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Hotels near Nanaimo Station
Search hotels near Nanaimo Station in Vancouver
Trending now in Vancouver
Hotels within 0.5 miles of Nanaimo Station
- Most bookedDays Inn - Vancouver Metro
101 people looked at this hotel in the last hour
Most recent review
"I like the hotel. One thing is the card to go into the room is not open the room. It cause a lot of problem for us. They need to change for the new card. No elevator, we have to carry the luggage to second floor. Breakfast is very good."
B, Jul 2016, CA
Hotels within 1 mile of Nanaimo Station
24 people looked at this hotel in the last hour
Most recent review
"A little old but very suitable for the price. Everyone was very helpful. Mike, from maintenance, set up a rope I could tie my dog to while I was sitting outside (my dog is very curious and loves to wander)."
A Traveller, Sep 2015, CA
Hotels within 2 miles of Nanaimo Station
86 people looked at this hotel in the last hour
Most recent review
"Great staff very pleasant and helpful. Older hotel with a great view of the mountains (even room numbers only) Price is reasonable for Vancouver but don't expect a five star hotel in the summer for their price If you want a clean basic accommodation on the skytrain route and don't mind walking..."
A Traveller, Jun 2016, CA
80 people looked at this hotel in the last hour
Most recent review
"Large room, comfortable, would stay again. Close to amenities and highway."
A Traveller, Jul 2016, CA
41 people looked at this hotel in the last hour
Most recent review
"We were very happy with the hotel. It's clean and close to restaurants. We will definitely book with them again."
A Traveller, Jul 2016, CA
39 people looked at this hotel in the last hour
Most recent review
"Jack the new guy was awesome and such friendly staff"
A Traveller, May 2016, CA
39 people looked at this hotel in the last hour
Most recent review
"It offered all we needed (two-night stay). Tea and coffee were provided."
A Traveller, Jun 2016, GB
17 people looked at this hotel in the last hour
Most recent review
"We stayed here with my family and it was a great location, super clean, and lots of space. The only down side is that this unit is the lowest floor, and we heard all the noise from upstairs. We had terrible sleeps because of it. "
A Traveller, Jun 2016, CA
Hotels within 3 miles of Nanaimo Station
130 people looked at this hotel in the last hour
Most recent review
"I would recommend this hotel as good value. The room was clean and comfortable and located close to downtown and a shopping plaza was next door."
K, Jul 2016, CA
88 people looked at this hotel in the last hour
Most recent review
"Great Hotel, The Hotel Staff is very kind and helpful. This was my fourth visit and I will go back next year."
A Traveller, Apr 2016, CA
Map of hotels near Nanaimo Station
- Stanley Park
- Vancouver Convention Centre
- Rogers Arena
- University of British Columbia
- Horseshoe Bay
- Holland Park
- Richmond Night Market
- Canada Place
- Granville Island Public Market
- Queen Elizabeth Theatre
- Richmond Centre Mall
- Capilano Suspension Bridge
- BC Place Stadium
- Metropolis at Metrotown
- Kitsilano Beach
- Steveston
- Vancouver Aquarium
- Simon Fraser University
- Richmond Olympic Oval
- Playland Amusement Park
Hotels in Vancouver near Nanaimo Station
Are you looking for a cheap Nanaimo Station hotel, a 5 star Nanaimo Station hotel or a family friendly Nanaimo Station hotel? You just landed in the best site to find the best deals and offers on the most amazing accommodations for your stay.
When you search for hotels near Nanaimo Station with Hotels.com, you need to first check our online map and see the distance you will be from Nanaimo Station, British Columbia.
Our maps are based on hotel search and display areas and neighborhoods of each hotel so you can see how close you are from Nanaimo Station and refine your search within Vancouver or British Columbia based on closest public transportation, restaurants and entertainment so you can easily get around the city. All the hotels details page show an option for free or paid onsite parking.
If you wish to see the hotels with the highest featuring discounts and deals near Nanaimo Station, simply filter by price/ average nightly rate. We recommend you filter by star rating and read our genuine guest reviews so you can get the best quality hotel with the best discount.
One of the new features on Hotels.com guest reviews is that also show reviews from Expedia for Nanaimo Station hotels and the TripAdvisor Nanaimo Station hotels reviews so you can make sure that you checking with a reliable source. See the review scores on our Vancouver hotel information pages.
Make the most out of your family vacation when you book your accommodation with Hotels.com – book your hotel near Nanaimo Station, Vancouver after reviewing the facilities and amenities listed for each hotel.
After booking your hotel near Nanaimo Station, expect to receive your reservation confirmation in the mail in less than 10 minutes. The confirmation email contains more information on all nearby attractions, local directions and weather forecast, so you can better plan the days during your trip.
After getting the best hotel rates you can still save more by winning 1 free night! That's right, book 10 nights in any hotel near Nanaimo Station, Vancouver and after you sign up for the Welcome Rewards program, you are eligible hotel you receive 1 night free*
The best hotel deals are here: We have Nanaimo Station hotel deals, Nanaimo Station last minute deals and offers to get you the cheapest Nanaimo Station hotel with our lowest price guarantee.
