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Bohemian wedding photos curated by Boho Circus on Pinterest. All sources of the photos may be found here. Wedding tassel necklaces may be found here.
Bohemian wedding photos curated by Boho Circus on Pinterest. All sources of the photos may be found here. Wedding tassel necklaces may be found here.
Posted at 02:00 PM in #BohoCircus, Bohemian, Boho Hair, Boho Wedding, Boho-Chic, Entertaining, Event Ideas, Gypsy style, Hipster Entertaining, Love | Permalink | Comments (0)
Tags: bohemian, bohemian alters, bohemian bride, bohemian brides, bohemian events, bohemian wedding dress, bohemian wedding events, bohemian wedding ideas, bohemian wedding planning, bohemian weddings, boho, boho alters, boho bride, boho brides, boho circus, boho circus blog, boho flowers, boho gypsy wedding, boho gypsy weddings, boho hipster wedding, boho hipster weddings, boho marriage, boho wedding, boho wedding dress, boho wedding events, boho wedding ideas, boho wedding parties, boho wedding planning, bohocircus, couples, couples in love, gypsy wedding ideas, hippie wedding ideas, love, outdoor wedding, outdoor weddings, rustic wedding, rustic weddings
1 Temporary Arrow Tats from HAPPYTATTS happytatts.etsy.com // 2 Arrow String Art from WOODLAND LANE woodlandlane.etsy.com // 3 Crossed Arrows Pillow from ORTOLAN HOME GOODS ortolanorganic.etsy.com // 4 Arrow Wrap Bracelet from COMEJEW comejew.etsy.com // 5 Set of Vintage Archery Arrows from GALLIVANTING GIRLS gallivantinggirls.etsy.com
Posted at 10:00 AM in #BohoCircus, A Rosy Outlook, BC Style, Boho Accessories, Boho Must Have, Boho-Chic, Etsy Finds | Permalink | Comments (0)
Posted at 10:00 AM in #BohoCircus, BC Style, Boho Accessories, Boho Jewelry, Boho Must Have, Boho-Chic | Permalink | Comments (0)
Tags: amelie mancini, arosyoutlook, bohemian apparel, bohemian home decor, Boho Circus, boho home decor, boho home goods, boho must have, cacti, cacti pouch, cacti purse, cacti shirt, cacti t shirt, cacti tank, cacti tee, cacti top, cacti tshirt, cactus, cactus apparel, cactus art, cactus iphone case, cactus necklace, cactus pillow, cactus pouch, cactus print, cactus purse, cactus shirt, cactus t shirt, cactus tank top, cactus tee, cactus top, cactus tshirt, confetti riot, decorart design, dessi designs, etsy finds, fableandlore, festival fashion, festival wear, hipster home decor, succulents, wanderlust, women, womens
Posted at 10:00 AM in #BohoCircus, @BohoCircus, Austin, BC Style, Bohemian, Bohemian Boutique, Boho Jewelry, Boho Street Style, Boho-Chic, Gypsy style, Lookbooks, Texas Fun, Traveler, What's New? | Permalink | Comments (0)
Tags: ACL, ARosyOutlook, ATX, austin, bohemian, bohemian apparel, bohemian chic, bohemian defined shirt, bohemian definition shirt, bohemian fashion, bohemian hipster, bohemian lifestyle, bohemian must haves, bohemian shirt, bohemian street style, bohemian style, bohemian t shirt, bohemian t-shirt, bohemian tee, bohemian tshirt, boho, boho apparel, boho chic, boho fashion, boho graphic shirt, boho graphic t shirt, boho graphic tee, boho graphic tee, boho graphic tshirt, boho hipster, boho lifestyle, boho must haves, boho street style, boho style, BohoCircus lookbook in Austin, demand the impossible shirt, festival fashion, festival must haves, festival style, free spirit shirt, free spirit t shirt, free spirit tee, free spirit tshirt, gypsy fashion, hipster graphic tees, hipster street style, home goods, jewelry, music festival shirt, old soul, old soul shirt, old soul t shirt, old soul tee, old soul tshirt, shop bohemian, shop boho, soco, south congress style, texas, Texas, urban hipster, volkswagen shirt, volkswagen tee, volkswagen tshirt, vw beetle shirt, vw beetle tee, vw beetle tshirt, vw bug shirt, vw bug t shirt, vw bug tee, vw bug tshirt
The classic cowboy essential has been upgraded to a boho accessory "must have." A signature look worn by all of the cowboy greats, bandanas are now found in collections by Free People, Spell, Madewell, Anthro, and many others.
Bandana Clutch They All Hate Us // Lonesome Dove Inspiration Red Branch Media // Gypsiana Shirt Dress and Bloomers Spell //Army Jacket and Pink Bandana Bliss and Mischief
All other photos: Free People Blog & Free People
Posted at 11:06 AM in Bohemian, Bohemian Boutique, Boho Accessories, Boho Must Have, Boho Street Style, Boho-Chic, Fashion | Permalink | Comments (0)
Tags: bandana clothing, bandana fashion, bandana inspiration, bandana style, bandanas, free people, spell & the gypsy collective
Which type of New Bohemian are you?
Lush with gorgeous photographs, our new favorite book, The New Bohemians, is considered a 300 page Bohemian Bible. Showcasing over twenty creative homes throughout the country, Justina's story is a discovery of how one can define the "New Bohemian." Have you noticed all the new terms that suggest the same eclectic mix of society? Boho hipster, gypster, hippie gypsy, boho hippie, prairie folk, gypsy nomad, and modern bohemian are terms that come to mind.
They seem to all refer to the same sort of person: THE NEW BOHEMIAN
Justina breaks down the New Bohemian into six categories: Modern, Earthy, Folksy, Nomadic, Romantic, and Maximal. Within each of these sections, she devotes pages to lovely home and lifestyle photographs, DIY projects, interviews with home owners, and healthy recipes. Here are a few photo teasers from the book that I found on Pinterest:
"Whether they live in a turn-of-the-century craftsman-style house, a cabin in the hills, an urban loft, or a dome in the desert, modern bohemians mix the clean lines and functionality of classic modern design with the decorative exhuberance of a bohemian." - Justina Blakeney.
The dining room of Josie Maran & Ali Alborzi speaks to the sleek style of the Modern Bohemian. Their 1920's cabin getaway in the hills of Beachwood Canyon, in Los Angeles, is full of items found during their world travels.
"Nature rules. The earthy bohemian home is full of foresty fancies: rattan and wool, plants and pets, earth tones and weathered woods. Relaxed and airy, informal with rustic details..." - JB
The above living room is the Portland, Oregon bungalow of Emily Baker & Kerby Ferris. "The bright white backdrop is offset by layered patterns and natural collections of foraged finds." The homeowners describe it as a sanctuary for yoga, meditation, rest, and creativity.
An Airstream nestled in an overgrown garden oasis in Los Angeles, and a recipe to create your own home fragrance, were our favorites in the Earthy Bohemian section.
"Packed with tales of adventures, treasure-hunting, and hand-me-downs, the folksy bohemian is a collector, a maker, an upcycler, and a storyteller." - JB.
This is the folksy bohemian casita of Mattie Kannard & Dennis Smith, Las Cruces, New Mexico. "A clutter of cats, wily little dogs, happy hens, and rogue roosters are all part of the Kannard-Smith household," - JB.
"...A nomadic bohemian, whose vibrant home is packed with textiles, ephemera, and anything else picked up during travels in both the actual and virtual worlds. Their homes are about movement: Objects, furniture, and even entire room are constantly being relocated." - JB
Prop stylists, interior designers, fashion designers, and casting directors are creative free spirits who would gravitate toward change and several are featured in this section.
Amhalise Morgan lives with her two kids, Harmony Cree and Rebel Spirit, in a Brooklyn brownstone. She spent a decade creating her dream home out of internet-found worldly treasures.
"The romantic bohemian home is a theatrical space, full of dramatic moments, music, and circuslike quirks...These homes are glamorous and faded, considered and whimsical, worldly and otherworldly." - JB
Arielle Pytka lives in a loftlike space in Venice, California. She describes her home as "a perfect mess." Justine describes it as "a playground for creative pursuits."
"Whereas the minimalist strips things down to the bare bones, maximal bohemians surround themselves with eye candy. Every surface of the home is abundant with art." - JB
This is Justina's home in Los Angeles nicknamed the "Jungalow." You may find more photos of Justina's home at her amazing Blog.
After reading The New Bohemians, you may get an idea as to which bohemian style you may fall into. We found ourselves in lots of the categories, and you may to! That just seems to sum it up: Bohemians are eclectic free spirits who are amazingly hard to categorize.
For more on Justina, and to purchase her wonderful book, please visit her at justinablakeney.com.
