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In the Olde Tradition of the Sacred Feminine, We Heal, Transform and Conjure Magic
Tarot Classes and Certification Courses beginning this September!
60 and 90 min Reiki sessions within large Crystal Portals
Tarot classes, Reiki Classes, Crystal Classes, Rituals...
Welcome to our Crystal Haven and Healing Centre! We are a spiritually based centre and mystery school located in the heart of Toronto. We specialize in crystals and minerals, books, decks and other products and services to support your individual healing, personal growth and journey on this Earth plane.
We are also the home of the Crystal Awakenings Certification Program, Astrology Fundamentals Workshop Series, Tarot Certification Courses with Jayne Marie and the Open Coven of TCOTM that honours the cycles of the Great Mother through the Wheel of the Year.
At The Rock Store, we follow the rhythms and flows of the Great Mother and journey around the wheel in communion with Her. We are devotes that surrender to Her mission and seek to merge deeper with her rhythms. Hope to see you soon!
This September many of our signature courses are starting again. These courses tend to fill early so register early to avoid disappointment.
THE ABUNDANCE CRYSTAL GRID TREATMENTS SEPT 8TH - OCT 1ST! Due to the popularity of this crystal grid, we have extended the finale date until Oct 1st. Call us to book a reiki session within the crystal grid designed to bring the harvest of abundance into your life. This is the time of the harvest. The time of the abundance of the Earth. This crystal grid works to align your energies with that of the season and charge up your life, your career, and your coiffures. Sept 28th - Oct 1st 60/90 mins avail. Reiki and Aromatherapy applied.
ASTROLOGY FUNDAMENTALS WORKSHOP SERIES starts again this Sept 29th! with Astrology Fundamentals Part 1. Astrology is such a fascinating way to explore more about yourself! Within the first class you'll learn that you are made up of a combination of all the signs and planets and they interact in a very specific and articulated way. We offer Astrology Fundamentals Part 1, 2 (Natal Chart) and Part 3 (Transits and Relationship Synastry), Private Astrology Sessions plus Continuing Studies throughout the year. CLICK FOR MORE INFOClick here for more info on..TAROT COURSES & CERTIFICATIONS
Click below on the link or on the Course Module to gain more information about the workshop or event. We require registration for all of our events due to limited space. Please review each policy before registration.
THE ABUNDANCE RITUAL SEPT 1ST 8 PM SOLD OUTINTRO TO TAROT SEPT 21ST 7 PM FALL EQUINOX GODDESS RITUAL SEPT 22ND 8 PM SOLD OUTCRYSTAL AWAKENINGS 101 SEPT 24TH 11 AM 2 spaces leftSHODEN REIKI LEVEL 1 SEPT 25TH 11 AM SOLD OUTTAROT FOR BEGINNERS 12 WEEK CERTIFICATION COURSE SEPT 28TH 7 PM 3 spaces leftASTROLOGY FUNDAMENTALS PART 1: 4 WEEK WORKSHOP SEPT 29TH 5 spaces left
THE UNDERWORLD CRYSTAL GRID OCT 15TH - NOV 1ST Booking Now!THE PROTECTION RITUAL OCT 22ND 8 PM 7 spaces leftUNDERSTANDING NUMEROLOGY & ITS MAGICAL APPLICATIONS OCT 25TH 7 PMSAMHAIN: THE WITCHES NEW YEAR GODDESS RITUAL 7:30PM & 10 PMTHE WITCHES NEW YEAR HALLOWEEN PARTY! OCT 30TH & 31ST
This November we start the Crystal Grid Chakra Series again with the Root Chakra. This series runs once monthly for two week intervals, over 8 months. This year we are offering a CHAKRA SERIES PACKAGE since so many of you commit to the full cycle already. This Package will be available up to your root chakra treatment.
ASTROLOGY CONSULTATIONS: NATAL CHART READING PROMOTIONS NOV 1 - 27TH Taking Bookings Now! Promotion: November 1 - 28th, 2016 Session Length: 60 MINS Promo Cost: $125 + HST (regular price $150 + HST) Astrologers: Emily T. and Samantha C. Please review policy on payment page.
Are you an Aries or a Libra? Most people are aware of their sun sign, which is found through the date of birth. It's common to hear people describe themselves as a Leo or a Pisces, and many horoscopes in...
We offer a broad range of services at the store to serve the Rock Store community, and Spiritual Counseling and Mentoring is just one. Here we highlight what this service is all about (and isn't). When people come to see...
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Pour essayer les milliers de modules disponibles ici, téléchargez Mozilla Firefox, une manière rapide et libre de surfer sur le Web !Fermer
Bienvenue sur les modules Firefox.
Choisissez parmi les milliers de fonctionnalités et styles supplémentaires pour personnaliser votre Firefox.Fermer
Walnut for Firefox 3.8 Redémarrage nécessaire
par alfredkayser
Walnut for a wooden look and feel. Walnut is a complete skin redesign so that all windows, widgets, panels, and many extensions have a wooden look and feel.
Install the About Home Themer extension to also theme the about:home page.
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À propos de ce module
I you like this theme, you can use the 'Donate' button on my homepage to show your appreciation!
To uninstall first make the default theme active (or the theme you want to remove) and then do 'uninstall' from the theme manager.
Please don't use the 'review' to report download errors. Download issues are not caused by the theme, but by one of the many AMO download mirrors.
Please don't use the review box for any bug reports. Contact me through the email address provided under the 'Support' heading. Using email is the quickest way to notify me, and generally I will try to make a fix available as quickly as possible.
Note, also Beta, Aurora and nightlies are supported, check the 'View all versions' at bottom of page to get the latest 'beta' release of my themes.
Commentaires du développeur
To uninstall: first select Walnut as the current theme in the Theme/Addons manager, restart firefox, then uninstall and then restart Firefox.
Please don't use the review box for any bug reports. Contact me through the email address provided under the 'Support' heading. Using email is the quickest way to notify me, and generally I will try to make a fix available as quickly as possible.
Note, as the AMO approval process takes ages, it may be that there is a newer version available as 'experimental' than the one currently displayed. Go to 'View All Versions' (at bottom of this page), and check if there is a newer (experimental) version available there.
Informations de version
Version 3.8
Added support for ForecastFox.
Enabled multiprocess support.
Lots of small fixes & improvements.
Bug 1278992 - Always show the URL bar dropmarker when in customization mode: Just show it always, and refer to normal styling
Bug 1277207 - HTML Tooltip arrow is mis-positioned when using RTL direction
Bug 1266456 - Create a HTML replacement for the autocomplete suggestion popup (part5)
Bug 1280642 - Name of 'Subscribe' and 'Share This Page' tools are greyed out if moved to panel and still in customization mode
Bug 1255989 - Cannot drag window from empty space in bookmarks toolbar items when customized to the menubar
Bug 1214801 - Hamburger menu always says "Sign into Sync" when in customization mode, even when already signed in
Bug 1249445 - There's a lot of empty space at the right of Storage Inspector in vertical mode if I select an item
Bug 1261477 - Values of long watch expressions are not visible
Bug 1281732 - Show network request JS stack traces in HTTP log message in Console
Bug 1283764 - Inspector computed view : "No CSS properties found" message is no longer displayed
Bug 1266420 - Create an HTML replacement for Sidebar toggle button
Bug 1204007 - Cross out permission icons in the control center when they have been blocked
Bug 1226898 - Autoscroll breaks the markup view if the height of the view is less than 100px
Bug 1241084 - It's hard to read text on Error pages in iframes (first letters in each row are completely clipped)
Bug 1269746 - bug 935640 removed only use of some social strings and CSS but not the strings/CSS
Bug 1267403 - Extend HTML tooltip API to be hosted in XUL panel if available
Bug 1260297 - [RTL][Eyedropper] prefix character "#" of color value moves to position start or end
Bug 1284851 - Unable to scroll if the focus from the filename is lost while the tooltip is visible
Bug 1269820 - Remove green lock with grey triangle when Mixed Active Content is Blocked
Bug 1253771 - Disabling mixed content blocking results in no indication of cert override
Bug 1286523 - Fix devtools-autocomplete-popup position in RTL locales
Bug 1113825 - console.table() column overflows when a cell contains a long string
Bug 870309 - Clean up customizableui CSS files
Bug 1226877 - [a11y] Invisible elements in devtools shouldn't be tabbable (aka accessibility is broken)
Bug 1285895 - When hovering "Cause" the mouse cursor should be "default" not "pointer"
Bug 1288238 - Improvement: Move computed devtools-toolbar just below the Box Model
Bug 1288375 - Icons missing in css Filter tooltip with Light theme
Bug 1262439 - Stop using a XUL panel for the eyedropper
Bug 1203292 - Replace permissions dropdown with 'x' icon in Permissions main view in Control Center
Bug 1273351 - Get rid of permission requirement for PointerLock API
Bug 1282189 - menu panel popup dynamic resize behavior is incorrect
Bug 1290094 - Clean up Ctrl-Tab panel styling
Bug 1290056 - Console: filename starting with underscore is incorrectly displayed due to direction: rtl
Bug 1285591 - [a11y] Fix accessibility in devtools autocompletes
Bug 1278797 - Fix requests-menu-stack-trace direction in RTL locales
Bug 1291358 - New console frontend: Add support for console.trace()
Bug 1170955 - Reader Mode: Give visited links a different colour
Bug 565718 - Show zoom indicator in UI if not at default zoom level
Bug 1295769 - Zoom indicator zoom effect could be smoother
Bug 1253327 - Fix direction of children-pointer in RTL
Bug 1269958 - Change the summary section for Multiple Files information in Downloads Panel
Bug 1218224 - Reader View bottom margin too big for
Bug 1206229 - Inform the user that changes may require a page reload
Bug 1297157 - Abstract away the visuallyselected attribute on anonymous tab nodes
Bug 1296207 - Remove billboard capability
Bug 1291642 - Part 1 - Add an optional checkbox to PopupNotifications
- Code source disponible sous la licence Licence personnalisée
- Qu'est-ce que c'est ?