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OSI-Approved Open Source (250)
- GNU General Public License version 2.0 (159)
- BSD License (24)
- GNU General Public License version 3.0 (20)
- GNU Library or Lesser General Public License version 2.0 (13)
- Apache License V2.0 (12)
- MIT License (8)
- Academic Free License (7)
- Common Public License 1.0 (7)
- Common Development and Distribution License (5)
- Mozilla Public License 1.1 (5)
- Open Software License 3.0 (5)
- Microsoft Public License (4)
- PHP License (3)
- Affero GNU Public License (2)
- Mozilla Public License 1.0 (2)
- Public Domain (21)
- Creative Commons Attribution License (17)
- Other License (11)
- Windows (315)
- Grouping and Descriptive Categories (180)
- Modern (118)
- Linux (79)
- Mac (64)
- Android (42)
- Other Operating Systems (39)
WWW/HTTP Software
- Programming Language: Visual Basic .NET ×
BoDo will convert any existing Web services written in dotNet (.Net) into fully functional php code. The generated code will handle all web service calls and will contain the whole functions from the original web service.
Bookmark-Manager 2006
Bookmark-Manager 2006 features a completely rewritten code base, optimized features and functions, and finally offers true international language support for your multi-browser bookmark management needs.
Canis Web Browser
Canis is a web browser for Microsoft Windows. Being a simple tabbed web browser, Canis eliminates annoying tool bars. Scripted in Visual Basic.NET, it is designed to help motivate beginner programmers by getting involved to help code the next version.
A collection of my programs and websites code
Concept SecureSurfer
Concept SecureSurfer is a filter based on the Microsoft .NET Technology. It gets from the Internet a list of WebSites containing pornography, violence and other bad things and if you go in one of these sites, you will see a default error page.
Cryo Login Center
Cryo Login Center also known as cLc is currently an ASP and ASP.NET based control panel interface for websites. It allows you to control many aspects of a website easily even for the novice web developers. Upload, Edit, Create, and more with cLc.
Crystal Explorer
Crystal Explorer is a browser based on Internet Explorer realized in Visual Basic .Net.
D20Database.Com D20 RPG Web System
To develop a database driven, membership based web interface for D20 based Role Playing Game Campaign and Player Character creation and management.
A .NET project to manage DKP (Dragon Kill Points) for World of Warcraft players.
Dark Torrent
Dark Torrent is a fully new BitTorrent Client developed in VB.Net. Since it use the .Net it will be extremly fast.
DemoGuard Free
DemoGuard Free will be an opensource software / script application suite for software developers. DemoGuard Free will simplify the creation of web aware demo programs and web based / program based registration.
Diablog is a web-based blogging tool that requires no databases. Uses XML for data storage.
DotNetNuke Ajax Chat
This project is a DotNetNuke Chat module using Ajax. It uses a database backend. It implements all of the best practices in DotNetNuke such as using DAL+ for the database layer.
DotNetNuke Module - Alphabetical Sitemap
Alphabetical sitemap module for Dotnetnuke 4.90 or above.
DotNetNuke News Module
The DNN News module is a core DotNetNuke module that allows you to create news channels of aggregate feeds as well as display news feeds (RSS, atom, etc) in a customized format.
DotNetNuke Oracle Data Provider
DotNetNuke Oracle Data Provider using ODP.NET.Which included an Oracle Helper that mimic Microsoft Data Access Block plus Ref Cursor,Clob etc.
DotNetNuke Portal Creation Wizard
Portal Creation Wizard simplifies the portal creation in the DotNetNuke CMS. With this wizard, users will be able to create their own portals without help from the administrator thus expanding the usability of DotNetNuke.
A free webmailer for .NET
DownOrNot 1.0
A Set of Tools for use by Experts at www.experts-exchange.com
Email Obfuscator
Libraries to obfuscate email addresses in html-output to fool spambots, so that they can not harvest your email addresses from your websites. Webmasters will like this project because it protects their privacy with just a single line of code.
Explore is a web browser written in Visual Basic. It uses a custom version of the Gecko control to provide a more safer browsing environment for everyone to use.
Favicon-Icon-Sync is the tool to synchronize your IE favorites Icons.
FeedShare provides a RSS/ATOM news service.
Film Canistr
This utility automatically refreshes the desktop wallpaper at a set interval with user-defined images from Flickr, the popular online image hosting service.

subset: commoncrawl


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Equipped with professional and productive multi core building wire factory, Xi'an Galaxy is one of the best China building wires, solid core building wiring, multi core electri building wires, pvc coated building wires and cables manufacturers, welcome to wholesale cheap products from us.
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Taxonomy: Tariff Line Affected - 2712
Petroleum Jelly, Mineral Waxes & Similar Products
EC: GSP for certain countries' sectors revoked for 2017-2019 period
DescriptionOn 9 March 2016, the European Commission announced regulation 330/2016 changing its Generalized Scheme of Preferences (henceforth: GSP) for a number of countries.
EAC: Impose of levy on all imports into the Community
DescriptionOn the 1 July 2014 the East African Community (EAC) imposed an infrastructure levy of 1.5% on all imports into the Community. This is with the exception of the United Republic of Tanzania, which will temporarily apply this levy for one year.
Kazakhstan: Reduction of export duties on oil and oil products
DescriptionOn 17 March 2015 the government of the Republic of Kazakhstan approved Decree No. 145. It introduces amendments to Decree No. 520 of 7 June 2010. Concretely, Decree No. 145:
Kazakhstan: Introduction of export duty on various products
DescriptionOn 7 July 2010 the government of the Republic of Kazakhstan approved Decree No. 520 of 7 June 2010. It introduced export duties on a number of goods.
Argentina: Introduction and subsequent termination of Prior Sworn Declaration Statements for Imports
DescriptionOn 1 February 2012, Argentina's Federal Administration of Public Revenues enacted Resolution 3252/2012.
Ecuador: The introduction of an import duty for Peruvian and Colombian products
DescriptionAccording to numerous and consistent press reports, on 5 January 2015 the government of Ecuador introduced a temporary duty of 7 percent for imports originating from Peru and of 21 percent for imports originating from Colombia.