Photography by Dabito
Posted at 04:01 PM in Art, Bohemian, Bohemian Family, Boho Accessories, Boho Icons, Boho-Chic, Book Obsession, Create It!, Design, Eco-boho, Good Karma, Gypsy style, Handmade, Home, Inspiration, Read, Vintage, What's New? | Permalink | Comments (0)
Tags: abrams books, abrams publishing, artists homes, bohemian book, bohemian crafts, bohemian DIY, bohemian family, bohemian home, bohemian home accessories, bohemian homes, bohemian interior decor, bohemian interiors, bohemian photographs, bohemian style, boho crafts, boho DIY, boho family, boho home, boho home goods, boho homes, boho interiors, boho recipes, coffee table book, Dabito, earthy bohemian, earthy boho, folksy bohemian, folksy boho, gypsy book, jungalow, justina blakeney, maximal bohemian, maximal boho, modern bohemian, modern boho, nomadic bohemian, nomadic boho, romantic bohemian, romantic boho, the new bohemians
One of the most impressive pieces of Southwestern Native American jewelry has made its way into the spotlight again as a staple in any bohemian wardrobe. This accessory has made an appearance in numerous photoshoots and editorial layouts, as well as a key statement in Leah Hoff's Nashville Wedding (pictured above, link below)-- wouldn't you love investing in a signature item like the squash blossom, instead of a bridesmaid dress?! We think yes!
"Turquoise is closely associated with Navajo jewelry, but it was not until 1880 that the first turquoise was known to be set in silver. Turquoise became much more readily available in ensuing decades. Coral and other semi-precious stones came into common use around 1900. One of the most important forms of Navajo and Southwestern Native American jewelry, is the Squash Blossom Necklace."
— Native American Jewelry Wiki Page
hoto Sources: 1 The Lane // 2 Sweet & Spark // 3 SRTA-PEPIS // 4Spell // 5 SRTA-PEPIS // 6 Spell // 7 District of Chic // 8 This Beadiful World
Posted at 09:01 PM in Bohemian, Bohemian Boutique, Boho Accessories, Boho Jewelry, Boho Must Have, Boho Wedding, Boho-Chic, Gypsy style, Inspiration, Native American, Vintage | Permalink | Comments (0)
Boho Circus Style on Instagram - Our Gypsy Soul tank courtesy of @_beretta, our Gypsy Soul Tambourine at LaceyFierce, our Bohemian Tshirt captured by Annika Keating, and our GS tambourine in tarot vignette by BelongWithWildflowers
Posted at 11:32 AM in #BohoCircus, @BohoCircus, Bohemian, Boho Street Style, Boho-Chic, Fashion, Gypsy style, Instagram, Join the Circus | Permalink | Comments (0)
I can only blame my gypsy soul for taking a break from posting here. My creative spirit veered in other directions for a while, but it's great to be back here posting again at Boho Circus. For the last couple of years, I've been busy writing a novel, curating my Pinterest, and creating a new line of Boho Circus bohemian apparel.
Here are a few shots from our Gypsy Soul photo shoot at Enchanted Rock State Park in Texas. According to legend, Tonkawa Indians named the popular 425-foot pink granite hill "Enchanted Rock" because they believed a Spanish conquistador cast a spell on the rock, making magical ghost fires glow at the top. We think that our model Avalon glows with the help of photographer Chyna Mason.
Check out our full line of goods at A Rosy Outlook.
Posted at 10:49 AM in A Rosy Outlook, Boho Hair, Boho-Chic, Etsy Finds, Gypsy style, Texas Fun | Permalink | Comments (0)
Tags: A Rosy Outlook, A Rosy Outlook Designs, arosyoutlook, blame my gypsy soul, bohemian apparel, bohemian shirt, boho circus, boho style, boho tee, Bren Mason, etsy, free people, gypsy soul, gypsy tambourine, gypsy top, hippie style, Inc., Llano, Texas, women's clothing
Taking this living space as inspiration from YoungHouseLove,
I found these amazing products at Anthropologie: Live Love Pillow, Quirky Heirloom Throw, Battersea Sofette in Fog Linen, Sari Throw, & Gloria's Round Garden Rug at Anthropologie...
Posted at 07:47 AM in Boho-Chic, Home, Inspiration | Permalink | Comments (1)


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subset: commoncrawl


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Health Care Administration Associate Degree
A career in healthcare administration will help you oversee the efficient operation of a medical facility. An associate's degree program can start you on your way. This article looks at the typical courses in administration and medicine that you will find in a 2-year program, along with career options upon graduation. Schools offering Health Care Administration degrees can also be found in these popular choices.
What Kinds of Associate's Degree Programs Are Available in Healthcare Administration?
According to the National Center for Education Statistics, there are nearly 100 schools that offer associate's degree programs in healthcare administration and management. You can earn an Associate of Science, Associate of Applied Science, Associate of Occupational Studies or Associate of Arts in healthcare administration or medical office administration. Programs can consist of 50-108 credits and usually take 18-24 months to complete.
|Program Length
|About two years
|Course Offerings
|Psychology, billing and coding, healthcare law, medical terminology
|Program Format
|Online or on campus
|Career Options
|Medical secretary, transcriptionist, executive assistant, office manager
What Classes Will I Take?
During a healthcare administration associate's degree program, you'll learn about medical office procedures and current healthcare issues. Typical courses that you can take include medical billing and coding, medical terminology, psychology, healthcare accounting and healthcare law. You also might develop keyboarding and database management skills. Most programs include an externship, which you can complete at an approved healthcare facility.
How Might I Learn Online?
You may be able to complete a healthcare administration program online. Technical requirements normally include a computer with high-speed Internet access and a DVD-ROM drive. You may need a microphone, headset and additional hardware plug-ins. If your online program contains an externship, you might need to mix online courses with in-person meetings. You also might need to take your final exams on campus.
What Are Some Common Career Possibilities?
With your associate's degree in healthcare administration, you might work as a medical secretary, executive administrative assistant, medical transcriptionist or medical office manager. Your job responsibilities and wages can vary, depending on the type and size of medical practice where you work. The U.S. Bureau of Labor Statistics reported that medical secretaries earned a median yearly salary $32,240 as of May 2014, while executive administrative assistants made a median salary of $51,270 per year (www.bls.gov). Medical transcriptionists received an annual median income of $34,750 during this same time.
To continue researching, browse degree options below for course curriculum, prerequisites and financial aid information. Or, learn more about the subject by reading the related articles below:


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We have been providing solutions for ethics compliance for 15 years. This experience allows us to provide effective solutions for your company. Navigating compliance issues can be complex and time consuming. With our help we can work with you to provide you with the resources you need to prepare your company.
As part of our consulting we work to assist companies with the implementation of ethics reporting systems. At times reporting systems can be met with confusion, frustration, and resistance. This can be avoided through proper communication and education. We will help you compile personalized information and materials that will aid in the adoption of a new anonymous reporting system.
No matter what your industry is, we help professionals of all different spheres navigate ethical issues. This starts with understanding the industry and the standards. Our experience provides insight into various industries to assist professionals in avoiding ethical issues and creating ethical guidelines for their business. Our consultation will help you avoid fines that lead to not only financial penalties, but also damage to your reputation.
We know ethics and we know training! Let our team of experts help develop your code of conduct and ethical standards into a world-class training system. You don't have to hire one outside firm to develop elearning and other who understands the ethics arena. We have experience in curriculum development, ethics/compliance, elearning development (using a variety of authoring platforms), and elearning LMS administration. Global Ethics Solutions can handle every stage of the process from conception to final rollout.
We are passionate about ethics and love to share our passion with your employees and leaders in-person! Global Ethics Solutions can facilitate one of our ethics program on-site or provide business ethics keynote speaking to groups, leaders, or conferences. One of company strategic partners are available to on-site speaking to meet your needs. If you are looking for any kind of professional training and speaking services, allow Global Ethics Solutions to take your training to the next level.
Contact us online or call 1-877-77ETHIC
Whether your company is large or small, Integri-Line can fit your company's unique structure.
Our ethics compliance software is easy to setup and use on any device. We will guide you from start to finish.
Our system scales to fit your needs! Get only the features you need and keep your costs down.
As a third-party, our experience and independence allows employees and stakeholders to trust the system.


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We are confident you will find Davinci Meeting Rooms with the highest quality and service, which provide affordable meeting space and executive business suites at great discounts. Please browse the following, if you are looking to book a specific type of meeting room in Marietta:
As a growing city in Georgia, Marietta is one of Atlanta's biggest suburbs in population and area. It is also the fourth largest city in the state of Georgia. The city's rapid growth is fueled by its proximity to many business places and other popular locations.
Marietta is an important economic area for Georgia. Its place near Atlanta is important for both small and large corporate investors seeking to expand their markets and service regions. The area is also growing in industry, health care, and manufacturing. Renting meeting rooms in Marietta, GA is a cost-effective solution to keep your company at the top of the list when it comes to doing business in the state.
Cheap deals on temporary, short-term, and long-term meeting rooms in Marietta are available throughout the year. You can find and reserve a venue conveniently while online. Book reservations and rent conference room facilities for meetings of all kinds to accommodate your clientele. Our private conference rooms in Marietta are found throughout all corners the city with convenient transportation and parking. Each location offers desirable features without the high rates or lengthy commitments of hotels. Finding inexpensive rental space in Marietta is easy with our many facilities.