Ce module n'est pas compatible avec votre version de Firefox à cause des éléments suivants :

subset: commoncrawl


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Do you struggle to pay attention at work? Is your child often caught daydreaming in the classroom? Inattention and trouble focusing are among the hallmarks of attention deficit hyperactivity disorder (ADHD), a condition that can make it harder for you or your child to get things done.
ADHD is one of the most common neurodevelopmental problems in childhood and adolescence. According to the Centers for Disease Control and Prevention, more than 1 in 10 kids in the United States live with ADHD. Many of them continue to deal with ADHD as they get older.
If your child is having trouble getting through homework or you can't seem to focus at work or around the house, you can find help right on your smartphone. These 14 apps do everything from setting up reminders to creating to-do lists to improve concentration.
Whether it's waxing your car, reading through emails, or finishing up a report for your boss, it's easy to get distracted when you've got a job to do. Put an end to procrastination with an app that's designed to keep you on task.
With 30/30, enter a list of jobs you need to complete. Then assign each job a set time. The app will keep track of how much of the task still needs to get done as you work, and how much time you have left to do it. When time is up, 30/30 will tell you to move on to the next task. Finish within the allotted time to complete the job, and feel a great sense of satisfaction in the process.
You probably juggle dozens of different tasks on any given day. A to-do list might help you keep track of them, but only if you remember to look at it. Written notes can't sound an alarm when it's time to walk the dog or go to your next meeting, but the Due app can.
This easy, fast app lets you set up reminders in a matter of seconds. Schedule one-time alerts, as well as regular notifications for repeated tasks. With distinct alert sounds, you'll know right away when it's time to take your medicine, go to the gym, or do the laundry. Sync your alerts across your iPhone, iPad, and Mac, so you'll be reminded of to-do items wherever you are.
Have you ever thought about keeping a journal, but couldn't invest the time it takes to write your whole life story? Momento makes memory keeping easy, by using your existing social media pages to create a complete personal history.
Momento records important memories as they happen. You can then incorporate those moments with photos and events you've already collected in your social media timeline. Weaving together your daily musings and social media history helps creates a complete record of your life. You can also tag events so you can easily find them later.
The road from idea to execution is often paved with mistakes and struggles. If you have trouble seeing projects to the end, check out MindNode, an app that organizes your thoughts for you, so you can focus on idea generation.
You start by writing down your thoughts into branching units called nodes, then connecting the dots to create a visual map of those ideas. Color-code the nodes to quickly identify similar themes. Drag and drop them into various patterns until a clear direction appears. You can cross-reference all your mind maps from your iPhone, iPad, or Mac, and publish them for everyone to see with the new MyMindNode service.
More than 2.5 million people have simplified their daily routine with Clear, the app that Gizmodo calls "perfect for busy people." Clear's interface is a collection of intuitive and easy-to-search lists. It's simple enough for anyone to use, yet powerful enough to organize even the most hectic schedule.
Create a list to remind you when it's time to watch your favorite TV show or buy your friend a birthday present. Build another list to remind you that you have to mow the lawn or wash the car. Organize your lists by theme, and then access them across your devices by syncing them up on iCloud.
Is your daily routine a little humdrum? Turn it into an adventure with EpicWin, the app that makes boring, everyday chores like dusting and laundry into an exciting game!
Here's how it works: Enter your to-do list into the app's easy-to-use interface and choose your avatar. Each time you "destroy" a chore in animated battles, you'll be rewarded with points that increase your character's stats and help you level-up. You'll never think of housework or homework in the same way again!
Have you ever gone on a frantic search for an important note or to-do list that you left lying around? With Evernote, you'll never lose track of your notes again. This app will organize and sync them across your devices and incorporate photos and audio notes so you don't have to write everything down.
See a recipe in a library book that you'd like to try at home? Take a photo of it and save it for later. Suddenly remember that you need to take the dog to the vet? Record a voice reminder so you won't forget to make the appointment.
Evernote lets you organize all your documents by tags, so you can easily search for them when you need them. You can also share your notes with friends and family via Facebook and Twitter.
Do you forget when to mop the kitchen floor or dust the curtains? HomeRoutines organizes your chores for you and breaks them up into manageable room-by-room chunks, so you never feel overwhelmed.
This app makes the mundane more fun by awarding you a gold star for each job you complete. Test yourself by setting the timer and seeing how much cleaning you can get done in 10 or 15 minutes. Once the house is sparkling, review your list of completed jobs and pat yourself on the back — or send it to your housemate as a reminder of just how hard you've worked to tidy up the place.
Convincing most kids to make the bed, do their homework, or take out the trash can quickly turn into a battle. But what if you could turn boring chores into a fun and rewarding game?
iRewardChart encourages kids to do more around the house by making them feel like they're in control. Even though parents choose the tasks, kids get to pick their own rewards. Whether you want your kids to do the dishes, say "please" more often, or be nicer to their siblings, this app can help you accomplish it with minimal arguing. What parent wouldn't want that?
Priority Matrix
If you're a busy person it can be hard to juggle everything, especially if you're the boss. Why not have a little electronic help? Many of us are already balancing a collection of different devices, from cell phones to computers and tablets (and maybe even more). Priority Matrix connects them all for the purpose of both time and team management.
The app divides your tasks into four quadrants from the most important, "Do now!" tasks to things that need to get done but can be delegated to others. You can track your progress, status, and due dates by getting emails and using colors, icons, and percentages. With Priority Matrix you can be sure tasks won't slip through the cracks.
Do you still keep all your important photos, videos, and documents on your computer's hard drive? If you need one of those documents while you're away from your computer, you could be out of luck. Or worse, your computer could crash and wipe out everything you've painstakingly saved.
With Dropbox, you never have to worry about missing or losing your most important files. This free app stores your documents, photos, and videos to all your devices. You can instantly share your files with anyone you choose, and they can share their files with you — without space-consuming attachments! Plus, Dropbox offers 2 GB of free space, just for signing up.
TeuxDeux is your traditional to-do list on steroids. You can make a list on your home or work computer and then take it on the road with the app. Create custom lists and repeating tasks, so you'll never forget a job.
The app was designed to be simple and easy to use, so all you need to do is type in your list. Then you can move things around if you need to and check them off when they're complete. It's as easy as writing a paper note, but much harder to lose.
Bogged down by all the time it takes to write emails and texts? Slay slow typing with the Dragon app. A voice recognition system lets you send messages or post online to Facebook or Twitter without having to type a single letter.
No matter how quick you are on the keyboard, Dragon is probably quicker. It can transcribe voice to text about five times faster than typing. Plus, it's completely hands-free so you can use it on the go. And if you make a mistake, you can easily fix it with the app's voice-driven editing interface.
Remember the Milk
How many times have you come home from a trip to the supermarket, only to realize that you've forgotten to buy one all-important item? Avoid those "oops!" moments with Remember the Milk, an organizational tool that will help you keep track of all your lists and tasks.
Despite its name, Remember the Milk does a lot more than just organize your grocery lists. Use it to set project due dates, prioritize tasks by importance, estimate how much time it will take to complete them, and then plan the best way to get them done. Remember the Milk also lets you back up your lists online, so there's no risk of misplacing them.
Allies in ADHD
None of the apps on this list take the place of medicines and therapy for managing ADHD. But they can help kids and adults focus better, get more organized, and stay on-track with their daily responsibilities. Use these apps as part of a well-rounded treatment plan to help you complete important tasks and improve your productivity!
We selected these ADHD apps based on their potential to aid people who have this condition. Additional factors included user ratings, affordability, accessibility, format, functionality, and relevance to patients' needs. This list represents a wide range of helpful iPhone and Android apps that make it easier to manage ADHD.
