be limited regarding the type of cells employed in experiments. We used only NP cells in this study, but both NP and AF cells are involved in the mechanisms of IVD degeneration. Moreover, the experiments were conducted using monolayer cultures with 1000 mg/L glucose and 10 % (v/v) FBS, 20 % (v/v) oxygen, and a neutral pH. However, in human IVDs, the central NP is hypoxic and the oxygen concentration is <1 % (v/v) [8]. Therefore, our culture conditions do not reflect the in vivo situation. It is now evident that nutrients, oxygen levels, and pH are powerful regulators of cellular activity and gene expression in IVDs, which can strongly influence the rates of ECM synthesis and degradation, and even cell viability [7, 39, 40]. This is a limitation of the present study. In addition, we chose a low serum environment of 1 % (v/v) FBS supplementation as a condition of nutrient deprivation because serum interacts with other nutrients and plays a major role in IVD cells [40]. Serum withdrawal to 1 % (v/v) FBS supplementation has been used as a positive apoptosis control for human AF cells in vitro and resulted in a significantly greater apoptosis incidence compared with the 20 % (v/v) FBS negative control [29]. However, it remains to be elucidated whether this serum concentration is an acceptably low nutritional condition for human IVD cells. Therefore, the effects of rhSIRT1 should be confirmed in vivo. In addition, SIRT1 is widely involved in the regulation of cellular processes [11, 12]. SIRT1 may also be directly involved in cell proliferation, as shown by the increases in cell number, or it may affect apoptosis through pathways other than autophagy. Further investigations need to be conducted.
We demonstrate that limited nutrition induced apoptosis through the mitochondrial pathway and that rhSIRT1 prevented apoptosis of human NP cells through acceleration of autophagy. Furthermore, the effects of rhSIRT1 on autophagy and apoptosis in human NP cells were stronger in Pfirrmann grade IV IVDs than in Pfirrmann grade II IVDs. The actions of rhSIRT1 in human NP cells depend on the degeneration grade of the IVD. SIRT1 may be a potential candidate as a novel treatment agent for degenerative IVD disease.
annulus fibrosus
- Annexin V-FITC:
annexin V conjugated with fluorescein isothiocyanate
- Atg :
autophagy-related gene
- Bax:
Bcl-2-associated X protein
- Bcl-2:
B-cell lymphoma
Dulbecco's modified Eagle's medium
extracellular matrix
fetal bovine serum
intervertebral disc
light chain 3
low nutrition
normal nutrition
- NF-κB:
nuclear factor κB
nucleus pulposus
phosphate-buffered saline
propidium iodide
- rhSIRT1:
recombinant human silent mating type information regulator 2 homolog 1
- siRNA:
small interfering RNA
silent mating type information regulator 2 homolog 1
sequestome 1
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We thank Drs Koki Uno and Teppei Suzuki for their generous assistance and Dr Mitchell Arico for help with preparing the manuscript. This work was supported by a Grant-in-Aid for Scientific Research and Young Scientists (B) 23791639 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (grant 1302-18300169).
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Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SM carried out all studies; participated in study conception and design, acquisition of data, analysis and interpretation of data; and wrote the manuscript. KK participated in study conception and design, secured funding, cowrote the manuscript, and helped to revise the manuscript. TY participated in the design of the study, conducted experiments, and contributed to drafting and revising the manuscript. KM and TT participated in the design of the study, conducted experiments, performed statistical analyses, and contributed to drafting and revising the manuscript. ZZ, YT, TK, MI, and TU acquired and interpreted data and helped with drafting the manuscript. TM, RK, and MK participated in study conception and contributed to drafting the final manuscript. KN participated in study conception and design and contributed to drafting and revising the manuscript. All authors read and approved the final manuscript.
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Cite this article
Miyazaki, S., Kakutani, K., Yurube, T. et al. Recombinant human SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human intervertebral disc nucleus pulposus cells. Arthritis Res Ther 17, 253 (2015). https://doi.org/10.1186/s13075-015-0763-6
DOI: https://doi.org/10.1186/s13075-015-0763-6
- Nucleus Pulposus
- Annulus Fibrosus
- Nucleus Pulposus Cell
- Annulus Fibrosus Cell
- Autophagy Activity


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Hotshot Trucking Co | Northport, AL
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text:
Happy Independence Day!