Davinci Meeting & Conference Rooms™ is a leading provider of short-term and long-term meeting rooms for professionals. We offer offices ranging from touch-down space for an hour to cost-effective workplaces for weeks at a time. With a global footprint spanning more than 850 cities, we maintain a thousands-strong portfolio of temporary meeting spaces.
We even provide value-added business support services in Marietta covering everything from receptionist services to catering. Davinci Meeting Rooms makes it easy for businesses with tight budgets and limited resources in Marietta to project a "big business" image without breaking the bank. To learn more about Davinci custom meeting space solutions available in Marietta, call us at 1-877-4BIZRMS (1-877-424-9767) or click on the above listing for more details on your preferred Marietta location.
You have chosen a time outside of normal buisness hours.
Your reservation will be PENDING APPROVAL
|Unlmited Hours/mo
|Board Room
|Small Conference Room
|Medium Conference Room
|Large Conference Room
|4 Hours/mo
|Day Office


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FACT #6 – Male survivors are not more likely to become sexual abusers
Simply put – the majority of males who are sexually abused do not become sexual abusers.
Otherwise know as the "Vampire Myth" this toxic stereotype is especially dangerous because it can create terrible stigma and shame, reinforcing fears in both the victim and his family that he is destined to become an offender. As a result survivors who report can sometimes be treated as potential perpetrators rather than victims who need help. In addition, adult men who experienced sexual abuse can delay disclosure for fear of being labeled as a potential risk to their own or other people's children.
Many studies investigating the link between victimization and perpetration show that it is a minority of offenders who report experiencing sexual abuse as children:
- "proportionally very few of these sexually abused boys (3%) went on to become sexual offenders; and, contrary to findings typically reported in retrospective clinical studies, proportionally few sexual offenders (4%) had a confirmed history of sexual abuse. – Leach, Chelsea, Anna Stewart, and Stephen Smallbone. "Testing the sexually abused-sexual abuser hypothesis: a prospective longitudinal birth cohort study." Child abuse & neglect (2015).
- 28.2% of the total 1,717 offenders reported that they had been sexually abused as children. Hanson, R. Karl, and S. Slater. "Sexual victimization in the history of sexual abusers: A review." Annals of Sex Research 1.4 (1988): 485-499.
- 31% of 348 men convicted of sexual assault crime reported "[e]vidence of some form of sexual trauma during their developmental years (ages 1 through 15)" Groth, A. Nicholas, and Ann W. Burgess. "Sexual trauma in the life histories of rapists and child molesters." Victimology 4.1 (1979): 10-16.
- 8 of 117 victims of CSA identified as potential pedophiles Bagley, Christopher, Michael Wood, and Loretta Young. "Victim to abuser: Mental health and behavioral sequels of child sexual abuse in a community survey of young adult males." Child Abuse & Neglect 18.8 (1994): 683-697.
- In 2 studies, Hindman and Peters report that 29% and 32% of convicted serial offenders reported being victims of CSA during a polygraph administered post conviction. Hindman, Jan, and James M. Peters. "Polygraph testing leads to better understanding adult and juvenile sex offenders." Fed. Probation 65 (2000): 8.
Again, while it is true that a significant proportion of perpetrators report having histories of sexual abuse, MOST victims DO NOT go on to commit acts of sexual violence against others.
Indeed, when child victims were followed prospectively, research by Loh and Gidcyz stated, "prospective analyses indicate that childhood sexual assault is not predictive of perpetration during the follow-up period" Loh, Catherine, and Christine A. Gidycz. "A prospective analysis of the relationship between childhood sexual victimization and perpetration of dating violence and sexual assault in adulthood." Journal of Interpersonal Violence 21.6 (2006): 732-749.
For persons seeking information on effective interventions for persons who perpetrate sexual abuse we suggest contacting https://www.atsa.com/


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This is a recent blog that I wrote for Marketing Executives Network Group or MENG.
Remember playing hopscotch? It was easy to play. All you needed was some chalk. Maybe you stole it from school.
Life in the New Normal is very much the same game.
Just like hopscotch it all depends when tile you land on.
Just like in hopscotch in the New Normal many people have one foot tied behind their backs. A balancing act of intention and resolve but precariousness.
Some companies are hiring and some companies are still firing.
It all depends on what tile you step on.
Stepping on the getting hired tile can be very difficult especially if you are over 50. Some are younger than Baby Boomers yet many people think they are out of the game. I am glad that I stopped looking for work in the Old Normal way a decade ago. I learned new games.
I always thought that getting through the eye of the needle was a tough parable.
Getting hired if you are a long term person in transition is even harder.
The irony is that the companies that fire people hire people slowly but they want you to like them on Facebook instantly. Please like me they say. Here is a contest. Here is a game you can play to win a gift certificate. Here is how you connect with us.
Many companies are sitting on piles of cash because you buy their products.
Companies don't look too often into the firing mirror. Some wouldn't see a reflection.
In the New Normal I hear from people that are killer on social media. They teach on how to harness its power.
However they send out emails saying they need work. They aren't landing on the right tiles.
In the depth of the melting of the world in the fall of 2008 as the financial markets crumbled I would be travelling to speak on Networking Your Way to New Business. I could have been in Des Moines, Duluth, or Oklahoma City. The world melted on the coasts first and not in those parts of the world. Business was tough. I came home and told my personal witch I felt like a fraud. "We teach what we need to know," she said. That is why she is my personal witch. I have lent her from time to time to other people who need a little brain alignment.
I spoke recently at a local college about how on why people shouldn't find a job. I had a follow up coffee in my hood with a young chap who was born on the other side of the globe. My sister was born in Beirut. He had some friends in Dubai and was heading there. He was younger than my kids. He asked me to connect him with somebody in my LinkedIn network from that part of the world. I never met her but she reads my blogs from time to time. She connected him with another contact in her LinkedIn game and he got a job with a Multinational Agency in her part of the world. He is probably 23.That is how Hopscotch is played today in the New Normal. Some people have one hand tied behind their backs and some people are free and growing.
It's your turn to hop in the New Normal.
You can connect with Hank Blank on Linkedin.
Follow his updates on twitter: @hankblank
Like Blank and Associates on Facebook
You might enjoy these articles.
Don't Let Your Business Cards Weaken Your Personal Brand
Why Reinvention is a Virtual Necessity
Watch Surviving the Great Recession.


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Do as the picture says and flash a smile because it's the weekend! Project "de-junk" spare bedroom continues this weekend for me plus spending time catching up with friends. What's everyone else up to?
- EAT. For DC locals, what's better than tasty sweet gelato? Flaky and savory biscuits to go with that tasty sweet gelato. Check out Mason Dixie Biscuit Co.'s pop-up at Dolcezza Union Market this Sunday from 11 am – 3 pm. Don't live in DC? Try this recipe from Bon Appetit (do yourself a favor and ignore the caloric information).
- SHOP. Peruse these eight different monthly fashion subscriptions and see which one suits you best. So hard to choose!
- DO. You've heard of Ikea Hacks but have you heard of Anthropologie Hacks? Try out these DIYs and save some cash.


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2012 proved to be a year of immense SEO growth in light of Google's algorithm changes. As an industry, evolution and innovation were keys to building high-quality content and adding as much value as possible for users, readers, customers, and potential customers.
SEO is still not perfect, nor will it ever be, but it appears as if the industry has learned, for the most part, how to work around Google's changes, regardless of how painful that may be. However, there are still some people and companies that are cleaning up after 2012, namely the ones who exploited loopholes in Google's algorithm that have since been closed, therefore rendering that SEO work useless.
Don't settle for short-term fixes anymore. If you want to build long-lasting and sustainable SEO success in 2013, you need to commit to a solid long-term strategy. How do you do this? Start with baby steps. We've compiled a list of short, do it yourself SEO tips you can use to greatly improve your SEO practices for 2013 while also making much more efficient use of your time. None of these individual tips should take any longer than an hour, thus the beauty of this list.
Research and Analysis
- Make a cup of coffee and read through Google's patents and figure out what they're going to do next.
- Take your client out for some drinks. Find out what else they are working on outside of your campaign. Odds are you'll find out some information about them that will be useful either now or down the road.
- Make sure to spend time with your development team. Get to know them personally as well as their individual problems and obstacles they are experiencing right now.
- Make a checklist of how well you know outreach targets. Plan out steps of how you can get to know them better.
- Find out who are the top bloggers for your locality. Know where they hang out so you can try to meet them. Do this at events though, don't stalk them home.
- Arrange your own meeting with key bloggers and journalists.
- Run a survey or poll within the industry to collect information. You can use this information later for content production.
Google+ and Authorship
- Set up Google Authorship for your website. If you have already, verify that it is working.
- Conduct a seminar to exhibit how your team can link up their Google+ author profiles.
- Promote and push your team to build upon their Google+ profiles and share other content.
- If you have a blog, find posts from guest bloggers and move them underneath the correct or relevant author.
Events and Sponsorship
- Sponsor some local events or sports teams.
- Submit a speaker pitch for an event within your industry.
- Create a content plan for liveblogging an industry event.
Content Strategy and Planning
- Brainstorm new product or service ideas that are link-worthy.
- Acquire a company.
- Get sued. (Just kidding, don't.)
- Relate your niche to a different subject to open up new content angles.