Please note: Healthline Networks does not imply warranty of fitness for a particular purpose or endorse any of these applications. These apps have not been evaluated for medical accuracy by Healthline Networks. The U.S. Food and Drug Administration has not approved them unless otherwise indicated.


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Pathfinder International Applauds Launch of First-Ever USAID Youth in Development Policy
On November 1, 2012, the United States Agency for International Development (USAID) released its first-ever policy on Youth in Development. Pathfinder International applauds USAID for taking this important step to advance the rights and opportunities for young people around the world.
Today's generation of young people is the largest in history. Through the sheer size of their numbers and where they live—predominately in developing countries—young people represent both the present and future of development.
Pathfinder believes that young people are our greatest asset for sustained development.Pathfinder believes that young people are our greatest asset for sustained development, but in order to succeed young people must be, as the policy's conceptual framework suggests, "supported, protected, prepared, and engaged." That is why, for over 30 years and in more than 30 countries, Pathfinder has been committed to advancing the sexual and reproductive health and rights of young people; empowering young people, particularly young women, as leaders and advocates; and meaningfully engaging young people in the design, implementation, and evaluation of programs. Pathfinder led the first-ever global youth program from USAID, Focus on Young Adults, which is highlighted in the policy on Youth in Development.
Pathfinder applauds USAID's Policy Task Team for the robust consultation process that led to the development of this policy. As co-chair of the Youth Health and Rights Coalition, Pathfinder valued the opportunity to provide input during the development of the policy over the last year and, most importantly, the opportunity to facilitate youth input into the policy. In October 2011, the Coalition hosted an online consultation between nearly 40 young people from Sub-Saharan Africa, Asia, the Middle East and North Africa, Latin America and the Caribbean, and the United States and members of the Policy Task Team.
Pathfinder looks forward to continued collaboration with USAID to support the implementation of the policy on Youth in Development.
Focus Area: Adolescent and Youth Sexual and Reproductive Health
Media Contact
Please contact Kate Stookey, Director of Public Affairs, at 617-972-1231 or firstname.lastname@example.com
Related News
What the US can learn from Ethiopia about birth control
What's more, women in Ethiopia are having fewer children (the fertility rate fell from an average of 6.5 children per woman in 2000 to 4.6 currently), maternal deaths are in decline, and more women are staying in school longer. Plus, more women are opting for long-acting reversible contraceptives (LARCs) instead of more traditional short-term methods like birth control pills or condoms.
International conference stresses youth's reproductive health rights
Youths are deprived of information and services related to sexual or reproductive health. These issues are considered taboo due to social, cultural or religious norms in the developing and middle-income countries, speakers discussed on the third day of the International Conference on Family Planning, held at the Bali Nusa Dua Convention Centre in Indonesia.


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Austin, TXUSA
May 18, 2011
As a former English major, I'm a sucker for Romantic and Victorian literature but I am also drawn to Modernist fiction as well. I generally stick to fiction when I read for pleasure but I'm trying to branch out.
Hey all! I am happy to say that I will be able to rejoin this group after an absence of a couple of months. Looking forward to getting back into some good fiction and the delicious soup at New World Deli!
Meetup members, Log in
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Vt. ponders community placements for inmatesThe Associated Press | September 17,2013MONTPELIER - A panel of Vermont lawmakers is grappling with a tough question: how the state can provide community placements for people with mental health or other disabilities who are coming out of the state's prisons.
State officials say it can cost $300,000 to $400,000 per year to provide one-on-one care for some people with what the Corrections Department calls "serious functional impairments" as they near the ends of their prison terms and move into the community.
A special legislative study committee is taking testimony on the issues on Tuesday.
Among those testifying are Robert Appel, a former executive director of the state Human Rights Commission, and law enforcement officials.MORE IN This Just InMONTPELIER A pending deal between the state and federal governments will give a big boost to... Full StoryWOODFORD A North Carolina man was killed when his motorcycle crashed Friday morning in a... Full StoryMANCHESTER Voters will be asked at town meeting to allow the use of $15,000 from a reserve... Full Story
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As are some of the throws you will witness and remember from the video below.Le'Veon Bell Jersey.
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We want him to continue to be harassed by Clay Mathews – and by his complement – Julius Peppers. We want him to continue to look confused and dismayed while wearing his stocking cap on the sidelines.


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Best. Prinny. Mis-spelling. Ever.Originally Posted by Darkprinny
Gotta love the resident dyslexic.
Ill dislex you in a in
wait ....................
Can that be a threat
Spoiler Alert!
TNOG.ORG - Games events in Manchester
MikeDBloke - PSN
I was into weird games before people knew what they where
@D_Bloke - My inane prattle


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1st MALAYSIA OPEN ROVERS CHALLENGE CAMP
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Don't forget! Please mention TrustATrader when you contact any of our trusted traders...
The Green Edge Gardens
The Green Edge Gardens is a Blockpaving & Driveways Covering St Albans, Harpenden and all areas within 10 miles.
- Tel: 07908 993549
- Mob: 07908 993549
51 reviews
- Initial impression : 4.98 out of 5
- Cleanliness : 4.96 out of 5
- Value : 4.94 out of 5
- Punctuality : 4.98 out of 5
- Quality : 4.98 out of 5
- Overall opinion : 4.98 out of 5
0 missed appointments in the past 6 months
About The Green Edge Gardens - Blockpaving & Driveways in St Albans
Green Edge Gardens is a leading landscaping, gardening and grounds maintenance business serving customers in St Albans, Harpenden and surrounding areas of Hertfordshire.
Over the past 12 years we've built a reputation for transforming gardens… creating outdoor spaces our customers love and add real value to their homes. We know how important it is to have a garden you can enjoy and use and we have both the vision and gardening knowledge to give you a special garden that's truly healthy and ready to thrive.
Much of our work comes from customer recommendations which we think is down to the strength of our great team. The director, Paul, has worked hard to make sure our staff enjoy working for Green Edge Gardens and in return they work together effectively and give their best, working incredibly hard to transform gardens quickly and tidily for happy customers.
We really pride ourselves on the quality of our work and strength of our team, and the outstanding feedback from our customers is testament to our success.
Our services are wide-ranging and include:
- Full landscape design service
- Driveways
- Patios
- Porches
- Building garages
- Pathways
- Fencing
- Decking
- Shed bases
- Garden clearance/tidy
- Turfing
- Lawns
- Block paving
- Garden maintenance
- Pruning
All Tree work
- Tree Felling
- Tree Reductions
- Stump Grinding
- Tree Removals
- Hedge Maintenance
- Shaping Crown
- Jet washing
- Pruning
- Free quotes & estimates
- Regular maintenance contracts service available, as well as 'one off' smaller jobs
We are fully insured and comply with health and safety regulations to keep our staff and customers safe.
Now you know a little about us we hope we can get to know you and help you get the best from your outdoor space. Just let us know what you're looking for and we'll be happy to drop by to give a free, no obligation, quote.
Please mention Trustatrader when calling. Thank you.
- C&G Level 1 Certificate in Practical Horticultural Skills
Reviews (51)
The Green Edge Gardens have received 51 customer reviews.
Mark Walters 2nd September 2016
"It was refreshing to work with The GreenEdge. Paul quoted for pruning all of our trees and undertook the work on time and without any problems. They were pruned so well its hard to believe they were pruned at all. There was an enormous amount of wood to remove and the garden was left in better condition then when they arrived. Absolutely faultless and a real pleasure to work with from start to finish. Paul gave us some great ideas for our garden which we will be doing next with them next year."
The reviewer found this trader while searching on TrustATrader.com
Frank Jenkins 24th August 2016
"Paul initially met us and spent some time going through our garden and offering great advise. Actually ways to make the garden look better and costing less. His attitude and approach was refreshing compared to other companies who came over. I accepted the quote and they arrived on time and on the right day and did an excellent job. We are extremely pleased and would highly recommend The Green Edge to anyone. I have already booked them in to do more work in the future. I am on their waiting list but its well worth the wait!!"
The reviewer found this trader while searching on TrustATrader.com
Fred Smyth 19th August 2016
"In all my years of dealing with tradesmen, The GreenEdge is by far the most professional, passionate and friendly bunch of hard working people I have ever met. What a splendid team. Paul was an absolute joy to deal with from start to finish. We understand now why they have so many fantastic reviews & so pleased we chose them. Thanks a lot guys!!!!"
The reviewer found this trader while searching on TrustATrader.com
Martin Seal 9th August 2016
"Paul and his whole team were friendly, trustworthy and above all delivered exactly what was agreed (at an unbelievable hard working turn around). They are the only people I would use and will recommend them to all."
The reviewer found this trader while searching on TrustATrader.com
Mike Jones 5th August 2016
"Absolutely brilliant from day one meeting Paul through to the work being undertaken. I would not hesitate to recommend The Green Edge to anyone they are 1st class in every possible way and you will not be disappointed. We are so delighted."