After the cookouts, parades and fireworks I have been reflecting on the meaning of Independence as it relates to my family.
Dr Jim Dobson has said that parenting is the only vocation in the world where you work your way out of a job just as your starting to get good at it. His meaning as I understand it is that our goal as parents is to raise up our children to the point that they are independent and don't need us anymore. This is a daunting task. It is all the more difficult when you are dealing with special needs kids.
Right now we are dealing with my son, Joshua and his struggle with time management. Sometimes it seems that Josh invented the concept of slow motion replay. Left on his own, he will finish his morning toilet, shower, dressing and breakfast just in time for… lunch. He has two speeds: "Stop" and "Plod."
We have tried reasoning, threatening, nagging, bribing and shaming. We've tried reminders, lists, prompts, voice recorders. We haven't found anything effective yet, but we will keep trying.
This is an area that is critical for his future success. And ultimately, that is what our goal is – self-reliance and independence.
Happy 4th of July.


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Style of Prince of Wales and Approach to Timeless Fashion
Prince Charles, Prince of Wales and heir to the British throne must have an irreproachable appearance. It must be said that the heir to the throne looked after his appearance from an early age. After all, the Prince of Wales has a duty to represent his country and the image of the nation.
Prince Charles for more than 30 years has remained true to the classic patterns that his trusty tailors Anderson & Sheppard, and Gieves & Hawkes have advised him and that he has always dressed in the absolute British style.
The cut of their costumes cannot obey more classic patterns. Wide cuts that help the suit rest on the body without insinuating too much the figure, wide flaps and low cut, borders whose length matches exactly the one of the knuckles of the hand and pants without turning in the two and three Pieces. Perhaps this explains a more remarkable difference in the dressing of Prince Charles and his royal acquaintances.
Although it has been common to see him wear simple suits or vest, in recent years the most frequent image of the Prince of Wales is the suit of crossed buttoned jacket (which, incidentally, is also a standard of Juan Carlos, The King of Spain).
Another note of his taste for true classic elegance is to wear a pocket square at all times. This complement is a faithful companion of all his jackets. No matter who he meets or what country to visit, he will always wear his handkerchief. In addition to his way of dressing, I have no doubt that he is proud of his wardrobe choice.
Prince of Wales has always used excessive colors in his outfits. His mono color or diplomatic costumes are his most frequent choice. Something similar happens with his shirts. These are usually also monochrome or in some cases, blended with fine lines. It will be difficult to see him in plaid shirts or striking stripes. His favorite color for these is the light blue. This color facilitates the correct combination of handkerchiefs and also allows him to choose a handkerchief for a more cheerful print.
All of his shirts have a double cuff, and there is not much fabric left after the cufflinks. Sometimes it can even give the impression that they are simple cuffs where the button has been replaced by a buttonhole. The cuff links are characterized by their simplicity and discretion with a part of gold in them.
This discretion can also be seen in his ties. He uses classic colors with small motifs. However, he still has a fondness wearing striped ties with bands of wide colors which denote his timeless style and appeal.
He is more likely to always wear a flower in the buttonhole, you can see him in various events without a flower. Perhaps his only shy wink to modernity are the bracelets that in some photo can be guessed under the double fist. All obeying once again the English classic patterns and colors.
As far as footwear is concerned, he continues to opt for the Oxford classic with the typical English wide width. With dark and diplomatic suits he always wears black shoes and in his gray suits he prefers brown or dark shades of wine.
If you look at the set formed by his shirt collar, tie knot and jacket lapels, you will appreciate that all these parts do not take any relevance and his face is the sole protagonist of the final portrait. You must not forget that as you have established on some occasion, all your clothing has as its final mission to highlight our face and our physique. The protagonist is you and not your tie knot or your coat.
Time passes for all and Princes are no exception. The face of Prince Charles has widened with the years and perhaps a second return to the tie would achieve a final result more in line with his current physiognomy.
I strongly believe that it will be a long time before you see a public figure of the relevance of the Prince of Wales wearing so many years according to the classical standards and giving the maximum sense to the word "elegance". Prince of Wales is a clear sign that it is not necessary to succumb to the most modern tendencies or to become authentic fashion victim to be able to be an exponent of elegance and style.