- Review old company reports, white papers, etc. for key statistics or statistics you may have missed or forgotten.
- Conduct a team brainstorm to develop new content ideas.
- Share a Google Calendar mapping out your website content plan. Make sure to schedule auto-send reminders to the people involved with the different areas of content creation.
- Shoot to split opinions with your content and promote to both audiences for a reaction. You don't need to be controversial, you just don't have to have a right or wrong answer.
Relationship Building
- Make sure you follow up! Make the effort to stay in touch with key contacts via social media, email, phone, and face-to-face.
- Establish friendships with key influencers and arrange meetings with them in person. Invite them to lunch or schedule a meeting.
- Reuse great outreach connections and build on-going relationships.
- Offer customer testimonials for your software, product, and service providers.
- See if you can get influencers to add you to their partner pages.
- Get your users involved! Build relationships and interaction with your brand by rewarding loyal fans.
- Have your friends and family link to you if they have personal websites.
Content Production
- Take photos and make them available under a Creative Commons license.
- Write topical content about a trend from that particular day.
- Interview experts within your niche.
- Craft an exclusive content pitch for a leading authority website.
- Instead of creating four average pieces of content, focus on one at a time and make it great.
- Develop a content ideation plan for an inforgraphic using data from within your company.
- Answer frequently asked questions within your industry. This can also be used to brainstorm content ideas.
- Set up Google Alerts for these questions, so when they are asked on blogs or forums you can reply with your opinion, and also link to your content.
- Create and syndicate video content to target new audiences.
- Provide a discount promotion to get people talking and to get them picked up on promo-code websites.
- Create a fun and interesting 404 error page, much like this one. Your users will love it and you might even get some links due to creativity.
Comment Marketing
- Make efforts to reply to comments on all of your content. This includes posts on your blog, guest posts, news coverage, etc. This is a great way to build lasting relationships.
- Find an active forum within your niche to start contributing and building profiles.
- Find two targeted blogs. Comment where you can add value and subscribe or follow them and their key writers on Twitter.
- Take photos of your office or store and upload a minimum of 6 high-quality photos to help your local listing stand out.
- Make sure that all of your company addresses are registered and up to date on Google+ Local.
- Design business cards that encourage customers review your brand on Google, TripAdvisor, Yelp!, etc. Also offer them a next-time discount incentive.
- Create landing pages with local intent. If you have multiple addresses, submit them to Google Local.
- Go to your local library and dig deep into historical information about your town or city. You may find a diamond in the rough that hasn't been written about online yet. This is great for picking up local citations and links.
- Create a list of power reviewers in your local sector and see how you can get onto their radar.
- Ensure that you are listed on key local providers such as Yell, ThomsonLocal, ReachLocal, etc.
Blogger Outreach
- Find two of the most authoritative and well-respected bloggers in your niche and see how you can connect with them. They have probably added how to do this in their "About" page.
- Pick up the phone and talk to top influencers.
- Hire writers within your niche and take advantage of their contacts for outreach.
- Spend your hour carefully creating a terrific content pitch. Make it personal and original.
- Give bloggers and journalists a product they can use or test in exchange for a review.
- Write for authority sites within your niche. Build strong relationships and strengthen your reputation by taking advantage of the audiences of well-respected industry blogs.
- Have your clients create a company email address for you with their domain so that it is clearer when you're sending emails out on their behalf.
Penguin and Panda Penalty Review
- Perform a content performance ratio analysis to find out how much content you need to clean up.
- Create a list of sites you want to remove links from.
- Clean up your own internal anchor text from over-optimization.
- Reduce cross-linking from other sites you own. Link from partner pages, not site wide.
- Clean up any links from your special profiles and author bios.
On-Site Optimization
- Prioritize a list of key actions and assign responsibilities and deadlines.
- Install SEO WordPress plugins to optimize your blog.
- Optimize page title tags. This is the oldest quick SEO fix in the book, but it still works.
- Test pay-per-click (PPC) and copy as title/meta description to increase organic click-through rates.
- Review navigation structure such as breadcrumbs and internal linking.
- Use tools. There is a ton of them, so make sure you figure out which two or three that will make you more efficient. Then really get to know them and learn how to use them.
- Document your delivery process. This will save you tons of time in the future when you are training your team.
- Conduct a knowledge share session with your internal team and/or client. Let them know what you do so you don't have to do it all by yourself.
Link Removal
- Analyze your backlinks to find off-topic or poor anchor text links.
- Analyze the market to find the percentage of exact-match anchor text for key competitors.
- Clean up any obvious paid or over-optimized links.
Link Reclamation
- Google your brand name and a key person within your organization, much like this "Brand Name" "Key Person's Name". Then ask to be credited with a link within each article if not already given.
- Google Image search for your infographics and/or photos. Ask for link credits where they are not given.
- Find broken links pointing out of site with this tool. This is another easy fix to clean up and also makes your site look much better.
Competitor Analysis
- Research competitors' top pages to get content ideas.
- Review brand traffic history in analytics to measure the impact of offline brand signals.
- Think of creative ways you can get more people searching for your brand by joining up with offline marketing.
Public Relations
- Bring your PR and social teams together to understand what they are doing and how they can help each other by working closer together.
- Sign up for PR service HARO (help a reporter out) to become a link source for news articles.
- Build a list of key journalists and media contacts you want to influence.
- Enter relevant industry and local awards competitions.
- Brainstorm ideas to connect offline with online strategies. Ask yourself, "How can I get more people searching for me and my company?"
- Create and submit an XML sitemap to keep this up to date in Google Webmaster Tools.
- Check up on how your website displays on mobile and tablet devices. Plan to create platform-specific sites if you don't already have them.
- Fix duplicate content homepage: www vs. non-www.
- Check for external duplicate content.
- Make sure the geo-targeting for your international site (if you have one) is set up correctly in Google Webmaster Tools.
- Optimize your site for mobile. Do it in less than an hour with a plugin
Analytics and Measurement
- Develop a strategy around your top 20 PPC spend/converting keywords.
- Find your top converting landing pages. Optimize them and update the content to attract new links.
- Talk to your clients and learn about their main business.
- Talk to your clients and learn about their main business.
- Spend your hour writing a report making a business case for the value of SEO to get extra resources allocated, because, let's be honest, one hour is just not enough.
(Information pulled from seomoz.org)
Certified Inbound Marketing Strategist | Content Marketing Specialist | Social Media Strategist | SEO


text:
A bill to be introduced in the state legislature next year would cut in half the time it takes lenders to sell abandoned properties.
Gov. Bill Ritter and state legislators announced the bill Tuesday at a news conference at the Clements Community Center in Lakewood. The bill, to be co-sponsored by Reps. Jeanne Labuda, D-Denver, and Dianne Primavera, D-Broomfield, and Sen. Mike John ston, D-Denver, will allow homes to be occupied more quickly so they don't become a safety hazard, a magnet for vandalism and other crimes, or a drain on nearby property values.
"Abandoned properties turn a family-friendly neighborhood into a hazard for children," Ritter said.
Current law calls for a minimum four-month sale process, but many foreclosure sales take seven to nine months to complete.
Currently, when a lender submits the paperwork to start the foreclosure process, the title for the property is still with the mortgage holder, even if the home is abandoned. That means there is no one responsible for maintaining the property, because the homeowner is gone and the home does not yet belong to the lender.
The bill would allow the bank to take over a property faster, giving municipalities and homeowners associations someone to hold accountable for maintenance.
"Before, we didn't discriminate between abandoned and non-abandoned," Ritter said. "When someone moves out and abandons their property, there's no reason not to move (the time frame) up and move it up quickly."
Colorado is on track to top the record of 39,900 foreclosure filings set in 2007 as widespread unemployment makes it harder for borrowers to make their mortgage payments. New foreclosure filings statewide during the third quarter reached a record high of 12,468, according to a report released last month by the Colorado Division of Housing. New filings for the first nine months of the year were up 18 percent to 35,112.
The number of completed foreclosures grew to 5,618 in the third quarter, the second consecutive quarter- over-quarter increase. But the total number of completed foreclosures fell to 14,971 during the first three quarters, compared with 16,265 during the same period last year.
The legislation would complement the $5.8 billion federal Neighborhood Stabilization Program designed to help foreclosure-blighted neighborhoods.
"More folks are fighting hard to maintain their mortgages," Johnston said. "The next critical step is to protect neighborhoods."
Margaret Jackson: 303-954-1473 or email@example.com


text:
Tuesday, December 4, 2012
When things happen that shouldn't happen...
It is greyish with a glimpse of blue behind those ugly clouds... The beautiful snow that came Sunday is almost all gone.
I am sweeping the leaves from my Wisteria at the front of my house. It is almost bare naked now. In winter the plowing truck throw all the snow in the fountain to melt, and if it is full of leaves it will clog up the drain. Madame Debonneville told me that when we moved in 5 years ago.
I am slowly walking my way to the back of the house to empty my bucket in the compost. We built a compost here behind the shed the same year we moved in. Madame Debonneville suggested we do so, as gardening produces lots of compost and it would save us numerous trips to the "Decheterie". She was right of course.