The reviewer found this trader while searching on TrustATrader.com


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Currently have pttrx at 50% in 401k and 20% in moneymarket, the remaining in stock index funds. At 47, should i just reallocate the moneymarket to pttrx or to stocks? Don't think i'll lose any sleep either way just looking for any take. Thanks.
Sentiment: Strong Buy
I have been almost exclusivly in (PTTRX) for the last 18 months. Ever since the market started climbing for reasons I did not understand. I have slept much better. I know there many people on this board that think Mr. Gross should leave but none of those people are as good at this as he is or they would have thier own funds. If the S&P drops back to around 1100-1050 I will move back into stocks. Until then I will be happy with a good safe return of 6%.
More like 10% !!
FYI, over the past 3 months, I converted almost 30% out of 35% cash into PTTRX in my 401K. Now holding 75% PTTRX in my 401K. I didn't see much likelihood that PTTRX would underform cash at around 1.5% yield. Also, fyi, I am totally opposiste with my brokerage accts. (100% in equities).
Thank you for all input. I''m going to put most in PTTRX now and wait for the fiscal cliff resolution.


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NBQX disodium salt
Cat. No. 1044
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Chemical Name: 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydr
Biological ActivityPotent, selective and competitive AMPA receptor antagonist. Neuroprotective and anticonvulsant; active in vivo. More water soluble disodium salt of NBQX (Cat. No. 0373). Also available as part of the AMPA Receptor Tocriset™.
Licensing InformationSold with the permission of Novo Nordisk A/S
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The technical data provided above is for guidance only. For batch specific data refer to the Certificate of Analysis.
All Tocris products are intended for laboratory research use only.
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Namba et al (1994) Antiepileptogenic and anticonvulsant effects of NBQX, a selective AMPA receptor antagonist, in the rat kindling model of epilepsy. Brain Res. 638 36. PMID: 8199874.
Sheardown et al (1993) The pharmacology of AMPA receptors and their antagonists. Stroke 24 146. PMID: 7504337.
Gill et al (1992) The neuroprotective actions of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX) in a rat focal ischaemia model. Brain Res. 580 35. PMID: 1504814.
Zeman and Lodg (1992) Pharmacological characterization of non-NMDA subtypes of glutamate receptors in the neonatal rat hemisected spinal cord in vitro. Br.J.Pharmacol. 106 367. PMID: 1382781.
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Crook et al (2014) A synaptic signature for ON- and OFF-center parasol ganglion cells of the primate retina. Vis Neurosci 31 57. PMID: 24801624.
Reinhard et al (2014) Step-by-step instructions for retina recordings with perforated multi electrode arrays. PLoS One 9 e106148. PMID: 25165854.
Bloodgood et al (2013) The activity-dependent transcription factor NPAS4 regulates domain-specific inhibition. Nature 503 121. PMID: 24201284.
Butti et al (2012) Subventricular zone neural progenitors protect striatal neurons from glutamatergic excitotoxicity. Brain 135 3320. PMID: 23008234.
Zheng et al (2010) SAP102 is a highly mobile MAGUK in spines. J Neurosci 30 4757. PMID: 20357126.
Shin et al (2006) Vasoconstrictive neurovascular coupling during focal ischemic depolarizations. J Cereb Blood Flow Metab 26 1018. PMID: 16340958.
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Very small embryonic-like stem cells (VSELs) isolated from bone marrow (BM) have been reported to be pluripotent. Given their nonembryonic source, they could replace blastocyst-derived embryonic stem cells in research and medicine. However, their multiple-germ-layer potential has been incompletely studied. Here, we show that we cannot find VSELs in mouse BM with any of the reported stem cell potentials, specifically for hematopoiesis. We found that: (1) most events within the "VSEL" flow-cytometry gate had little DNA and the cells corresponding to these events (2) could not form spheres, (3) did not express Oct4, and (4) could not differentiate into blood cells. These results provide a failure to confirm the existence of pluripotent VSELs.
During normal development, pluripotent cells from the inner cell mass give rise to several types of tissue-committed stem cells (TSCs), restricted in their differentiation potential. TSCs can self-renew and produce downstream progenitors and mature cells throughout life. It remains controversial (Wagers et al., 2002; Wagers and Weissman, 2004) whether some pluripotent cells self-renew as pluripotent stem cells (PSCs) and persist after birth (Beltrami et al., 2007; Check, 2007; Jiang et al., 2002; Kögler et al., 2004; Krause et al., 2001; Serafini et al., 2007).
One group has recently identified bone marrow (BM)-residing very small embryonic-like stem cells (VSELs) in both human and mouse as a putative nonembryonic source for PSCs (Drukała et al., 2012; Kucia et al., 2006b; Wojakowski et al., 2009; Zuba-Surma et al., 2011). Mouse VSELs have been characterized to: (1) be very small (3–5 μm), (2) have a CD45−Lineage(Lin)−SCA-1+ phenotype, (3) express pluripotent marker genes (e.g., Oct4, Nanog), and (4) when cultured on the myoblast C2C12 cell line, form embryoid body-like spheres and then differentiate into multi-germ-layer cells. Moreover, these cells were reported to be directly obtainable from BM or mobilized peripheral blood without culture. Thus, their pluripotency should be reproducible in independent laboratories, a requirement for a scientific finding to be generally accepted (Jasny et al., 2011). However, recent studies have shown the lack of stem cell characteristics in VSELs isolated from mouse (Szade et al., 2013) or human cord blood (CB) (Danova-Alt et al., 2012), while another study confirmed the existence of mouse VSELs and their ability to give rise to lung cells (Kassmer et al., 2013).
We sought to recapitulate the previous findings on relating to mouse VSELs, focusing on the DNA content of the small-sized fraction and on its hematopoietic lineage potential. We discovered an anomaly in separating cells by forward scatter (FSC) using different types of cell sorters and, more significantly, could not find PSCs within the VSEL fraction of mouse BM.
We sought to identify VSELs from mouse BM by using commonly reported VSEL characteristics (Kucia et al., 2006a; Ratajczak et al., 2011; Zuba-Surma et al., 2008). We fractionated BM samples using fluorescence-activated cell sorting (FACS). Dead cells and debris were excluded as Ratajczak and colleagues previously reported (Zuba-Surma et al., 2008). To minimize the risk of missing candidate cells, SCA-1+ cells (including SCA-1lo) beyond the threshold defined by the fluorescence minus one (FMO) method (Herzenberg et al., 2006) were included, and only the obvious Lin+ cells were excluded from the Lin− fraction (which included Linlo cells). Consequently, the Lin−SCA-1+ gate was more inclusive than that described elsewhere for VSELs and included all cells reported to be in the VSEL fraction (Kucia et al., 2006a; Ratajczak et al., 2008a, 2011). Lin−SCA-1+ events were subdivided into three fractions according to CD45 expression (Figure 1A; Table S1 available online).
We used the FSC intensity of 10 μm microspheres to define the cutoff point between FSClo and FSChi events. As previously reported (Zuba-Surma et al., 2008), the CD45− fraction contained many more FSClo cells than FSChi cells. Conversely, the CD45hi fraction contained many more FSChi cells than FSClo cells (Figure 1B; Tables S1 and S2).
FSClo events could include erythrocytes, vesicles, or cell fragments and/or debris. Since the original group has stated several times that VSELs are diploid (Kucia et al., 2008; Ratajczak et al., 2007, 2008b), we analyzed DNA content of FSClo events using SYTO16, a cell-membrane permeable DNA dye. We found that SYTO16hi events, representative of diploid cells, were present in about 98% of the CD45hiLin−SCA-1+ fraction but only ~10% of the CD45−/intLin−SCA-1+ fraction (Figure 1C). The remaining events in the CD45−/intLin−SCA-1+ gate showed much lower intensity for SYTO16, indicating that these were likely to be cell fragments and not diploid cells. Furthermore, most of the CD45−/intLin−SCA-1+SYTO16hi cells were in the FSChi gate (Figure 1C). This indicated that our VSEL candidates were the relatively larger cells in the population analyzed, unlike the VSELs described in previous reports (Kucia et al., 2006a; Ratajczak et al., 2011; Wojakowski et al., 2009; Zuba-Surma et al., 2008, 2009).
We hypothesized that this discrepancy was due to the type of flow cytometer used: Ratajczak et al. (2011) mainly used a MoFlo (Beckman Coulter) (Zuba-Surma et al., 2008, 2009), but we used a FACSAria (BD Biosciences). Thus, we analyzed the same BM sample on a FACSAria and MoFlo. Based on the FSC intensity of unfractionated BM, 10 μm microspheres were positioned to the left of lymphocytes on the FACSAria but in between lymphocytes and granulocytes on the MoFlo (Figure 2A). Using the FACSAria and the MoFlo, we sorted the BM sample into three subpopulations, based on the FSC intensity of defined-size microspheres. We then reanalyzed each of these subpopulations on both the FACSAria and MoFlo. Critically, a given population of cells seemed smaller (based on microsphere reference FSC intensities) on the MoFlo than on the FACSAria (Figures 2B and S1A). However, fluorescence intensity of the cell-to-cell comparison did not differ between the sorters (Figure S1B). Finally, we evaluated the FSC of candidate VSELs and hematopoietic stem/progenitor cells on FACSAria and MoFlo. Notably, more than 70% of candidate VSELs were detected in the FSChi gate on FACSAria, whereas >80% of them were in the FSClo gate on MoFlo (Figure 2C).