Sri Lanka: Duty surcharge on imports withdrawn
DescriptionOn 26 May 2010, the Ministry of Finance and Planning issued a notification rescinding an earlier order that charged a 15% surcharge on imports of all products (except certain specified commodities).
UK: Introduction of numerous trade finance schemes
DescriptionOn 9 February 2011, the UK Export Finance authority announced it would introduce new export support measures. These included: Introduction of the Bond Support Scheme
UK: Introduction of the Export Refinancing Facility
DescriptionOn 30 April 2014, the UK government launched its Export Refinancing Facility (ERF) with a volume of GBP 5 billion and the aim to allow UK exporters to provide favourable financing conditions to overseas customers.
Afghanistan: Introduced export duties on natural resources
DescriptionOn 1 February 2010, the government of Afghanistan introduced export duties on certain natural resources (Letter CS2-538570 of the Ministry of Finance of the Government of the Islamic Republic of Afghanistan).


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Operations based out of a tiny office building? They have a cheap phone recorder and don't bother to respond to emails. There is no disclosure regarding something as simple as HOW and WHERE specifically are they producing these alleged cameras? I hear alot of talk with no back up!!! Anyone????
Hey Genius, its in the 7/21 8-K filing. Do you know how to read? Here it is: Item 5.03 – Amendments to Articles of Incorporation or Bylaws; Change in Fiscal Year.
On July 19, 2016, at the Annual Meeting of Stockholders, the stockholders of the Company approved an amendment to the Company's Third Amended and Restated Certificate of Incorporation, as amended, to implement a Reverse Stock Split of the Company's Common Stock with the ratio to be determined by the Board of Directors (the "Board") of the Company, within a range of one (1) share of Common Stock for every two (2) to twenty (20) shares of Common Stock. If the Board of Directors continues to believe that a Reverse Stock Split is in the best interests of the Company and its stockholders, the Board will determine the ratio, within the range approved by the Company's stockholders, of the Reverse Stock Split and implement such Reverse Stock Split by filing a Certificate of Amendment to the Company's Third Amended and Restated Certificate of Incorporation (the "Certificate of Amendment") with the Secretary of State of the State of Delaware (the "Reverse Stock Split"). The Board of Directors may delay effecting the Reverse Stock Split without re-soliciting stockholder approval. The Reverse Stock Split, if any, will become effective after the filing of the Certificate of Amendment on the effective date set forth in the Certificate of Amendment. Beginning on the effective date of the Reverse Stock Split, each certificate representing pre-split shares will be deemed for all corporate purposes to evidence ownership of post-split shares.
Sentiment: Strong Sell
1- Dilution of more shares and 2- effecting a 20-1 Reverse Split!!! Management lining their pockets with more stock awards!! Wow, how horrible and pathetic but typical!!! Common shareholders lose again!!
Sentiment: Strong Sell
Headquarters in a tiny office building in NC? When you call the 800# you get a cheesy poor quality message on a cheap voice message machine. How many employees? 3 maybe? LOL
They are milking everything they can with unjustified & unwarranted over compensation, to the detriment of the common shareholders.
Ditto to that!
What is the source for the Lassiter details? I cannot locate it and have already checked the sec site. Thanks.
Luke, It just seems like there are those out there "in the know" selling for some reason, while retailers like us are blindly holding & averaging down. I'm not convinced that any blackout period itself would cause this consistent erosion. What is going on?
Drops every day a little without fails and with no news!! What is going on???????????????
This garbage dribbles down every day! About ready to give up and sell. I regret the day I first noticed this POS!
Hoping that this slow drip death of the PPS reverses itself soon with some real volume. I am holding from higher levels. So far a very foolish move on my part.
Do you think that this is the reason for the consistent erosion in in PPS?
You are WRONG. The chart says that we are on the verge of an uptrend instead! Technically, according to the chart we are at the cusp of and on the verge of a positive MACD cross over.
Nice pick up at 46 cent but it may have been prudent to take your profits last week. LMFAO regarding 5.00-6.00. This thing is not a buy and hold, its a pump and dump. Like I mentioned last week we are still in the dump phase.
Doing a hell of a job, down 12%. How is that making money? Also curious. What is your average pps which you are holding (you current loss)?
LOL!!! You are clearly desperate now by pumping with out of date and irrelevant clips from the past. Trying to get more bagholders? I guess misery does love company.
Already down 11%. My guess is that we should see 30 to 40 cent range soon.
Yes you must compare with STEM and other tiny bios like HTBX. Statistically most trials fail. Look it up genius. Tiny bios typically fail & die out because they cannot survive bad results, run out of money, delist, etc.. Explain how HTBX is any different???.
Q4 is a LONG way off Mr. Pumper. Apparently your strategy is to buy and pray? If results are not overwhelming positive, this stock is toast & company is done, like STEM! IMHO.


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Temple showed great improvement from its earlier meeting with Connecticut, but a poor offensive night contributed to the same outcome.
The Owls shot just 19 for 56 (33.9 percent) from the field and committed 16 turnovers during a 68-55 loss to the No. 21 Huskies in an American Athletic Conference matchup Thursday night at the Liacouras Center.
Coming off Sunday's 71-64 win over then-No. 23 SMU, Temple fell to 7-18, 2-11, while the Huskies are 21-5, 9-4.
On Jan. 21, Temple lost at UConn, 90-66, but this time the Huskies had to work much harder on offense and shot 2 for 13 from beyond the arc.


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Other Sellers on Amazon
+ FREE UK delivery
+ £1.45 UK delivery
STABILO EASYgraph for left-handers pack of 6 - Ergonomic pencil
|£5.80 FREE UK delivery.
|You Save:
|£2.72 (32%)
- Enter your model number above to make sure this fits.