For a good 60 years she has been growing her own vegetables here, Madame Debonneville. She has taught us so much, helped us when plants been attacked by slugs or pests, showing us how to plant seeds the best, when and where. How to rake the beds, harvest fruits from their plants. Without Madame Debonneville we wouldn't have this beautiful garden and be able to grow our own vegetables as good as we do.
The house is old, built sometime in the 1750's. We are the first family to live here that is not a Debonneville family. We feel very special and lucky to have been chosen to take care of this beautiful place.
Madame Debonneville loves us and we love her. She is a part of our family, an old gracious lady with 86 years behind her. She scrubs the fountain, turns the soil, picks the walnuts from the trees and the apples off the ground. She chats with us and the kids, takes care of Charlie, the cat, when we are on vacation.
Madame Debonneville got hit by a car Sunday when on her daily walk around the village. She is in hospital and we don't really know what is going to happen. Some stupid idiot (sorry for my vocabulary) decided to drive his car with summer tires on in the first big snow storm of the year, lost control of his vehicle and BOOM! She flew a good 10 meters like a projectile her family says. She is bruised but have no fractures although she has hurt her cord and her head. I am devastated. I am sad. I can't really see the blue glimpses in the sky today. For me it is all grey... I am not religious but I will pray my way. I will pray for our dear and loved Madame Debonneville to soon get better and laugh with us again. Things like this should not happen...
26 comments :
So sorry to hear this I will pray for her and for you!!ReplyDelete
Oh no. Hope she recovers soon.ReplyDelete
- AnonymousDecember 4, 2012 at 5:48 PM
What a shocking thing to happen. I can see that you care a lot about this lady. I know what it is to befriend an elder lady. I had such a friend and unfortunately lost her this year, she was very special. Now I and others take care of the friend that she lived with and make sure we visit her and take her dog out for walks, she is a lovely lady of 86. The elderly are precious and have much to share with us if we give them that time. It sounds like you have a very special and respectful friendship with each other and I hope very much that Mrs Debonneville will recover very soon. I am sure with your love, support and yes prayers that she will. I am thinking of you and especially Mrs Deboneville, and hoping that she will be fit and well very soon.ReplyDelete
Take care.
Dear AnonymousReplyDelete
Thank you for your encouraging words and support. it is highly appreciated and warms my heart.
Que triste isso! Que tudo fique bem! beijos,chicaReplyDelete
Poor Madame Debonneville. I hope that she will heal and be able to enjoy life again. One wonders why the sweetest people have to have bad things happen to them. Sending postive thoughts her way.ReplyDelete
oh I hope Madame Debonneville recovers. We all need a Madame Debonneville and it sounds like she needs you. I hope for the best, Heather xReplyDelete
Oh, it is sooo sad situation :-( I hope, your old friend will be soon in good condition again! JolanaReplyDelete
Oh how horrific, she sounds so lovely and full of Grace. My thoughts and prayers are with you and this lovely woman. Praying is good, you don't have to be a woman of Faith.ReplyDelete
Hugs to you,
hi annette.!I can imagine how sad you are,iam not a religious person either,but i belive in pray .from bottom of my heart I pray for this nice lady ,hope she be fine and comes home from hospital&this christmas you all be together ,xo zohrehReplyDelete
I am so sorry (and upset), I hope she will get better!ReplyDelete
Love, Anu
What a special friend this lovely old lady is for all your family Annette. No wonder you are devastated.ReplyDelete
I really hope she makes a full recovery.
Thinking of you
Jacquie x
I am so sorry to hear about your friend Annette! Please know she is in my thoughts & prayers!ReplyDelete
I work in an elder care facility and we have a dear lady who turned 105 yrs old this past Saturday!
When you see your friend again, give her a huggle from all of us!
Prayers for your dear, sweet friend. xoxo DebbieReplyDelete
So sorry to hear about this accident....I hope that Mme Debonneville recovers soon,ReplyDelete
xxx Alessandra
So sorry to hear about this sweet lady! Will keep her in my prayers!! xo HeatherReplyDelete
What a terrible accident..and worse because it was preventable. I hope the old lady recovers well. It must have been an awful shock to all her family as well.ReplyDelete
- AnonymousDecember 5, 2012 at 11:40 AM
Dear Annette!ReplyDelete
Oh, how sad this is! It makes me sad and also angy, because it was an preventable accident! I wish she will get back home and live here happy live again soon! My thoughts are with her and with you!
Oh my how horrible! I do hope Mme Debonneville is going to fully recover.ReplyDelete
It's been snowing here for the last few days. Lots of unprepared people, despite a very precise weather forcast, skidding everywhere and driving as fast as they would in the summer time!
Oh no poor Madame Debonneville, sending her best wishes.ReplyDelete
She sounds like the loveliest lady, and what a great name.
Love your gorgeous cat pic..so beautiful.
oh forgot to add, I chopped down our wisteria, because I was so fed up with the leaves in my kitchen!!ReplyDelete
What an awful thing to happen. Poor lady - I will also pray in my own way for her recovery. It is so special to be able to have close relationships with older people. They have so much to offer and yet are often dismissed by society (well certainly ours in the UK). I am sure your love for her will help her at this time. xxxReplyDelete
I'm so sorry to hear about your sweet old lady. I'm thinking of my lovely grandmother (89)... Madame Debonneville is a very strong lady but she needs your love and your presence to be better. I hope she will recover soon.ReplyDelete
Sincerely thinking of your friend and you.
What a terrible thing to happen and how awful you must all feel about it. I do hope that she makes a good recovery. Sending best wishes to you both.ReplyDelete
S x
Oh no!! I am praying for her. I am so sorry!! Trish xoReplyDelete
That's so sad. I hope she's okay.ReplyDelete
Thank you so much for visiting my world. I love reading your comments and I do my utterly best to respond to questions and sweet messages. Thank you again for popping by.


text:
With My Fitness Coach: Dance Workout, enjoy complete and fun fitness workouts combining Fitness, Tonic Latin Dance, Boxing and Aerobic. Create your own workout program and challenge yourself or your friends with the balance board or the Wii remote to get the higher score.
Game Features
- Complete Cardio program including Latin dance Fitness and Cardio Boxing:
- 14 different choreographed dance routines using popular Latin dance styles such as Salsa, Samba, Rumba, Flamenco, Reggaeton and Meringue
- 9 different choreographed dance routines featuring boxing moves
- 8 Fun mini games targeting specific muscles: Rodeo, Karate, Sword Fighting, Marathon/Jogging, Jump Rope, Canoe, Boxing, Bull Dodging
- A Personal Coach gives you personalised advice on your performance:
- Your coach motivates you and increases the intensity of your workout based on your performance. Your Coach follows your fitness calendar, calories burned and BMI.
- A multiplayer mode to enjoy your workouts with a friend!
- Attractive in-game tracklist:
- Just Dance by Lady Gaga
- I Will Survive by Gloria Gaynor
- Cosmic Girl by Jamiroquai
When will my order be dispatched?
This item will usually be dispatched within 24 hours (Monday – Friday)*. Orders placed outside of these days will be dispatched on the next working day.
* Excluding UK public holidays
How will my order be sent?
Our standard delivery option is FREE in the UK via Royal Mail First Class, with large or high value orders sent via Parcelforce courier service. Delivery to Europe (from an additional £2 per item) and other Worldwide countries (from an additional £4 per item) are sent via Royal Mail International who work with trusted delivery partners to deliver to the destination country.
We also offer a guaranteed Special Next Day service* on this item for UK delivery at an additional cost from £5.95, which is guaranteed to be delivered before 1pm the following day or we'll refund the postage cost!
* Available until 3:00pm (Monday – Friday) excluding UK public holidays.
When can I expect it to arrive?
- UKDeliveries to destinations in the UK usually arrive within 1-2 working days after the order is dispatched with the vast majority arriving on the next working day.
- Europe
Deliveries to destinations in Europe take on average 3-5 working days, but times will vary depending on the import regulations and postal service in your country. Please check with your local postal service for an estimate on how long it takes to ship items from the UK to your country.
- Worldwide
Deliveries to destinations outside of Europe take on average 5-7 working days, but times will vary depending on the import regulations and postal service in your country. Please check with your local postal service for an estimate on how long it takes to ship items from the UK to your country.
If your order has not arrived by the expected delivery date this is usually no reason for concern - see our delivery help pages.
|Nintendo Wii
|Release Date
|27th Aug 2010
|Age Rating
|Age 7+
|Fitness and Health
|Sale Offers
|3 for £20


text: BBC Pushes and Pulls The BBC website has an exploration for pushes and pulls too. Students can experiment with changing the force that is applied to the horse toy and observe the results. They can also place cones or brick walls in front of the toy too. I also made an observation sheet for this one too. (click here to download) Advertisements Share this:Click to share on Twitter (Opens in new window)Click to share on Facebook (Opens in new window) Related


text:
- Store
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- Pot Book: A Complete Guide to Cannabis edited by Julie Holland, M.D.
Pot Book: A Complete Guide to Cannabis edited by Julie Holland, M.D.