To address the concern that flow cytometer FSC based on microspheres is not a reliable indicator of absolute size, we measured the size of cells with an image flow cytometer (FlowSight, Amnis). We captured 2 × 106 BM cells and identified 34 events having the immunophenotype corresponding to the candidate VSELs (Figure S1C). Their mean diameter was nonsignificantly smaller than that of a randomly selected population of 100 CD45hiLin−SCA-1+SYTO16hi events (7.06 versus 7.51 μm; p = 0.82; Figure 2D). However, only the candidate VSEL fraction contained few very small-sized (<5 μm) particles, as previously shown in fetal liver-resident VSELs (Zuba-Surma et al., 2009).
To determine whether the CD45−/intLin−SCA-1+ fraction contained any pluripotent cells, we compared Oct4 expression levels among freshly purified CD45−/intLin−SCA-1+ and CD45hiLin−SCA-1+ cells and a mouse embryonic stem cell (ESC) line using quantitative RT-PCR with four different primer pairs. We could not detect Oct4 expression in CD45−/intLin−SCA-1+ (both SYTO16hi and SYTO16lo) cells at all (Figure 3A). These results contrast with previous reports (Kassmer et al., 2013; Kucia et al., 2006a; Ratajczak et al., 2011; Shin et al., 2010).
Next, we performed a sphere-forming assay using a mouse myoblastic C2C12 cell line (Kucia et al., 2008; Shin et al., 2010). The C2C12 cell line is known to initiate its own differentiation and form muscle tubules when cultured in low serum concentrations (Yaffe and Saxel, 1977). We observed that, when cultured alone, C2C12 cells aggregated spontaneously and sometimes formed sphere-like structures (Figure S2A). Therefore, to distinguish between spheres originating from candidate VSELs and spontaneous aggregation of C2C12 cells, we isolated candidate VSELs from Actin-EGFP transgenic mice and cocultured them with C2C12 cells. Some GFP+ cells proliferated and formed small clusters (Figure 3B) but never the spheres described in previous reports (Kucia et al., 2008; Shin et al., 2010). Eight-day progeny was harvested and analyzed for the absolute number of GFP+ cells by flow cytometry. The mean number of GFP+ cells initiated from the CD45−/intLin−SCA-1+ fraction was significantly lower than that from the CD45hiLin−SCA-1+ fraction: 27.2 versus 1764; p = 0.0002, respectively (Figure 3C). We repeated this assay with BM samples harvested from Oct4-EGFP transgenic mice. However, we could not detect any GFP+ cells in day 8 cultures of the following originally plated cells: 1,000 CD45−/intLin−SCA-1+, 5,000 CD45hiLin−SCA-1+, or 105 unfractionated BM cells (Figure 3D). The addition of leukemia inhibitory factor and/or usage of mouse embryonic fibroblasts instead of C2C12 cells did not affect the results (data not shown).
Grown GFP+ cells from both CD45−/intLin−SCA-1+ and CD45hiLin−SCA-1+ fractions had similar morphologies: an unlobulated nucleus and many vacuoles in voluminous cytoplasm, suggestive of macrophages (Figure S2B). Furthermore, flow cytometry analysis confirmed these GFP+ cells expressed high levels of CD45 (a hematopoietic lineage marker) and CD11b (a macrophage marker; Figure S2C). Since C2C12 cells are reported to secrete macrophage-colony stimulating factor (M-CSF) into the culture supernatant (Ghosh-Choudhury et al., 2006), it is possible that they may induce macrophage differentiation to either CD45−/intLin−SCA-1+ or CD45hiLin−SCA-1+ cells via M-CSF signaling.
To demonstrate the pluripotency of a particular cell, clonal expansion without differentiation from a single cell in vitro is an indispensable step. However, the C2C12 cells did not work to induce this expansion in candidate VSELs. Therefore, we sought another way to demonstrate that CD45−/intLin−SCA-1+ cells are pluripotent or at least could even generate any tissue-specific lineage. We focused on testing hematopoietic potential because (1) a previous report showed that transplantation of cultured VSELs could reconstitute the hematopoietic system (Ratajczak et al., 2011) and (2) methods of purification and functional analyses of hematopoietic stem cells (HSCs) have been established (Spangrude et al., 1988; Uchida and Weissman, 1992). If hematopoietic potential cannot be recapitulated, the cells in the VSEL fraction must not be pluripotent.
Freshly isolated CD45−Lin−SCA-1+ BM cells (including both FSClo and FSChi) did not proliferate under hematopoietic culture conditions (Figures S3A and S3B). A recent report indicated that 5-day coculture with the OP9 stromal cell line (i.e., OP9 priming) is critical for VSELs to be designated for a hematopoietic lineage (Ratajczak et al., 2011). In accordance with previous reports (Nakano et al., 1994; Seiler et al., 2011), we found that, when cocultured with OP9 cells, an ESC line was capable of hematopoietic differentiation (data not shown); this served as a positive control. However, CD45−Lin−SCA-1+ cells could not generate hematopoietic colonies in the methylcellulose assay, even after OP9 priming (Figures 4A and 4B). The addition of various hematopoietic cytokines during the OP9 priming did not affect the results (data not shown). Furthermore, CD45−Lin−SCA-1+ cells failed to produce any hematopoietic cells under the following additional conditions: (1) serial plating (Ratajczak et al., 2011); (2) including SYTOX-Blue positive fraction; (3) using only the SYTO16hi cells obtained from ten mice; (4) using cells from H2K-BCL-2 transgenic mice (Domen et al., 1998); or (5) using cells sorted on a MoFlo machine (data not shown). These observations indicate that the FMO-defined CD45−Lin−SCA-1+ fraction, at least in vitro, lacks hematopoietic potential, as recently described both for mouse VSELs (Szade et al., 2013) and their human counterparts (Danova-Alt et al., 2012).
Within the remaining CD45hiLin−SCA-1+ fraction, 38.0% of CD45hiLin−SCA-1+FSChi cells but no CD45hiLin−SCA-1+FSClo cells formed hematopoietic colonies. Also, 1.86% of CD45intLin−SCA-1+FSChi cells formed hematopoietic colonies (Figure 4B). Many colonies from the CD45intLin−SCA-1+FSChi fraction contained fewer cells than those from the CD45hiLin−SCA-1+FSChi fraction, and their differentiation potential was restricted to nonerythroid cells and tended to skew to the monocyte/macrophage lineage (Figures 4C and S3C). In addition, when compared to CD45hiLin−SCA-1+FSChi cells, CD45intLin−SCA-1+FSChi cells showed a more indented nucleus, lower levels of SCA-1, and higher levels of Lin and side scatter (Figures S3D and S3E). These findings suggest that the CD45intLin−SCA-1+FSChi cells are at a lineage stage downstream of the CD45hiLin−SCA-1+FSChi cells. However, the exact lineage stage from which granulocyte-macrophage progenitors can develop may vary depending on conditions such as the type of cytokine cocktail (Rieger et al., 2009). Therefore, we cannot definitively determine the stage of the initially plated cells that gave rise mainly to macrophages in this assay.
To directly evaluate whether the colony-forming cells in the CD45intLin−SCA-1+FSChi fraction were progeny of HSCs or independent of hematopoietic lineage cells, we engrafted EGFP-HSCs into uncolored mice. The experimental design, summarized in Figure 4D, was similar to that described in a previous report (Hall et al., 2007). Three months after the mice underwent transplantation of GFP-expressing HSCs, 98% of their peripheral blood cells (not shown) and 96.8% of their BM granulocytes were GFP+ (Figure S4A). The frequencies of CD45−, CD45int, and CD45hi fractions within Lin−SCA-1+ gate in the chimeric mice were comparable to those in age-adjusted nonirradiated syngeneic mice (Figure S4B). We evaluated the frequency of colony-forming cells in each fraction. As in the experiments with mice that did not receive transplants, a limited number of colony-forming cells were detectable in the fraction of CD45intLin−SCA-1+FSChi and CD45hiLin−SCA-1+FSChi cells (Figure 4E). Analysis by fluorescent microscopy and flow cytometry confirmed that all of the colonies derived from CD45intLin−SCA-1+FSChi cells (81/81) and CD45hiLin−SCA-1+FSChi cells (926/926) expressed GFP (Figure 4F). This indicates that the CD45intLin−SCA-1+FSChi cells that demonstrated hematopoietic potential originated from HSCs. Furthermore, recipient CD45−/int cells, including the previously reported highly radiation-resistant VSELs (Ratajczak et al., 2011), failed to respond to this critical myelosuppressive condition and generate HSCs and/or their downstream progenitors in vivo. In other words, HSCs, reported to have little developmental plasticity (Wagers et al., 2002), are the only contributors to postnatal mouse hematopoiesis.