- Ergonomic HB graphite pencil designed especially for left-handers^Triangular shape and moulded grip zones promote children to hold the pencil with the correct grip^Moulded grip zones along the whole shaft of the pencil mean even after sharpening, the child can benefit from the grip^Varnish coating prevents pencil getting dirty^Box of 6 pencils
Frequently Bought Together
Customers Who Bought This Item Also Bought
What do customers buy after viewing this item?
Product Description
Product Description
Pencil EASY GRAPH HB VE=6
Pencil EASY GRAPH HB for left-hander 321HB6
Manufacturer's Description
STABILO EASYgraph, the first ergonomic graphite pencils especially designed for left or right handers to ensure that the correct grip is found intuitively from start. This means that the fingers on the writing hand stay relaxed when writing. The triangular shape for perfect fit with the non slip grip moulds for left or right handers ensure that the correct grip is formed. The great mix of colours and the attractive design makes it fun to use. The first years of pre-school are an exciting time for children; finally they can get to grips with numbers, letters and the world of grown ups.
As a manufacturer of writing instruments, STABILO want to accompany children on this journey of discovery in the best way possible. That is why, for several years now, the company has been working together closely with educationalists, scientists and designers to develop optically appealing and ergonomically suitable pens for children to write with. This is important, as research has proved that holding a pen incorrectly can impair a child's performance at school and even lead to health problems.
STABILO's ergonomic designed products focus on comfort and efficiency. Using an ergonomic product helps to avoid unhealthy stress, tiredness and potential damage to the posture of the hand. Therefore, it is vital to have the correct tools during the most important part of a child's life, SCHOOL. On the basis of these findings STABILO have developed a range of ergonomic products, STABILO EASY, for left and right handers, including the brand NEW EASYcolors and EASYgraph
Customer Reviews
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bought for my left handed son. Seems he holds this pencil better and writing has gone betterPublished 23 days ago by kaustav mukherjee
A great help for my grandson who is left handed. Good strong pencil and coloured top so that right and left handedness can be clearly identified.Published 5 months ago by Fufa
You may thing how can a pencil make that much difference? But it does, it makes it far easier for left handed children to grip it better so a thumbs up from mePublished 5 months ago by Ms. C. M. Hillary
This is much more comfortable to hold so makes my grandaughters life easier sent some to school who were verr impressed.Published 8 months ago by Sheila Goodchild
I like it. My son loves this pencil. It s a good quality and fast delivery.Published 8 months ago by TULAY
Arrived quickly. Massive improvement in my daughters writing, worth every penny.Published 9 months ago by missc
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Foursquare © 2016 Lovingly made in NYC & SF
Club Habanos
Nightclub · $$$
6759 N Palm Ave (at Herndon Ave)
"I love this place ! The music is great ! The bartenders make a damn good cocktail and the place feels like fam , thanks to Adela ! Order the margaritas !! So good !!"
Arena Nightclub
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This profile was last updated on . .
Is this you? Claim your profile.
Direct Phone: (302) ***-****
Email: j***@***.org
118 Ashwood Street
Bethany Beach, Delaware 19930
United States
The McClenny (Clenny, McClenny, McClenney and various other spellings of our name) family is widely disbursed and the purpose of this website is to provide a central, easily available family data storage location for all who are interested in our family. ... more
McClenny Webmaster
www.mcclenny.org [cached]
Connection Newspaper Article About Tony McClenny
www.mcclenny.org [cached]
Tony McClenny is a heck of a popular guy.
After all, people from all parts of the world call
Bethany Beach News | Coastal Point
www.bethanybeachnews.com [cached]
McClenny Family Website
www.mcclenny.org [cached]
If you have family information, pictures you wish to share or if you have corrections to offer, please e-mail them to: firstname.lastname@example.com . You can also mail them to: Tony McClenny, 118 Ashwood Street, Bethany Beach, DE 19930-9699.
McClenny Family Website
www.mcclenny.org [cached]
Among all of the conventioneers at last year's ONE BBSCON in Colorado Springs, getting the latest on one of the fastest growing industries on the globe, two sysops, Andrew Bilski and Tony McClenny, set the foundations to do some growing of their own.
John McClenny
Stillwater Public Library
Ian McClenny
Navigant Consulting, Inc.
Shaun McClenny
Capitol Rehab Inc
Cynthia McClenny
Cornerstone Assistance Network


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|Message view
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|Apache Wiki <wikidi...@apache.org>
|[Jackrabbit Wiki] Update of "JackrabbitOnJBoss" by JukkaZitting
|Mon, 19 Apr 2010 13:35:25 GMT
Dear Wiki user, You have subscribed to a wiki page or wiki category on "Jackrabbit Wiki" for change notification. The "JackrabbitOnJBoss" page has been changed by JukkaZitting. The comment on this change is: Note about JCR-2344. http://wiki.apache.org/jackrabbit/JackrabbitOnJBoss?action=diff&rev1=30&rev2=31 -------------------------------------------------- }}} It's a hack, because the code shouldn't need the JNDI environment details when deployed inside JBoss, but that's all it took to get the WebDAV servlets working in JBoss 4.0.5. - 5. In JBoss 5, there's a conflict between xml-apis and xercesImpl bundled with Jackrabbit and Jboss libraries. + 5. In JBoss 5, there's a conflict between xml-apis and xercesImpl bundled with Jackrabbit and Jboss libraries. This problem affects Jackrabbit versions <= 2.1 (see [[https://issues.apache.org/jira/browse/JCR-2344|JCR-2344]]) {{{ Caused by: java.lang.ClassCastException: org.apache.xerces.jaxp.DocumentBuilderFactoryImpl cannot be cast to javax.xml.parsers.DocumentBuilderFactory
|View raw message


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Perry Ellis Men's Saddle Oxford Reviews
Rated 4.3 out of 5 by 49reviewers.