Encompassing the broad spectrum of marijuana knowledge from stoner customs to scientific research, this book investigates the top ten myths of marijuana; its physiological and psychological effects; its risks; why joints are better than water pipes and other harm-reduction tips for users; how humanity and cannabis have co-evolved for millennia; the brain's cannabis-based neurochemistry; the complex politics of cannabis law; its potential medicinal uses for cancer, AIDS, Alzheimer's, multiple sclerosis, and other illnesses; its role in creativity, business, and spirituality; and the complicated world of pot and parenting. As legalization becomes a reality, this book candidly offers necessary facts and authoritative opinions in a society full of marijuana myths, misconceptions, and stereotypes.
579 pages, 2010, paper


text:
- Research article
- Open Access
Glutamate, aspartate and nucleotide transporters in the SLC17 family form four main phylogenetic clusters: evolution and tissue expression
© Sreedharan et al; licensee BioMed Central Ltd. 2010
- Received: 6 March 2009
- Accepted: 8 January 2010
- Published: 8 January 2010
The SLC17 family of transporters transports the amino acids: glutamate and aspartate, and, as shown recently, also nucleotides. Vesicular glutamate transporters are found in distinct species, such as C. elegans, but the evolutionary origin of most of the genes in this family has been obscure.
Our phylogenetic analysis shows that the SLC17 family consists of four main phylogenetic clades which were all present before the divergence of the insect lineage. One of these clades has not been previously described and it is not found in vertebrates. The clade containing Slc17a9 had the most restricted evolutionary history with only one member in most species. We detected expression of Slc17a1-17a4 only in the peripheral tissues but not in the CNS, while Slc17a5- Slc17a9 are highly expressed in both the CNS and periphery.
The in situ hybridization studies on vesicular nucleotide transporter revealed high expression throughout the cerebral cortex, certain areas in the hippocampus and in specific nuclei of the hypothalamus and thalamus. Some of the regions with high expression, such as the medial habenula and the dentate gyrus of the hippocampus, are important sites for purinergic neurotransmission. Noteworthy, other areas relying on purine-mediated signaling, such as the molecular layer of the dentate gyrus and the periaqueductal gray, lack or have a very low expression of Slc17a9, suggesting that there could be another nucleotide transporter in these regions.
- Dentate Gyrus
- Bony Fish
- SLC17 Family
- Vesicular Glutamate Transporter
- Slc17a9 mRNA
Membrane proteins constitute about one third of all proteins encoded in the human genome [1]. The largest family of membrane-bound proteins consists of over 800 G protein-coupled receptors [2, 3] while the second largest is the solute carrier (SLC) family including 384 human genes [4]. The SLC genes encode proteins related to passive transporters, ion-coupled transporters and exchangers. SLCs were functionally grouped into forty-three subfamilies [5]. Since then additional families have been added including five new subfamilies, SLC44 - SLC48, according to the Hugo Gene Nomenclature Committee [4].
A systematic phylogenetic analysis of the entire repertoire of SLC genes reveals that 15 of the SLC subfamilies, along with synaptic vesicle 2 (SV2) proteins, can be clustered into 5 major groups, named α-, β-, γ- and δ-groups [4]. The α-group is largest with SV2 proteins and seven SLC subfamilies (SLC2, 16, 17, 18, 22, 37 and 46). The main common features of the members of the α-group are the presence of 12 putative transmembrane (TM) regions, N- and C-termini at the cytosolic side, a large extracellular loop between TM1 and TM2 (except SLC17) and a large third intracellular loop.
The SLC17 family belonging to the α-group is known as the type I phosphate/vesicular glutamate transporter family [6]. The SLC17 family consists of nine genes that have previously been functionally divided into four subgroups: (i) type I phosphate transporters, SLC17A1-4, (ii) vesicular excitatory amino acid transporter, SLC17A5 (previously known as sialin) (iii) vesicular glutamate transporters (VGLUT), SLC17A6-17A8, and (iv) vesicular nucleotide transporter (VNUT), SLC17A9 [7, 8].
The type I phosphate transporters are known to cotransport sodium (Na) and phosphate (Pi), with a capacity to also transport organic anions. Their ionic coupling properties have not been determined and the identity of their endogenous substrates remains unresolved. Moreover, data on the tissue distribution of the type I transporters seem rather limited. The SLC17A1 expression has been studied by northern blot and it has been identified in the kidney, liver and, at very low levels, in the brain [9, 10]. The SLC17A2 has a different expression pattern, with relatively high levels in the heart and skeletal muscle and lower levels in the kidney, liver, lung, placenta, pancreas and brain [11, 12]. SLC17A3 is limited to the liver and kidney [13], whereas SLC17A4 is expressed in the intestine, colon, liver, and pancreas [14].
The second group of SLC17 proteins consists of the vesicular excitatory amino acid transporter (VEAT/SLC17A5), previously known as sialin. It was first identified as a lysosomal sialic acid transporter implicated in the Salla disease and infantile sialic acid storage disorder [15]. However, a recent study showed that SLC17A5 serves as a vesicular protein transporting aspartate and glutamate and, hence, the name vesicular excitatory amino acid transporter (VAT) was suggested [7]. Slc17a5 shows ubiquitous expression [15]; in the brain it is predominantly expressed in the hippocampus, striatum and cerebral cortex [16, 17].
The third group contains vesicular glutamate transporters involved in loading glutamate into synaptic vesicles of glutamatergic cells. In the SLC nomenclature the three identified VGLUT transporters, VGLUT1 through 3 are classified as Sl17A7, A6 and A8, respectively. Slc17a7 and Slc17a6 are predominantly expressed in glutamatergic neurons of the central nervous system (CNS). Interestingly, these genes have a complementary expression pattern with limited overlaps [18, 19]. Unlike Slc17a7 and Slc17a6, Slc17A8 is expressed in CNS neurons not classically considered as glutamatergic [20, 21]. The peripheral expression of Slc17a6 and Slc17a7 is limited to the pancreatic islets whereas Slc17a8 is expressed in the liver and, at lower levels, in the kidney.
The fourth type of the SLC17 genes is represented by a single gene in humans and it is named SLC17A9. Recently it has been identified as the first vesicular nucleotide transporter. The vesicular storage of this protein is confirmed by studies on PC12 cells. Functional characterization of SLC17A9 in liposomes containing the purified protein revealed that it transports nucleotides, such as ATP and ADP [8]. This is likely a major breakthrough in understanding neuronal signaling because ATP co-transmission is crucial in many neurons. The purinergic receptors that respond to ATP, UTP and adenosine serve as important drug targets and this transporter is likely to draw large attention for pharmaceutical development. Northern blot analysis showed wide expression of SLC17A9 in various organs, but predominantly in the brain, adrenals and the thyroid gland [8]. However, the detailed expression profile of SLC17A9 in the brain is unknown.
Vesicular glutamate transporters are fundamental components in glutamate signaling and are evolutionarily old. A vesicular glutamate transporter has been characterized in the tunicate Ciona intestinalis [22] as well as in Drosophila melanogaster [23] and Caenorhabditis elegans [24]. Also the presence of SLC17A9 in many animal species has been suggested [8]. However, despite the obvious biological importance of the SLC17 family, the evolutionary history of these genes has not been systematically mapped.
In the current project, we thoroughly mined the entire SLC17 family and explained the evolutionary events that shaped different branches of this family. We also performed comprehensive expression analysis of the nine SLC17 genes using quantitative real-time PCR (RT-PCR) in the rat. We then looked in more detail at newly discovered Slc17a9, the only known vesicular nucleotide transporter, and performed a comprehensive expression profiling of the Slc17a9 in the mouse brain by in situ hybridization.
Animal handling and tissue isolation for quantitative real-time PCR
Adult male Sprague-Dawley rats were housed in the controlled environment with optimum ventilation, temperature of 21°C, 12:12 LD cycle, and free access to standard chow and water. After the acclimation period of 7 days, the rats were sacrificed by decapitation. The peripheral tissues and brain regions of interest were isolated. These tissues were then immersed in the RNA-later solution (Ambion, USA) for 1 h at room temperature to remove RNases, and then stored at -80°C until further processed. All animal procedures were approved by the Uppsala Ethics Committee and followed the guidelines of European Communities Council Directive (86/609/EEC).
RNA isolation and cDNA synthesis
RNA isolation and cDNA synthesis were performed as described previously [25]. In brief, the tissue was homogenized by sonication and the RNA was extracted using the TRIzol/chloroform method as previously reported [26]. Genomic DNA contamination was removed by adding DNaseI (Fermentas, Sweden). The cDNA was synthesized by priming with random hexameres and MMLV reverse transcriptase (GE Healthcare, Sweden)
The expression profiles of all the nine rat genes (rSlc17a1- rSlc17a9) were tested in a rat tissue panel containing cDNA of 16 different brain regions and various peripheral tissues. All the primers were designed using Beacon Primer Design 4.0 software (Premier Biosoft, USA) and all the sequences of primers used are shown in Additional File 1. The RT-PCR reaction mixture contained cDNA synthesized from 25 ng of total RNA, 0.25 pmol/μl of each primer, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 4 mM MgCl2, 0.2 mM dNTP, SYBR Green (1:50,000) (Invitrogen, USA) and 0.02 U/μl Taq DNA polymerase (Biotools, Spain). The reaction conditions were as follows: initial denaturation at 95°C for 4 min, followed by 50 cycles at 95°C for 15 s, 55-62°C for 30 s (optimal annealing temperature) and 72°C for 30 s. This was followed by a melt curve between 55°C and 97°C (10 seconds per step and 0.5°C increase per step) to identify non-specific amplification. All RT-PCR experiments were performed in duplicates. A negative control for each primer pair and a positive control with 25 ng of rat and mouse genomic DNA, respectively, was included on each plate. MyiQ thermal cycler (Bio-Rad Laboratories, Sweden) was used to perform RT-PCR.