Prince of Wales is the ultimate proppant of British elegance in its maximum. If you have the opportunity to go to an English bookstore and look at the many biographies that exist on Prince of Wales, you can see in the photos of the interior that his dress has hardly undergone remarkable changes in all these years.


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Applesoft BASIC
Applesoft BASIC (sometimes called "Applesoft II") was the second dialect of BASIC supplied on the Apple II computer, superseding Integer BASIC. Applesoft BASIC was supplied by Microsoft and its name is derived from the names of both Apple and Microsoft.
Apple's customers were demanding a version of BASIC that supported floating point calculations. As Steve Wozniak, the creator of Integer BASIC and the only person who understood it well enough to add floating point features, was busy with the Disk II drive and device controller, and with Apple DOS, Apple turned to Microsoft, who was the BASIC vendor of choice after their success with Altair BASIC, and licensed a 10 KB assembly language version of BASIC dubbed "Applesoft."
It was similar to (and indeed shared a common code base with) BASIC implementations on other 6502-based computers, such as Commodore BASIC: it used line numbers, and spaces were not necessary in lines.
While Applesoft BASIC was slower than Integer BASIC, it had many features that the older BASIC lacked:
- Atomic strings. A string is no longer an array of characters (like in Integer BASIC and C); it is instead a garbage-collected object (like in Scheme and Java). This allows for string arrays;
DIM A$(10)resulted in a vector of eleven string variables numbered 0 to 10.
- Multidimensional arrays.
- Single-precision floating point variables with an 8-bit exponent and a 31-bit significand and improved math capabilities, including a trigonometry library.
- Commands for high-resolution graphics.
CHR$, ASC, STR$,and
VALfunctions for converting between string and numeric types
LETstatement optional
- User defined functions (simple one-line functions written in BASIC, with a single parameter)
- Error-trapping, allowing BASIC programs to handle unexpected errors by means of a subroutine written in BASIC.
Relatively few action games were written in Applesoft BASIC, for several reasons:
- In this era of carefully counting clock cycles and limited memory, it was inefficient to write speed-dependent programs that ran on a runtime interpreter.
- The use of real numbers for all math operations created unnecessary overhead and degraded performance. Applesoft actually converted integer numbers to real before performing operations on them, then converted them back to integers if necessary.
- So-called shape tables are a slow alternative to bitmaps. No provision existed for mixing text and graphics, except for the limited "Hardware split screen" of the Apple II (four lines of text at the bottom of the screen). Many graphics programs thus contained their own bitmap character generator routines. No provision was added in the 128 KB Apple IIe and Apple IIc models' BASIC interpreters for the new machines' extra memory and double-resolution graphics, or for the Apple IIGS's 16-color mode. (Beagle Bros offered machine-language workarounds for these problems.)
- The program was stored as a linked list of lines; a
GOTOtook O(n) (linear) time, and although Applesoft programs were not very long compared to today's software, on a 1 MHz 6502 this could be a significant bottleneck.
- No sound support aside from a PEEK command that could be used to click the speaker. The language was not fast enough to produce more than a baritone buzz from repeated clicks anyway. However, compiled assembly language was fast enough, and music spanning several octaves could be played by repeated calls to a machine-language tone generator.
Here's Hello World in Applesoft BASIC:
Multiple commands could be included on the same line of code if separated by a colon (:). The ? can be used in Applesoft BASIC as a shortcut for "PRINT", though spelling out the word is not only acceptable but canonical -- Applesoft converted "?" in entered programs to "PRINT", which would appear when a program was listed. So the program above would actually appear in a LIST command as shown below:
This article includes text from Everything2, licensed under GFDL.
- There was a well-documented bug in Applesoft BASIC that could actually crash the interpreter if ONERR GOTO was in effect and numerous program errors occurred. Apple provided a short assembly-language routine which could be POKEd into RAM and CALLed to ameliorate the problem to an extent. Later it was discovered by an enterprising hacker that the required code was actually in the Applesoft ROM (though it was never executed) and could be called there instead.