Rated 4 out of 5 by KcDurbin Great looking shows! True to size. True to color. Great price! May 25, 2016
Rated 5 out of 5 by Jbottillo Great product Great and easy ordering, fast shipping. May 24, 2016
Rated 4 out of 5 by 2720rc Great shoes.Excellent value. December 1, 2015
Rated 4 out of 5 by Globalworks Just a bit big Nice shoe, nice design. Unfortunately it's just a bit too big. I had high hopes for it. September 28, 2015


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Top Definition
When an artist has a lot of inspiration at one time, they will draw many different pieces of art, seemingly nonstop. Often times, this can lead to developing a different style, or sometimes stopping altogether if the artist loses their inspiration afterward.
That guy made like 8 pictures in a day! He's on a serious art binge.
by guyNUM01 July 15, 2010
Trending Now


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|Reviews for Amorously Minded
| Manny-Sama chapter 1 . 7/27/2011
Yeah, I was the one that initially put this fic on the Fanfic Recs page for Marvel vs. Capcom... But I don't think it has its own page. : If it does, link it to me!
More to the point, this is a great chapter that was worth the wait! Keep it going!
| Prominence Flare chapter 6 . 7/26/2011
Heheh. Vroom vroom.
| Liber-T.E.A chapter 6 . 7/25/2011
I saw that on TVTropes. Nice.
Very funny chapter, though I feel really bad for Pete.
| Evowizard25 chapter 6 . 7/25/2011
Thank you for updating. This was another great chapter. I just love Lilith. She's so cute. And I love the thought of Tony being protective of her. I think it makes him the better man for it. I hope you update soon.
| Anmynous chapter 6 . 7/25/2011
This is going to be a bit disjointed from all my scrolling back and forth, but here goes!
If they're going to the mall, shouldn't Peter ask for a credit card? Ought to be good...
It's a little weird to pull up the Civil War story in this chapter on top of the previous, even if 'it is from Peter's perspective this time. Maybe have him go into (even) less detail would be appropriate?
''Little succubus'' looked a bit strange, should I imagine Peter emphasizing the 'little'?
Man, I'd love to see that youtube video. You know you got a good scene when the next chapter can get away with referencing it!
I haven't seen that ''not the last guys I'd send'' phrase before, though it's easy enough to infer what it means. Is it just a more different usage of ''not the first thing I'd do''?
Tony assuming things about demons went over my head. Just a jab at him being shocked over having a half-demon kid?
Morrigan uses 'play' twice in as many sentences, it looks a little off in the context.
I can't tell if the rewrite has improved the previous chapters, as I already thought they were pretty awesome, and I still do. It's just an amazing piece of work! One of those stories where both premise and execution are impressive.
| MorenoX25 chapter 6 . 7/24/2011
I re-read the whole story and it was really enjoyable.
I loved the chris hansen reference.
Also when peter said "Oh yeeaaah," I can't help to picture Happy Hour 1 when Peter saw the batmobile.
I can't wait for next chapter and the shenanigans that are sure to come.
| Icezera chapter 6 . 7/23/2011
Very nice update and revisions. Still some spelling errors in chapter 5 like toe instead of tow (in tow) but overall, it's progressing pretty well. Please keep this great story up!
I hope Morrigan comes back into the picture though :(
| ADD Kyuubi Naruto chapter 5 . 7/18/2011
you my good sir, are awesome thank you for this gem
| Karg42 chapter 5 . 7/17/2011
Someone watches the Hero's Bar and After Hours videos. Awesome. This is going to be good.
| xazavier009 chapter 5 . 7/13/2011
| Icezera chapter 3 . 6/27/2011
I noticed that you're writing style is currently paragraphs of exposition followed by straight dialogue in the latter half of each chapter. This isn't the best way to write. Most people can get easily bored with exposition and if the subject matter wasn't as interesting as it is, I'm sure they would simply skip the paragraphs.
So, try to turn parts of the first half of the story, the exposition part into exposition mixed with dialogue. The latter part is usually good with dialogue and exposition mixed well.
| Icezera chapter 1 . 6/27/2011
Very nice to see that this isn't a dead fic, and I can clearly see places where you revised. There are less grammatical errors, too, although there's still one place where for is used instead of from (IIRC).
Can't wait to keep reading your other chapters!
| Morality0duality chapter 5 . 6/14/2011
The is so funny i shattered my windows I beg of you continue.
| Zexs chapter 5 . 6/8/2011
God as soon as I saw the summary I went a cracked open a cold one because I knew this one was going to be duzzy, and you did not disapoint
| Bookish Cupcake chapter 1 . 5/23/2011
Loving it :D


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Look 100062 BK 13-Inch Fry Pan
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Frying Pan, 13-Inch or 32cm, non-stick. Featuring a unique insideoroutside non-stick surface, reinforced with titanium for superior non-stick performance and durability. This makes it ideal for low or fat-free cooking for a healthier lifestyle. PFOA free. All LOOK cookware is ultra-dense, making 100 percent warp-free. With extra heavy 3or8-Inch bases this ensures rapid and even heat distribution with no -Inchhot spots-Inch. The ergonomic handles remain cool on the stovetop and are guaranteed oven proof to 500F. Hand washing recommended.
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I had used LOOK cookware for decades. It was made in Iceland! For whatever reason that plant closed and I could not buy LOOK at all. They have since moved production to Europe. They have a few pieces on Amazon and I look forward to buying a new set.