Data analysis and expression calculation
RT-PCR data were processed with the MyiQ software v1.04 (Bio-Rad Laboratories, Sweden). The melting curves were analyzed to confirm that only one product with the expected melting point had been amplified. The threshold cycle (Ct) values for the transcripts of interest were obtained and all samples with a signal, required to be at least 2 Ct values from the Ct value in the negative control (if any), were considered as expressed. LinRegPCR was used to calculate PCR efficiencies for each sample and Grubbs' test (GraphPad, USA) was applied to exclude the outliers and to calculate the average PCR efficiency for each primer pair. The GeNorm [27] protocol was used to identify the most stable housekeeping genes for each tissue and normalized quantities were calculated.
cDNA Probe Synthesis
Antisense probe for mouse Slc17a9 was generated from EST clone 4986674 (Invitrogen). Plasmid preparation was performed using JETSTAR Plasmid Midi Kit (Genomed, USA). The plasmid were linearized with EcoRV (Fermentas, Sweden) and used as template for antisense digoxigenin (DIG)-labeled probe synthesis and T7 polymerase was used for probe synthesis. The sense probe was generated from the same EST clone and the plasmid was linearised with BstXI. Sp6 polymerase was used for digoxigenin (DIG)-labeled probe synthesis. The probe synthesis was carried out as described earlier [28].
Tissue preparation for in situ hybridization
Adult male C57Bl6/J mice were housed in similar conditions as described above for the rats. The mice were anesthetized by an intraperitoneal injection of 1:1 mixture of Domitor (Medetomidine hydrochloride, 70 μg/g body weight, Orion, Finland) and Ketalar (Ketamine hydrochloride, 7 μg/g body weight, Pfizer, Sweden). Transcardial perfusion through the left ventricle with phosphate buffered saline (PBS) followed by freshly prepared 4% paraformaldehyde (PFA) was performed to obtain fixed brain tissue. The brain was excised and incubated in 4% formaldehyde overnight, thereafter the tissue was dehydrated and infiltrated with paraffin (Tissue Tek vacuum infiltration processor; Miles Scientific, Elkhart, IN). Paraffin-embedded brain was sectioned (7-μm thick) on the Microm microtome onto Superfrost slides (Menzel-Gläser, Braunschweig, Germany) and stored at 4°C until usage.
In situ hybridization
The paraffin sections were deparaffinized in X-tra solve (Medite Histotechnic, Burgdorf, Germany) and rehydrated with the series of ethanol washes (100%, 90%, 70%, 50%, 25%); followed by washing in PBS. The rehydrated sections were fixed with 4% PFA for 10 min, washed with PBS and treated with proteinase K (Invitrogen, Germany); 27 μg/ml diluted in 10 mM Tris-HCl/1 mM EDTA, pH 8.0) for 15 min. The sections were refixed with 4% PFA for 5 min and acetylated for 10 min in 1.3% triethanolamine (Sigma-Aldrich, USA) 0.2% acetic anhydride (Fluka, Neu-Ulm, Germany), and 0.06% HCl diluted in RNase free water. The sections were then permeabilized with 1% Triton-X 100 (Sigma-Aldrich, USA) for 30 min and washed with PBS. The slides were subsequently incubated in the hybridization buffer [50% formamide (Fluka, Germany), 5× SSC, 5× Denhardt's, 250 g/ml yeast transfer RNA (Sigma-Aldrich, USA), 500 g/ml sheared salmon sperm DNA (Ambion, Austin, TX) diluted in RNase free water] without the probe for 1-2 h. The probe (0.5 μg/200 μl) was denatured in the hybridization buffer at 80°C for 5 min and then cooled in ice. The prehybridized sections were hybridized with the cooled denatured probe in the hybridization buffer overnight at 65°C. Next day the slides were transferred to a 0.2× SSC and incubated at 65°C for 1-3 hrs. The slides were then washed first with 0.2× SSC at room temperature and then in the B1 solution (0.1 M Tris-HCl, pH 7.5, and 0.15 M NaCl). The sections were immunoblocked with 10% fetal calf serum in B1 and incubated overnight at 4°C in peroxidase conjugated anti-digoxigenin Fab fragments (Roche, Mannheim, Germany) diluted 1:500 in B1 containing 10% fetal calf serum. The following day the slides were washed with PBS and then incubated in 300 μl of the amplification buffer (Molecular probes™ Tyramide Signal Amplification Kit, Invitrogen, USA) containing 3 μl of the labeled tyramide solution (Molecular probes™ Tyramide Signal Amplification Kit, Invitrogen, USA ) for 10-15 min. The sections were washed with PBS and stained with DAPI. They were mounted using DTG (2.5% DABCO (Sigma), 50 mM Tris-HCl pH 8.0, 90% glycerol) and analyzed using the fluorescent microscope (Zeiss XBO75). The Slc17a9 probe covers the 1178 bp of the entire cDNA.
Data mining
The sequences for the 9 human SLC17 family proteins were downloaded from the NCBI http://www.ncbi.nlm.nih.gov/ using the Entrez data retrieval tool with the accession numbers as follows: SLC17A1, NP_005065.2; SLC17A2, NP_005826.1; SLC17A3, NP_001091956.1; SLC17A4, NP_005486.1; SLC17A5, NP_036566.1; SLC17A6, NP_065079.1; SLC17A7, NP_064705.1; SLC17A8, NP_647480.1; SLC17A9, NP_071365.3. The sequences from each family were aligned using Tcoffee 5.72 [29]. From the alignments, sequence Hidden Markov Models (HMMs) were constructed using the HMMER 2.2 package [30]. The models were constructed using HMMbuild with default settings and calibrated using HMMcalibrate. The models were searched against the following protein datasets: Mus musculus (NCBI build 37.50, pep all); Gallus gallus (Hashington University build 2.50, pep all); Tetraodon nigviridis (build 8.5, pep all); Danio rerio (build 7.50, pep all); Takifugu rubripes (build 4.50, pep all); Ciona savigny (build 2.0.50, pep all); Branchiostoma floridae; Drosophila melanogaster (4.50, pep all); Caenorhabditis elegans (build 190.50, pep all); Schizosaccharomyces pombe (pompep); Saccharomyces cervisiae (build 1.01.50, pep all). All hits with an e-value better than 0.1 were extracted and non-SLC17 proteins were removed using phylogenetic analysis. The analysis included all human SLC16, SLC17, SLC18 and SLC22 proteins in order to represent the SLC families most similar to SLC17 [4].
Phylogenetic analysis
Phylogenetic trees were calculated for the SLC17 family using the following procedure. Amino acid sequences in Fasta format were aligned using T-Coffee 5.72. The alignment was bootstrapped 1000 times using SEQBOOT from the Win32 version of the Phylip 3.6 package [31]. Maximum-parsimony trees were calculated on the bootstrapped alignment with PROTPARS from the Win32 version of the Phylip 3.6 package. The trees were un-rooted and calculated using ordinary parsimony, and the topologies were obtained using the built-in tree search procedure. Majority-rule consensus trees were constructed using CONSENSE from the Win32 version of the Phylip 3.5 package. Neighbor joining trees were calculated on the same bootstrapped sequences using the Phylip 3.6 programs PROTDIST followed by NEIGHBOR. For the maximum likelihood tree, the topology obtained from the maximum parsimony tree was used as a user-defined tree in TreePuzzle [32] and maximum likelihood branch lengths were estimated in TreePuzzle using the following parameters: Type of analysis: Tree reconstruction; Tree search procedure: User defined trees; Compute clocklike branch lengths: No; Location of root: Best Place (automatic search); Parameter estimates: Exact (slow); Parameter estimation uses: 1st input tree; Type of sequence input data: Amino acids; Model of substitution: JTT. Amino acid frequencies: Estimate from data set; Model of rate heterogeneity: Mixed (1 invariable + 8 Gamma rates); Fraction of invariable sites: Estimate from data set; Gamma distribution parameter alpha: Estimate from data set; Number of Gamma rate categories: 8. The trees were plotted using TreeView [33].
RT-PCR measurements
Expression analysis of all nine members of the SLC17 family was performed on a panel of rat tissues. The rat panel consisted of cDNA synthesized from the RNA isolated from seven coronal sections of the rat brain [25], different regions of the brain, various peripheral tissues in total of 35 samples. The normalization factor was calculated based on the expression of four housekeeping genes and the relative expression values are the fold increase from the minimum detected expression in the rat panel. The validation of the method and use of normalization factors are explained elsewhere [36].