- Applesoft could be extended by two means: the ampersand (&) command and the USR() function. Machine-language routines could be attached to the ampersand and USR hooks in the computer's memory and call Applesoft ROM code to retrieve the values of parameters that followed them. A number of third-party packages were produced to add functionality to Applesoft by one or the other of these mechanisms. Additionally, CHRGET, a vector at location $B1, fetches the next character from program text and provided a hook that allowed external routines to change the interpreter's behavior.
- Applesoft, like Integer BASIC before it, did not come with any built-in commands for dealing with files or disks. The Apple II disk operating system, known simply as DOS, thus intercepted all input typed at the BASIC command prompt to determine whether it was a DOS command. Similarly, all output was scrutinized for a Control-D character (ASCII 4), which BASIC programs would send before printing a disk command to get DOS's attention. ProDOS followed this lead, although the BASIC command interpreter was placed in a separate program called BASIC.SYSTEM.
- Neither Apple nor Microsoft ever made source code for Applesoft BASIC available. However, Glen Bredon included a program with his Merlin assembler that would generate a commented copy of a BASIC disassembly from the ROMs in a user's computer (using the ROMs to decrypt the provided comments and so forth), cleverly skirting copyright law.
- Both Integer BASIC and Applesoft used the technique of tokenizing to reduce the memory requirements of programs and to speed their interpretation. As code was entered, BASIC keywords would be converted to single-byte tokens; the process was reversed when the program was listed. Integer BASIC used characters with codes above 127 for normal text and codes below 128 for tokens; Applesoft used exactly the reverse.
- Due to Applesoft BASIC's slow performance, BASIC compilers were much sought-after tools for BASIC programmers. The first, Microsoft's The AppleSoft Compiler (TASC), was actually written in Applesoft and then used to compile itself. A later product, the Einstein Compiler, was somewhat more sophisticated and offered better performance both in compilation and in execution. The ultimate BASIC compiler was the Beagle Compiler, written by Alan Bird and published by Beagle Bros; it integrated closely with ProDOS (the current Apple II OS at the time of its release), compiled programs in seconds rather than minutes, and achieved superior code execution performance by optimizing integer math and pre-computing addresses of GOTO targets, among other tricks. Unlike previous compilers, the Beagle Compiler did not truly compile BASIC programs to machine code, but rather converted them to a highly optimized bytecode that was interpreted by a runtime module, much like the UCSD p-System.


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In essence, the Right to be Forgotten derives from a Spanish case concerning Mr Mario Costeja Gonzalez, who took issue with a search result that was returned by Google when his name was searched. The offending result was an advert for a home of his that had been re-possessed. It represented a time when Mr Gonzalez had serious financial concerns and by it being returned by Google cast negative aspersions on his current financial position, which was markedly improved.
In determining the case, the Spanish Court decided that any material which is deemed irrelevant, excessive or outdated has no place being readily available to members of the public. The Court deemed that Mr Gonzalez's old repossessed home notice was excessive, outdated and irrelevant and therefore ordered Google to remove it.
This was not material that was defamatory, harassing or even untrue. The Court found that it was merely irrelevant, excessive and outdated. Three words for the legal test – irrelevant, excessive and outdated.
In real life this means that if you have old material that is no longer relevant, but that may not be defamatory, harassing or offensive it can still be removed. However, you have to know how to apply to Google and how to deploy the correct Right to be Forgotten application. At the moment Google just have a form, which goes into a queue and gets looked at as when Google get through their other thousands of application.
As specialists with an historical and on-going relationship with Google, we at Pinder Reaux are able to bespoke each removal application and most importantly ensure that any application made is the correct one that will result in a positive conclusion. Google often come back with further questions and sometimes even rebuff an initial removal application. However, with us on your behalf, being able to properly draft your application and immediately deal with any further information, queries or rebuttals Google may have, we will make sure that you are in the best possible position to have the negative material finally removed from Google.
Google do not entertain numerous repeated requests and so you HAVE TO get it right first time, and we at Pinder Reaux can help you do that. Call us immediately on 0208-252-7373 to discuss your requirements with our specialist team.


text: Looks like the SP500 fund is by far the lowest cost. About 80% of the funds in VTSAX are in the SP500. You could add another 15% mid cap and 5% small cap to approximate VTSAX, but I don't think it is worth it with those expense ratios.If I were in that situation I would go 100% SP500. Later on when you have a little extra to invest you can open an IRA at Vangaurd and invest in VTSAX there.