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I purchased an 8" pan probably ~6 years ago at least (I think at a much more favorable price back then). It still works great for eggs, single size pancakes, etc. The coating has held up better than Calphalon Commercial purchased at a similar time. I don't put it in the dishwasher but everything wipes right out anyway.
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Achievement Trading Thread (Co-op Partners) - PC
Post here if your looking for poeple to play anarchy with on PC.
I am.
So am I, add me GT: JeftahX
I've been queueing for a game and even after 30 mins, no one pops up!
I would love to play this with people so just let me know if you'd like to! thanks!
It doesn't seem...
It doesn't seem like a lot of people still play this game. If you do and would like boost, send me a PM. UTC/GMT +2.
I am.
People who add me, please tell me you are from this thread.
ill be getting this soon in the next few days so ill be up for getting some played before on xbox
GT DarkFiendDoku
"the gamertag does not exist"
you sure you entered that right? Anyway, Add me for PC, looking to get the Anarchy cheevos out of the way. GT: Giptuc
sorry meant to be spaces Dark Fiend Doku
Desperately looking for a partner to get 50,000 points with as well as completing 200 team challenges. Message me on live. Thanks!
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text:
Kendrys Morales hit a pitch to center in the eighth inning last night that would be a base hit 99% of the time. But unfortunately for Morales, Mike Trout was playing center field which meant that last 1% was in play.
Mike Trout makes a no-look leaping catch, Kendrys Morales isn't impressed
Hunter Pence's f-bomb bonaza after the Giants clinched a playoff berth was caught on live TV
CSN Bay Area cameras were present as the San Francisco Giants celebrated a playoff berth with the usual playoff t-shirts, champagne, and googles to protect everyone's eyes from said champagne.
Tommy Lasorda: If you don't cheer for the Dodgers, you're going to Hell
Tommy Lasorda was feeling pretty good last night when the Dodgers beat the Giants 9-1 to clinch the NL West. Lasorda was feeling so good that he openly questioned how anyone could not be a Dodgers fans after watching that Dodgers win.
Derek Jeter may be on the hook for $16,000 in taxes on retirement gifts
As the Derek Jeter retirement tour winds down, Jeter will be walking away from baseball with a Hall of Fame résumé and $33,000 worth of gifts.
San Francisco Giants pitcher Sergio Romo and coach Shawon Dunston had a heated exchanged during batting practice
The San Francisco Giants are still battling for a playoff spot and the pressure might be starting to get to them. The Giants defeated the Los Angeles Dodgers yesterday in 13 innings 5-2 which leaves them 3 1/2 games behind the Dodgers. The team celebrated last nigh together but earlier that day, the mood was […]
Ron Washington breaks silence, says he was not true to his wife
Ron Washington held a press conference on Thursday to address his sudden resignation as manager of the Texas Rangers.
David Ortiz says he won't go on a retirement tour like Derek Jeter and Mariano Rivera
Boston Red Sox slugger David Ortiz is 38 years old and knows that retirement is right around the corner. Ortiz has played well this season, but also realizes that his body is starting to wear down. And with the Red Sox out of playoff contention, Ortiz is starting to discuss his future.
Rangers' comments add more mystery to Ron Washington's resignation
Texas Rangers manager Ron Washington resigned abruptly on Friday citing an off-the-field personal matter. Washington didn't give many details in his resignation statement other than making it clear that he wasn't walking away because of "the disappointing performance of the team this season."
The San Diego Padres pay homage to Bud Selig by creating a plaza in his name
As we all know, MLB commissioner Bud Selig will step down after this season. Even though his farewell tour hasn't been as epic as Derek Jeter's, some teams have shown their appreciation for Selig. None more so than the San Diego Padres.
John Rocker has agreed to join the cast of 'Survivor'
When you think of former Atlanta Braves pitcher John Rocker, the first word that comes to mind is "controversy". As we all know, Rocket made some disturbing comments in a Sports Illustrated article back in 1999 which still follows him to this day.


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Connect any celebrity with Jim Taylor to see how closely they are linked... romantically!


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Great sunscreen
Fantastic sunscreen offered myself and the family protection from getting burnt
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the best
very happy with this natural sunscreen with no nasties and no white ghost look. Little oily for the face so I just use a little under my day cream
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Great for the kids
We used this last year on all the kids all through summer in an out of the pool and no one got burnt! Great protection for the family.