The Slc17a1 and Slc17a2 were expressed in the skeletal muscle, kidney and liver. Slc17a3 was expressed in the skeletal muscle, kidney and uterus, whereas Slc17a4 was expressed in the kidney, liver, stomach and intestine. Slc17a5 had ubiquitous expression in all tissues tested. Slc17a6 and Slc17a7 showed expression at different yet detectable levels in all brain regions tested and in the spinal cord. However, in the periphery Slc17a6 was expressed in the eye, skeletal muscle, liver, spleen and thymus, while SLC17A7 was detected in the eye and heart. Slc17a8 was predominantly expressed in the choroids plexus and - at low levels - in the cortex, hypothalamus, hippocampus, brainstem, spinal cord, stomach and eye. Slc17a9 expression appeared ubiquitous with the highest level in the stomach, intestine, liver, skeletal muscle, spleen and blood; relatively lower levels were detected in all brain regions studied, spinal cord, adrenal gland, eye, skin and skeletal muscle.
In situ hybridization analysis for Slc17a9
We have performed detailed mining and phylogenetic analysis of all SLC17 family proteins from eight species. The analysis shows that this family consists of four main phylogenetic clades, which were present before the divergence of the insect lineage from those leading to mammals. We denote those clades I to IV. Three of these main clades include all the human members while one clade contains only sequences from C. elegans. No member of this entire clade has previously been described in the literature. We propose the order of events for the evolutionary history for the SLC17 family from the nematodes to human and this is schematically presented in Figure 1. We did not find any SLC17 family genes in any species more basal than the nematode, although we found sequences for predicted proteins in Nematostella vectensis that have similarity to the SLC17 family as well as to other SLC α-family members. Our current data suggest that the four main ancestral groups, denoted in four main colors in Figure 2, originated from an MFS type transporter [4] before the divergence of nematodes.
Clade I contains only the newly discovered vesicular nucleotide transporter, VNut [8]. The evolutionary history of this gene is unique within the SLC17 family as it is the only gene that has one member in almost all the species. For multiple sequence alignment of the VNuts discussed in this paper, see Additional File 3 and Additional file 4. An exception is however among bony fish, where T. nigroviridis and D. rerio have retained an extra copy, most likely as a result of the extra genome duplication. The new clade II was only found in C. elegans. Our searches and phylogenetic analysis suggest that these sequences are indeed members of a distinct clade that does not exist in the vertebrate linage. Clade III contains the vesicular glutamate transporters, VGluts, which were present already in C. elegans. A common ancestor to the three mammalian VGluts appears to have existed before the divergence of gnathosomes and this common ancestor likely gave rise to the three VGluts found in current vertebrates (Figure 2). The invertebrate species and also B. floridae have more than one gene in Clade III; the phylogenetic analysis (Figure 1) suggests that this could be the outcome of local duplications. Clade IV has the most complex evolutionary history of the four SLC17 clades. The first members of this clade appear in D. melanogaster having five local copies of the original Slc17a5 sequence, which we name Slc17a5B. This gene gave rise to another Slc17a5 subtype, Slc17a5A, before the split of the B. floridae lineage. The original copy, Slc17a5B, was lost before the split of bony fish. Also, before the split of bony fish, Slc17a5 was duplicated to form the ancestor of Slc17a1 - a4 that we named Slc17a1/2/3/4, which we found in bony fish.
The long evolutionary history of the VNut suggests that nucleotides as neurotransmitters are fundamental to neuronal signaling and that this transport mechanism is likely to have been present already in the most primitive nervous systems. In vertebrates, nucleotides are known to function as co-transmitters to classical transmitters, in particular GABA [25]. Purines have also been shown to be released as neurotransmitters on their own and from their own vesicles, but at the same synapses as other transmitters, inducing excitatory postsynaptic currents. This has been most clearly demonstrated in fish [37]. Nucleotides as functional neurotransmitters act on two classes of purinergic postsynaptic receptors, the G protein-coupled P2Y [38] and the ionotropic P2X receptors [39]. Among these, the P2Y receptors have the shortest evolutionary history, as being present in fish [40], but not in B. floridae [41]. The evolutionary history of the P2X receptors is, however, much longer. P2X receptors are found in algae [42] as well as in Dictyostelium discoideum [43]. Interestingly, these receptors appear to have been lost independently in both the arthropod and nematode lineages [44]. Here we show that although neither arthropods nor metazoans appear to have purinergic receptors, at least not the type present in vertebrates, these lineages have retained a single copy VNut transporter. These data suggest that purinergic signaling was present already in early eukaryotes before the divergence of the plant lineage and lineage leading to metazoans.
We observe that rat Slc17a9 in the present study shows a differential expression profile when compared to the mouse and the human counterpart in an earlier study [8]. This discrepancy can be due to the species specific expression pattern of the Slc17a9 gene which has also been observed earlier between mouse and human [8]. The RT-PCR data infers that the expression of the adrenal gland is 34-fold higher than the tissue that showed lowest expression. Further, the difference in the method of detection of the mRNA level could account for some changes in the expression levels.
Postsynaptic currents induced by stimulation of purinergic receptors have been demonstrated functionally in the locus coeruleus, hippocampus, hypothalamus, dorsal horn, habenuclear nucleus of the thalamus and somatosensory cortex in mammals [45]. It is however unclear whether all brain regions and most neurons use ATP as a cotransmitter. Our in situ hybridization shows that SLC17A9 mRNA is widely expressed in the mouse brain, suggesting that neurons from most brain regions may have the capacity to store ATP in vesicles. This widespread distribution is in concert with involvement in multiple processes suggested for purines, including those related to feeding regulation [46], stress and anxiety responsiveness [47, 48], capacity of receptors to bind amino acids [49], and hippocampal neural network formation [50]. It also shows that regions where purinergic neurotransmission has previously been demonstrated, for example, medial habenula and the dentate gyrus of the hippocampus, have particularly high expression of Slc17a9 (see Figure 4). It is however interesting to note that some regions, such as the molecular layer of the dentate gyrus and the PAG, seem to lack or have very low expression of Slc17a9. Since purinergic neurotransmission has been reported within these areas [47, 50, 51], it is likely that SLC17A9 does not serve as the sole vesicular nucleotide transporter in these regions. The remaining orphan transporters in the SLC17 family are Slc17a2, a3, and a4 which are expressed only in the periphery (Figure 3) and hence it is unlikely that these would be the additional transporters for purinergic neurotransmission. One possibility is that one of the six orphan transporters phylogenetically closely related to the SLC16, SLC17 and SLC18 families, that we recently reported [4], could serve as an additional vesicular nucleotide transporter. Another possibility is that SLC17A9 accounts for the only vesicular nucleotide transporter and those neurons that do not express this gene do not use purinergic signaling.
The three orphan transporters Slc17a2 - 4 [6] and Slc17a1 were found only in mammals and originated from the common ancestor, Slc17a1/2/3/4, which we found in all three species of teleost fish. The duplications from this common ancestor occurred most likely after the split of the bird lineage as we did not find the common ancestor or any of the duplicates. The basal member of this clade IV; Slc17a5 was recently shown to be a vesicular transporter for glutamate and aspartate, hence it is possible that SLC17A1 to A4 could also be vesicular transporters. In fact, all transporters from the SLC17 family except SLC17A1 which transport p-aminohippuric acid across renal apical membrane[52] that have been functionally investigated are to our knowledge - a vesicular transporter, which suggests that the location to vesicles is an ancestral functional feature common for the SLC17 family. Our quantitative RT-PCR data show that Slc17a1, a2, a3 and a4 are not expressed in the brain, which was confirmed by searches in the Allen Brain Atlas database [53]. These transporters are mainly expressed in the muscle, liver and kidney, though it is still possible that these are expressed in secretory granules or other vesicles in these tissues. The SLC17A9 protein was, for example, shown to be expressed on chromaffin granules in the mouse adrenal gland [8].
We have performed extensive mining of a cluster of solute carriers, named SLC17, that transport glutamate, aspartate and nucleotides. We show the presence of four independent evolutionary clades among these genes which correspond well to the currently known substrates. Surprisingly we found a new branch that forms one of these main clades including eight novel genes in C. elegans. The overall tissue distribution in central and peripheral panels provides for the first time a comprehensive overview of the expression patterns for this family. The detailed in situ hybridization of SLC17A9 shows high expression throughout the cerebral cortex and distinct patterns in, for example, the medial habenula and the dentate gyrus of the hippocampus where purinergic neurotransmission is important. Other regions implicated in purine signaling, such as the molecular layer of the dentate gyrus and the PAG, seem to lack or have very low expression of Slc17a9 suggesting that there could be another nucleotide transporter in these regions.
The studies were supported by the Swedish Research Council, Svenska Läkaresällskapet, Åke Wikberg Foundation, Lars Hiertas foundation, Novo Nordisk, Magnus Bergvall Foundation, Byggmästare Engkvist foundation, Tore Nilsons foundation, Gunvor och Josef Aners foundation and Åhlens foundation. R.F. was supported by the Göran Gustafsson foundation.
Authors' Affiliations
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