The existence of pluripotent cells after the preimplantation blastocyst stage has not been demonstrated, except in the case of germline stem cells. Therefore, VSELs and other PSCs in postnatal mice must be independently verified to change how we view events in embryogenesis and fetal development. PSCs in postnatal humans, if they exist, would have an extremely significant impact on society because of their potential applications in research and regenerative medicine. In fact, the discovery of VSELs has already led to a commercial venture, Neostem, to bring their potential to human therapies. One of its financial backers, the Vatican, has announced in two symposia at the Vatican that these cells represent an ethical alternative to ESCs derived from humans (International Vatican Conference, November 9–11, 2011 and April 11–13, 2013, http://www.stemforlife.org/vatican-initiative).
In contrast to these optimistic findings, some groups have recently reported on their failure to detect VSELs. To accept a discovery as a scientific fact in the stem cell field, we previously proposed that all of the following three criteria are critical (Weissman, 2007): (1) the initial discovery must be published in fully peer-reviewed journals; (2) the experiment as published must be repeatable in many independent laboratories; and (3) the phenomenon described should be so robust that other experimental methods must reveal it. And in the case of stem cells, the regeneration derived from their transplantation should be rapid, robust, and lifelong.
It is not infrequent that, although the initial findings are peer-reviewed, subsequent attempts to independently replicate the findings fail (Begley and Ellis, 2012). We therefore sought to employ a logical strategy to examine all the cells within the CD45−/intLin−SCA-1+ population in mouse BM, even going as far as to include dead cells and cell debris in the analysis. Besides surface markers, the main criteria for the VSELs as defined by the original group are the following two points: (1) very small in size (3–5 μm) and (2) pluripotency. First, we tried to clarify whether this fraction contains any viable cells of the reported small size. We found that about 90% of all the events in this gate are indeed relatively small and have much less DNA than that of diploid cells according to SYTO16 staining measured on FACSAria (Figure 1C). This suggests that these relatively very small events are simply cell debris or fragments; subcellular particles, such as exosomes and blebs from dying cells, could be included. As mentioned above, it is unreliable to determine absolute cell size by FSC intensity (Figure 2). Furthermore, any modifications such as fixation or cytospin have the potential to affect the cell size. Therefore, we used an image flow cytometer and directly measured the size of candidate events without any modification. This experiment revealed that most of the SYTO16lo events lack the round shape of cells and are jagged, suggestive of dead cells and/or cell debris. SYTO16hi cells, both from CD45−/intLin−SCA-1+ and CD45hiLin−SCA-1+ are not statistically different in size and the majority are not small (<5 μm). However, we were still able to detect an exceedingly rare population of CD45−/intLin−SCA-1+SYTO16hi events, which we sought to evaluate for pluripotency.
Since the original group reported that VSELs can survive and differentiate to the hematopoietic lineage on OP9 cells, we sought to evaluate these candidate VSELs by the same assay, namely blood colony formation. When we cultured all the SYTOX-Blue-positive cells in the CD45−/intLin−SCA-1+ fraction (without size discrimination), no colony formation was detected. This indicates logically that SYTOX-Blue staining method can separate dead cells functionally; there are no viable VSELs in the SYTOX-Blue-positive fraction. We were unable to detect any hematopoietic cells among the SYTOX-Blue-negative CD45−Lin−SCA-1+ cells—the ideal candidates for VSEL cells—under the following additional conditions: (1) serial plating (previous reports showed serial plating would recover cell growth; Ratajczak et al., 2011); (2) using only the SYTO16hi cells obtained from ten mice; (3) using cells from H2K-BCL-2 transgenic mice (to improve viability by inhibiting programmed cell death; Domen et al., 1998); or (4) using cells sorted on a MoFlo machine (data not shown).
Since we have shown that size may not be a reliable parameter in isolating VSEL, we broadened our search by not using the size criteria while maintaining the immunophenotype (CD45−/intLin−SCA-1+FSChi). Compared to CD45hiLin−SCA-1+FSChi cells, CD45intLin−SCA-1+FSChi cells showed much less hematopoietic differentiation potential, and it never matched CD45hiLin−SCA-1+FSChi cells, even after serial plating, in contrast to the original group's report (Figure 4B; data not shown; Ratajczak et al., 2011). However, given our observation of some hematopoietic output from the CD45intLin−SCA-1+FSChi fraction, we sought to determine the developmental origin responsible: HSC or candidate VSEL. All of the hematopoietic differentiation potential in the CD45intLin−SCA-1+FSChi fraction were derived from prospectively isolated and transplanted HSCs (Figure 4F), suggesting that the only cells with hematopoietic differentiation potential in the CD45intLin−SCA-1+FSChi cells are hematopoietic stem/progenitor cells.
Failure to detect hematopoietic lineage potential seriously calls into question the existence of pluripotent stem cells in CD45−Lin−SCA-1+ cells. However, this finding does not deny the existence of other multi- or oligopotent stem cells with other lineage potentials. For instance, Morikawa et al. (2009) reported that CD45−Lin−SCA-1+platelet-derived growth factor receptor α+ cells in adult mouse BM can give rise to mesenchymal stromal cells both in vitro and in vivo.
At the time of our writing this report, a few independent groups have replicated select aspects of VSEL: Bhartiya et al., 2012 reported VSEL-derived TSCs existed in mammalian ovary or in human BM and CB (Parte et al., 2011) and Sovalat et al. (2011) claimed VSELs could be isolated from human BM or granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood. However, all of these other groups evaluated only the existence of VSEL by the analysis of the cell size and immunophenotype without evaluating the pluripotency criteria, as criticized by the others in the field (Ivanovic, 2012). In fact, other recent reports failed to detect the stem cell properties of mouse (Szade et al., 2013) or human VSELs (Danova-Alt et al., 2012). When only one or a few markers of cells rather than the function of cells themselves are assayed, artifactual or nonphysiological expression of single markers can lead to the interpretation that a cell type rather than a marker is being studied. The only assays acceptable for functionally identifying stem cells are those that assess (1) self-renewal; (2) differentiation to clonal progeny of the cell types inferred from the name of the cell; and (3), for adult tissue outcomes, robust and sustained regeneration. The transfer of donor markers to host cells could occur by cell fusion and, theoretically, by exosome transfer, to the extent that exosomes are shown to be physiological entities and not cell culture artifacts.
We conclude that most CD45−/intLin−SCA-1+ cells in mouse BM were not as small as previously reported. Moreover, even despite broadening our search to include larger cells as candidates, we could not find a single instance of CD45−/intLin−SCA-1+ cells that (1) expressed Oct4, (2) proliferated to form spheres in cultures, or (3) demonstrated the ability to generate cells of the hematopoietic lineage—three functional aspects of pluripotency that were previously reported. These results suggest the absence of embryonic-like pluripotent cells in postnatal mouse BM.
Additional rigorous data would be needed to demonstrate their existence both in humans and mouse before clinical application, including the derivation of the same repertoire of normal tissue cells currently demonstrated with ESC and/or induced pluripotent stem cells.
C57Bl/Ka-Thy1.2 Ly5.1 (B/Ka), C57Bl/Ka-Thy1.1 Ly5.1 (BA), C57Bl/Ka-Thy1.1 Ly5.1-Tg (actin-EGFP), and C57Bl/Ka-Thy1.1 Ly5.1-Tg (pH2K-BCL-2) mouse strains were derived and maintained in our laboratory. The Oct4-EGFP transgenic strain (Tg[Pou5f1-EGFP]2Mnn/J) was purchased from the Jackson Laboratory. The C57Bl/6J-W41/W41 strain was kindly provided by Dr. Susan L. Hall, Loma Linda University. We used 4–12-week-old female and male mice. All animal procedures were performed in accordance with the International Animal Care and Use Committee and the Stanford University Administrative Panel on Laboratory Animal Care.
BM cells harvested from bilateral femurs and tibias with a flushing method were treated with BD Pharm Lyse Buffer (BD PharMingen). These cells were stained for 30 min with phycoerythrin (PE)-conjugated lineage antibodies (Lin), which consisted of anti-CD45R/B220 (clone; RA3-6B2, final concentration; 4 μg/ml), anti-T cell receptor (TCR) β (H57-597, 4 μg/ml), anti-TCR γδ (GL3, 4 μg/ml), anti-Ly-6G/C (RB6-8C5, 8 μg/ml), anti-CD11b (M1/70, 4 μg/ml), and anti-Ter119 (TER-119, 4 μg/ml); allophycocyanin-Cy7-conjugated anti-CD45 (30-F11, 8 μg/ml); and biotin-conjugated anti-Ly6A/E (SCA-1) (E13-161.7, 10 μg/ml) followed by streptavidin-PE-Cy5 (1 μg/ml). All the antibodies were purchased from BD. One micromolar SYTO16 (Life Technologies) was added to evaluate DNA quantity.