Written by Simone on 19 September 2015


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Golden year for Palestine couple
June 29, 2015 EAST PALESTINE — Bill and Barb Webb of Pine Tree Circle, East Palestine, recently celebrated their golden wedding anniversary. more »»
Equizis celebrate 65th year
June 27, 2015 SALEM — George and Marcia Equizi, former residents of Salem, recently celebrated their 65th wedding anniversary in Sarasota, Florida. more »»
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June 1, 2015 HANOVERTON — Roy Harlan and Phyllis Ann Stacy, state Route 30, celebrated their 50th wedding anniversary on May 29. The couple met while growing up near Ary, Kentucky. Mr. more »»
Lisbon couple married 65 years
May 21, 2015 LISBON — Margaret and Ken Williams, Sunset Drive, are celebrating their 65th wedding anniversary May 21. They were married May 21, 1950, at First Presbyterian Church by the Rev. John G. Waeld. more »»
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April 3, 2015 EAST PALESTINE — Jerry and Helen Barnhouse, South Washington Street, will be celebrating their 50th wedding anniversary by renewing their vows at 9:30 a.m. more »»
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March 19, 2015 EAST LIVERPOOL — Jerry and Donna Wines, East Liverpool, celebrated their 60th wedding anniversary on March 10. more »»
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March 11, 2015 EAST ROCHESTER — Twylla and George "Jack" Hawk of East Rochester will observe their 60th wedding anniversary with an open house where all are welcome to attend from 2-6 p.m. more »»
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December 18, 2014 COLUMBIANA — Donald and Jacqueline Leonard, Columbiana, celebrated their 65th anniversary on Nov. 11 with a family celebration at the Dutch Haus. The former Jacqueline G. Snyder married Donald E. more »»
60th year for Salem couple
November 14, 2014 SALEM — Roberta and George Bell Jr. of Salem, will celebrate their 60th anniversary on Dec. 5. The former Roberta Honaker, daughter of the late Mr. and Mrs. more »»
Golden year for Leetonia couple
November 10, 2014 LEETONIA — Leonard and Margret DiLudovico Sr., Leetonia, celebrated their 50th anniversary on Oct. 3. The former Margret Stepanian married Leonard DiLudovico on Oct. 3, 1965 at St. more »»
60th year for Columbiana couple
October 30, 2014 COLUMBIANA — The Rev. Carl and Palma Blakeman Jr., Columbiana, celebrated their 60th anniversary with a vow renewal and dinner reception on Oct. 3. more »»
Golden year for Chester couple
October 30, 2014 CHESTER, W.Va. — Charley and Carol Butler, Chester, West Virginia, celebrated their 50th anniversary with a surprise wedding ceremony on Sept. 2. more »»


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Within 2–3 months of in vitro culture-expansion, mesenchymal stromal cells (MSC) undergo replicative senescence characterized by cell enlargement, loss of differentiation potential and ultimate growth arrest. In this study, we have analyzed DNA methylation changes upon long-term culture of MSC by using the HumanMethylation27 BeadChip microarray assessing 27 578 unique CpG sites. Furthermore, we have compared MSC from young and elderly donors. Overall, methylation patterns were maintained throughout both long-term culture and aging but highly significant differences were observed at specific CpG sites. Many of these differences were observed in homeobox genes and genes involved in cell differentiation. Methylation changes were verified by pyrosequencing after bisulfite conversion and compared to gene expression data. Notably, methylation changes in MSC were overlapping in long-term culture and aging in vivo. This supports the notion that replicative senescence and aging represent developmental processes that are regulated by specific epigenetic modifications.
Mesenchymal stromal cells (MSC) are precursors for mesodermal cell lineages such as osteocytes, chondrocytes, and adipocytes. They can be isolated from various tissues by plastic adherent growth under specific culture conditions. MSC are heterogeneous, and reliable markers for the definition of the multipotent subset of 'mesenchymal stem cells' remain to be elucidated (Horwitz et al., 2005; Dominici et al., 2006; Wagner & Ho, 2007). Nevertheless, the ease of isolation and the possibility of culture expansion to very high cell numbers as well as the absence of critical side effects of MSC in clinical trials raise hopes for a variety of therapeutic applications (Wagner & Ho, 2007).
Mesenchymal stromal cells have a restricted lifespan in vitro such as any other somatic cell and can therefore only be expanded for a limited number of cell divisions before entering a senescent state and unequivocally stopping proliferation. This is accompanied by enlargement and morphologic changes referred to as 'fried egg morphology'. Furthermore, many groups have shown that long-term culture of MSC impairs their differentiation potential (Banfi et al., 2000; Baxter et al., 2004; Bonab et al., 2006; Noer et al., 2007; Wagner et al., 2008). Therefore, the senescent state needs to be taken into account for quality control of MSC in cellular therapy.
The phenomenon of replicative senescence was first described in the 1960s by Leonard Hayflick (Hayflick, 1965) and since then a scientific discussion is ongoing whether the so-called 'Hayflick limit' resembles the aging process of the whole organism. We have recently demonstrated that replicative senescence induces reproducible gene expression changes in MSC (Wagner et al., 2008; Schallmoser et al., 2009). Interestingly, these changes closely resemble age-associated gene expression changes in adult stem and progenitor cells of young vs. elderly donors (Wagner et al., 2009). This overlap in differential gene expression indicates that long-term culture and aging might be regulated by similar mechanisms.
Two different basic principles are conceivable for cellular aging: (i) it might either be due to accumulation of damage that leads to cellular deterioration (Kirkwood & Austad, 2000) or (ii) it results from a cellular program that is regulated by a biological clock (de Magalhaes & Church, 2005; Prinzinger, 2005). Various molecular pathways have been shown to be relevant for the process of aging and senescence such as telomere shortening, DNA damage, accumulation of the cyclin-dependent kinase inhibitor p16INK4a and oxidative stress (Kiyono et al., 1998; O'Hare et al., 2001; Di Donna et al., 2003; Ho et al., 2005; Janzen et al., 2006). Furthermore, it has been proposed that epigenetic changes such as DNA methylation play a central role in senescence and aging (Wilson & Jones, 1983; Young & Smith, 2001; Nilsson et al., 2005; Chambers et al., 2007; Shibata et al., 2007; Suzuki et al., 2008).
DNA methylation is the best characterized epigenetic modification. CpG dinucleotides in the mammalian genomic DNA may be methylated at cytosine moieties. Upon replication the same methylation pattern is established on the newly synthesized DNA strand by DNA methyltransferase 1 (DNMT1). Thereby, epigenetic modifications are inherited to both daughter cells (Jaenisch & Bird, 2003). Methylation may impact gene transcription by direct interference with transcription factors or with methyl-CpG-binding proteins that modify histones and thereby inactivate the respective promoter region. Other authors have previously suggested that methylation levels gradually decrease upon long-term culture (Wilson & Jones, 1983; Nilsson et al., 2005). This study is based on the hypothesis that long-term culture and aging are associated with epigenetic modifications at specific CpG sites.