Sorting and analyses were performed on multilaser-equipped FACSAria II and, where otherwise indicated, on a MoFlo cell sorter or FlowSight image flow cytometer. Dead cells were distinguished by adding 1 μM of SYTOX-Blue dead cell stain (Life Technologies). To minimize contamination, a second round of sorting was performed. The automatic cell deposition system was used for single-cell assays. Data were analyzed with FlowJo software (Tree Star) or IDEAS software (Amnis).
By comparing the FSC of defined-size microspheres (Flow Cytometry Size Calibration Kit, Life Technologies) and that of BM cells, we evaluated relative cell size. The SCA-1 channel was separated between negative and positive by the FMO method. We further divided Lin−SCA-1+ cells into three subfractions according to the CD45 expression level: the upper limit of the CD45− subpopulation was defined by the FMO method; the CD45hi subpopulation by the obvious positive peak; and the CD45int area as that between CD45− and CD45hi. HSCs and candidate VSELs were expected to reside in the CD45hiLin−SCA-1+FSChi and CD45−Lin−SCA-1+ (and/or CD45intLin−SCA-1+) FSClo populations, respectively.
A C2C12 mouse myoblast-derived cell line was maintained in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal calf serum (FCS). For the sphere formation assays, freshly isolated 1 × 103 BM Lin−SCA-1+ cells from Actin-EGFP mice were: (1) plated on the 1 × 105 C2C12 cells seeded the previous day on a 24 well plate and (2) cultured for 8 days in DMEM with 2% FCS. Half of the culture media was changed every day. The grown cells/clusters were trypsinized, harvested, passed through a 100 μm filter, and analyzed by FACSAria. The cells were also observed by fluorescence microscopy (DMI 6000B, Leica Microsystems).
For liquid cultures, purified populations were suspended in 12 well plates with the following medium: Iscove's modified Dulbecco's medium (IMDM; Life Technologies) supplemented with 20% FCS, antibiotics, 10 ng/ml of mouse (m) recombinant interleukin 3 (IL-3), 10 ng/ml mouse stem cell factor (SCF), and 10 ng/ml mouse Flt3 ligand (R&D Systems). For clonogenic analyses of CD45hiLin−SCA-1+ (including HSCs) and CD45−/intLin−SCA-1+ (including VSELs) faction, cells were cultured 10 days in IMDM-based methylcellulose medium (Methocult GF M3434; StemCell Technologies), which contained FCS, bovine serum albumin, 2-mercaptoethanol, recombinant human (h) insulin and transferrin, as well as recombinant mSCF, mIL-3, hIL-6, and human erythropoietin. All cultures were incubated in a humidified chamber in 5% CO2. Colonies were scored and picked up for making cytospin preparations to define cell components.
OP9 coculture was performed as previously reported (Ratajczak et al., 2011). OP9 cells were maintained in α-MEM (MEMα Nucleosides Powder, Life Technologies) with 10% FCS. Freshly isolated Lin−SCA-1+ cells (CD45−, CD45int, or CD45hi) were plated on OP9 cells in 12 well plates with α-MEM plus 20% FCS and cultured for 5 days. Cultured cells were then trypsinized, centrifuged, and replated in methylcellulose medium with OP9 cells. For serial passage, methylcellulose cultures were trypsinized, centrifuged, and replated in new methylcellulose medium. Colonies were scored and picked up to make cytospin preparations and stained by the May-Giemsa method.
BM mononuclear cells from actin-EGFP mice were enriched for c-kit+ cells by using anti-c-kit microbeads (Miltenyi Biotec) and sorted for propidium iodide−Lin(CD3/CD4/CD8/Mac-1/Gr-1/B220/CD19/Ter119)−CD34−CD150+Flk2−c-kit+SCA-1+ long-term HSCs. Eight-week-old W41/W41-recipient mice (Reith et al., 1990), hematopoietic-deficient due to mutations in the W locus encoding the c-kit gene, received 400 cGy total body irradiation followed by intravenous injection of 100 sorted long-term HSCs via the retro-orbital vein. Peripheral blood and BM cells were analyzed by flow cytometry after transplantation to evaluate chimerism.
Prior to quantitative RT-PCR, 1 × 103 of each of the following cell types were sorted by flow cytometry: CD45−/intLin−SCA-1+SYTO16lo, CD45−/intLin−SCA-1+SYTO16hi, CD45hiLin−SCA-1+SYTO16hi, and SSEA-1hi ESCs. RNA extraction, reverse transcription, and PCR amplification were performed by using CellsDirect One-Step qRT-PCR Kit (Life Technologies) according to the manufacturer's instructions. Gene-specific products were monitored by the 7900HT Fast Real-Time PCR System (Life Technologies). Four different design of primer pairs for Oct4 messenger RNA were used: (1) designed by Niwa group (Toyooka et al., 2008), (2 and 3) designed by Ratajczak group (Kucia et al., 2006a; Liu et al., 2009), and (4) TaqMan probe set Mm.PT.51.7439100.g (Integrated DNA Technologies). Primer sequences and amplicon sizes are listed in Table S2.
The nonparametric Mann-Whitney test was applied to all comparisons of mean values after F-test evaluation of variances. All statistical analyses were performed on Prism 5 software (GraphPad).
We thank L. Jerabek and T. Storm for laboratory management; T.J. Naik for technical assistance; A. Mosley, C. Wang, and A. McCarty for animal care; Dr. S.L. Hall, Loma Linda University, for providing W41/W41 mice; and J. Ooehara, Y. Yamazaki, and M. Otsu for supporting experiments of side-by-side comparison between MoFlo and FACSAria.
This study was supported by fellowships from the Toyobo Biotechnology Foundation, the Uehara Memorial Foundation, Human Frontier Science Program (to M.M.), and the Japan Society for the Promotion of Science (to Y.M.) and by grants from the US National Institutes of Health (R01 CA086065 and U01 HL099999 to I.L.W.), the California Institute for Regenerative Medicine (RT2-02060 to I.L.W.), and the Leukemia & Lymphoma Society (700709 to I.L.W.). H.N. is a shareholder and a member of the scientific advisory board of ReproCELL and Megakaryon and a member of the scientific advisory board of Shionogi. I.L.W. is a member of the scientific advisory board of StemCells.
This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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This review is from: EVGA GeForce GTX 980 04G-P4-2986-KR 4GB FTW GAMING w/ACX 2.0, 26% Cooler and 36% Quieter Cooling Graphics Card
Pros: It is a 980
Cons: This card has had issues since the first day that I installed it. I have completely rebuilt my computer since having this card, so I know that it is the card that has issues. Also, I put it into my son's computer - same issues. When the display goes to sleep, sometimes, it cannot be brought out of sleep mode (no display and monitor is in sleep mode). A computer restart is required. When the computer is started up from being completely powered off, there is no display. Have tried different DisplayPort cables, including expensive ones, and nothing helps alleviate these display issues. Contacted EVGA for a new firmware. Installed the new firmware, which did not resolve the issues. Now the fans have stopped working completely. Even at 95 degrees Celsius, the fans do not spin at all. I submitted an RMA for this card now, but I am still thinking about just purchasing a new card from a different vendor, just so I do not have to deal with these issues anymore.
Other Thoughts: I would not recommend this card to my worst enemy! This will be my last EVGA card. I have been buying EVGA exclusively since I bought a brand-new EVGA 8800GT.READ FULL REVIEW
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Pros: Awesome performance. Geforce GTX 570M graphics and a great LED-backlit display. Quad-core/hyper-threaded i7. Steel Series keyboard is very durable and works very well. I have been able to run all of my games at the highest settings with the beta Nvidia 285.62 driver installed. The Dynaudio sound system produces the best sound I have ever heard, from a built-in sound system on a notebook! The Cinema Pro mode makes movies and games sound great! I also like the switch that turns the touch pad on and off, so you do not accidentally hit it while playing games.
Cons: Battery life is not great (I expected this, as this is a gaming machine, not a ultra-portable low-power laptop).
Other Thoughts: This laptop is full of hardware potential! In a price vs. hardware comparison (what you get for your money), this laptop has to be at the top. Quality hardware at a very competitive price.READ FULL REVIEW


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How GPL fits in with the future of antitrust regulation
Perspective: Attorney Eric J. Sinrod explains the ramifications of a recent case on the future of open-source software.
Tips for new Gentoo users
Gentoo is one of the most difficult distributions to learn, though veteran Gentoo users might point out that its friendly community and extensive documentation can help new users. Here are some tips that might make Gentoo easier for anyone who wants to give it a try.
Firefox password manager is not secure
The automatic password manager in the Firefox web browser is not secure, according to a report on the software maker's website.
Jboss World Berlin: riding with Red Hat
This week, Berlin is hosting the European JBoss World Conference. Red Hat is now the organizer of the three-day conference for developers and users of the JBoss Enterprise Middleware Suite (JEMS). In April, the Linux distributor took over the middleware specialist and recently began offering its products, which are still published under the independent JBoss brand, as part of its own the Red Hat Application Stack in combination with Red Hat Enterprise Linux (RHEL).
Vista's EULA Product Activation Worries
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Portable Apps Suite Lets Software Programs Run on iPod, Flash ...
PortableApps.com has launched Portable Apps Suite and the latest version of its Mozilla Firefox Portable Edition. With more than seven million downloads to date, PortableApps.com claims to bring computing to the pocket, letting users take their favorite applications anywhere and access these on a PC of choice, without installing software or leaving data behind, as per a statement issued by the company.
Mini-itx Specialist Finds Linux A Good Fit
Microsoft Windows may be the dominant operating system in businesses and homes, but there are some places it just doesn't fit. That is particularly true for the mini-ITX systems that are gaining popularity in single-purpose applications.
Enterprise Open Source
Why open source is ready for the enterprise: beyond free stuff
BasKet makes organization easy
BasKet is a multipurpose note-taking application that allows you to collect and organize text, pictures, files, and more. BasKet 0.6.0, released last month, sports a new interface and improved features over previous releases.
Zen Engineering And CHILI GmbH Partner To Market Linux-Based ...
CHILI U.S.A To serve As The U.S. Support And Distribution Center For CHILI PACS Radiology Software Products
Microsoft's Ballmer forced to eat his Linux words
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Banned Linux World of Warcraft Players Get 20 Days Free
As a follow-up to our previous story "To be or not to be banned...", we have nothing but good news for the "very small percentage" of World of Warcraft players running the game on Cedega under Linux!
Protest the Microsoft-Novell Patent Agreement
Bruce Perens of Debian and OSI fame has written an open letter to Novell concerning their agreement with Microsoft.
Berry 0.76 Screenshot Walkthrough
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The slides are now available for the Chinese standards/open source conference I wrote about on November 8. The most interesting news I learned there was that China has been activelyl developing its own open document specification, which it calls Uniform Office Format. You can now see the full presentation of WU Zhi-gang here.
Vista's EULA product activation worries
Walking on thin ice? The terms of Microsoft's End User License Agreement (EULA) for its upcoming Vista operating system raises the conflict between two fundamental principles of contract law. The first, and more familiar, is that parties to a contract can generally agree to just about anything, as long as what they agree to doesn't violate the law and isn't "unconscionable".
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Good karma for Red Hat
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The database abstraction framework strikes back
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Faculty Highlight: Dr. Miriam Segura-Totten
Dr. Miriam Segura-Totten is collaborating with biology education students Kristen Howard, Kelsee Ryan and Emma Thomaswick, and Dr. April Nelms in the Teacher Education Department on a project to better understand how students read biology literature. The study centers on investigating the obstacles that undergraduates encounter when reading biology research articles. The ultimate goal of this research is to create ways of analyzing the literature that allow students to overcome these obstacles. To attempt to determine these barriers, the research team will conduct focus groups and "think aloud" exercises with biology students at UNG.
Learn more about the Biology Department and get to know some of our faculty here:
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#1 - zysolyn
Reply +16(02/16/2013) [-]
Actually, stars do not burn. They are giant balls of gas where the pressure induces atomic fusion, and the process is so hot that it glows. (If anything gets hot enough, it will glow) The Sun is actually too hot for burning to take place, as oxygen cannot be maintained in that kind of heat.
#14 to #1 - hylebus
Reply 0(02/16/2013) [-]
"Too hot for burning" isn't exactly the phrase you're looking for. Burning is generally a reaction of Carbon with Oxygen, with the result of product of Carbon Dioxiode, e.g. Methane + Oxygen, CH4 + 2 O2 -> CO2 + 2 H20. Point is, most of Sun's mass is Hydrogen and Helium, as it's fairly "cool" and young star. When it becomes hotter by aging, the fusion temperature will increase to that extent that it will create heavier elements, like Oxygen or Carbon, eventually Iron.
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Hendon, soon to be Halfords Autocentre. Call [02082058792] to enquire about our services at this centre. Maximise the lifespan and value of your car with regular car servicing at Halfords Autocentre...
Mei Quan Academy of Taiji - HendonHendon School, Golders RiseNW4 2HP London 07909856490Closed
With over forty venues around London, the Mei Quan Academy offers a range of traditional Tai Chi and Qi Gong exercises to all levels of student, from complete beginners through to advanced...
SCRAP MY CAR NOW CASH HENDONLondon 07775 451526Closed
... SCRAP CARS WANTED FOR CASH, car breakers, vehicle salvage, scrap yard, scrap cars, vehicle...
Hendon Locksmiths 020 8099 1938, 24h LocksmithSunningfields RoadNW4 4QR London Hendon 020 8099 1938
... types of jobs and are available 24 hours 7 days a week. Our locksmiths work throughout Hendon, West Hendon, Colindale, Cricklewood and the local areas. Locksmith Hendon NW4 | Call: 020 8099 1938 -...
Glaziers HendonChurch RoadNW4 4EA London Hendon 02032 808946
Glaziers hendon ExpressServe our glazier can replace your glass today window repair, glass repairs, replacement windows professional window repairs company Glaziers hendon.
Locksmiths HendonChurch RoadNW4 4EA London Hendon 020 8535 1143
Local locksmiths Hendon. 24 hour emergency locksmith in Hendon for Hendon lock repair, lock installation and lock change. Hendon locksmiths can lock pick and gain entry.
Premier Inn London Hendon The HydeHyde HouseNW9 6LH LondonOpen
We're ideally located for Wembley Stadium and Wembley Arena. Before taking in a game or concert, be sure to pay a visit to Wembley Park with its exciting range of bars and shops ... premier inn london hendon, hotels in hendon, hendon hotels, hendon premier inn, london hendon the...
RAF Museum LondonGrahame Park WayNW9 5LL London Colindale 020 8205 2266Closed
Visit the Royal Air Force Museum today - based in both London and Cosford West Midlands a great free day out for all the family and aircraft enthusiasts too.
Tesco Stores Hendon Metro10 Sentinel SquareNW4 2EL London HendonClosed
Operating over 2, 900 stores nationwide, Tesco is one of the leading merchandise retailer in the UK. Whether you prefer the traditional face-to-face or the online shopping you are offered different...
Hendon Central Tyres22-24 Parson StreetNW4 1QB London Hendon 020 3302 8131Closed
Hendon Central Tyres NW4 are experts in automotive repair including puncture repairs, wheel balancing and alignment, car servicing, brakes and battery. Our aim and business objective is clear, to...
Mike Cousins Foam Supplies274 West Hendon BroadwayNW9 6AE London West Hendon 020 8202 8433Closed
Mike Cousins & Sons Upholsterers And Foam Supplies can have your home cushion foam cut to size for you, while you wait! Call into our workshop or phone us for details. ... upholstery services, foam cushions in hendon, upholstery foam in neasden, reupholster, Foam...
Hendon Therapies Clinic100 Colindeep LaneNW9 6HB London 020 8205 9777Closed
If youre troubled by musculoskeletal disorders or a painful sports injury, call 07957 976 865 to start natural and non-invasive osteotherapy treatments.
The Hendon377 Hendon WayNW4 3LP London Hendon 020 8202 6691Closed
... pub
Hendon Stationers20 Sentinel SquareNW4 2EL London Hendon 020 8202 3493Closed
... stationery suppliers
Hendon MinicabSt Georges AvNW9 0JT London Hendon 02034758952Open
Hendon minicab provide a cab service to all over UK. We are specialized in Airport transfer, Cruise Transfer and Railway station transfer. Get your instant quote and pre-book your cab through online...
Kadiris Of Hendon26 High RoadNW10 2QD London Willesden 020 8459 0936Open
Kadiri Foods Limited (KFL) has been trading as Kadiri Refreshment House, Kadiri Kitchen and now Kadiri's. Incorporated by the founder, Mr Jamal Kadiri, the company first opened in 1974 and has since...
Hendon LibraryThe Burroughs, HendonNW4 4BQ London 020 8359 2628Closed
... books for sale, public library, Libraries, Library, buy books, library books
Sainsbury's HendonUnit 4, Hyde Estate RoadNW9 6JX London The Hyde 020 8201 3078Closed
Sainsbury's was founded in 1869 and today operates over 1, 200 supermarkets and convenience stores and employs around 161, 000 colleagues. We put our customers at the heart of everything we do and...
Hendon Pharmacy52 Vivian AvenueNW4 3XH London Hendon 020 8202 7889Closed
... pain killer, pharmacist, pharmacy, medication, chemists, prescription, Chemists Dispensing, health,...
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