Mesenchymal stromal cells were isolated from bone marrow aspirates from young donors (21–50 years) or from bone marrow aspirates from elderly donors (53–85 years) upon hip fracture as previously described (Wagner et al., 2009). All cell preparations fulfilled the criteria of MSC such as typical growth morphology, immunophenotype and in vitro differentiation potential (Fig. S1) (Dominici et al., 2006; Wagner & Ho, 2007). Within 2–3 months of long-term culture the cells underwent replicative senescence accompanied by a decreased adipogenic differentiation potential and an increased propensity for osteogenic differentiation (Wagner et al., 2008) whereas no spontaneous differentiation was observed under nondifferentiation conditions even at later passages. The proliferation rate decreased gradually until the cells finally stopped to proliferate (Fig. S2A). These senecent passages were associated with expression of senescence-associated beta-galactosidase (SA-beta-gal) and increased p16 gene expression (Fig. S3).
To determine effects of long-term culture on DNA methylation patterns we harvested MSC of early passages (2nd passage; P2) and late passages (P8–P15) from eight donors. Methylation status at 27 578 CpG sites was simultaneously analyzed using the HumanMethylation27 BeadChip microarray (Illumina). Scatter plot analysis of methylation in early vs. late passages revealed that DNA methylation remains overall constant upon MSC culture expansion (Fig. 1A). However, several CpGs showed a highly significant degree of differential methylation between early and late passage in all eight MSC samples. Only differential methylation of more than 20% was considered to be relevant and these CpGs were considered for further analysis. Twenty-nine CpGs were hypermethylated upon long-term culture whereas 55 CpGs were hypomethylated (Fig. 1C; Table S1). Hypermethylated CpGs were distributed in the promoter regions and within 23 nonredundant and functionally characterized genes including distal-less homeobox 5 (DLX5), runt-related transcription factor 3 (RUNX3), and chromosome 10 open reading frame 27 (C10orf27). Hypomethylated CpGs were localized in the promoter regions and within 43 protein-coding genes including cycline-dependent kinase inbibitor 2B (CDKN2B), bone morphogenic proteins 10 and 15 (BMP10/15), left-right determination (LEFTY1), and distinct CpG sites within the RUNX3 promoter. The function of all differentially methylated genes was further classified by Gene Ontology analysis. Hypermethylated genes were significantly over-represented in the functional categories cell-matrix adhesion, embryonic morphogenesis and development whereas hypomethylated genes were rather associated with epidermal differentiation (Table S2). There was only a moderate enrichment of methylation changes in specific chromosome regions: Three of the hypermethylated genes were located at chromosome band 10q22 (P = 0.0003) whereas four of the hypomethylated genes were enriched at chr21q22 (P = 0.0012). Overall, our results inevitably demonstrate that long-term culture induces reproducible and significant methylation changes at specific CpG sites and especially seem to affect genes involved in development.
To further elucidate methylation changes in MSC upon aging, we next analyzed methylation profiles in MSC that were taken from the second passage of young or elderly donors, respectively. Several CpG sites revealed age-associated methylation changes (Fig. 1B). Two hundred and ninety-five CpGs were hypermethylated, whereas 349 CpGs were hypomethylated upon aging (Fig. 1D; Table S3). Genes associated with CpGs that were hypermethylated upon aging included HOXA2, HOXA5, HOXA6, HOXB2, DLX5, aristaless-like homeobox 4 (ALX4), T-box 5 isoform 2 (TBX5), RUNX2, RUNX3, O-6-methylguanin-DNA methyltransferase (MGMT) and C10orf27. Genes associated with CpGs that were hypomethylated upon aging included HOXB1, HOXB3, HOXB4, HOXB6, HOXD3, HOXD12, paired-like homeodomain transcription factors PITX1 and PITX2, sirtuin 6, BMP4, mesoderm specific transcript (MEST), paired box genes PAX1, PAX8, PAX9, TBX1, S100A4, and also RUNX3 and ALX4. Differentially methylated genes were further classified by Gene Ontology analysis. Hypermethylated genes were significantly over-represented in various signal transduction pathways and limb morphogenesis. In contrast to this, hypomethylated genes were most significantly over-represented in genes involved in sequence-specific DNA binding and developmental processes (Table S4). Furthermore, there was an enrichment of hypermethylated genes at the chromosome regions chr21q22 (9 genes; P = 0.0006), chr2p25 (5 genes; P = 0.0011), chr6q27 (4 genes; P = 0.0013), and chr12q24 (7 genes; P = 0.0073). Hypomethylated genes upon aging were enriched at chr20q13 (9 genes; P = 0.0032). Interestingly, methylation changes were highly enriched in genes with simian-virus-40-protein 1 (SP1) binding sites (hypermethylation: 15 of 258 genes, P = 7.8 × 10−11 and hypomethylation: 31 of 273 genes, P = 6.0 × 10−5).
Differential methylation of six selected CpG sites (DLX5, HOXA5, S100A4, C10orf27, RUNX3, and CDKN2B) was validated by pyrosequencing after bisulfite conversion. Candidate genes were selected based on the correlation between gene expression and CpG island methylation as well as their attributed biological function. Methylation levels were overall slightly lower in pyrosequencing results as compared with microarray analysis. However, differential methylation upon long-term culture or upon aging was very consistent between the two methods (Fig. 2A,B). Moreover, differential methylation was also verified in three additional independent donor samples (Fig. S4A). Comparative analysis of different passages revealed that methylation changes are continuously acquired with every passage upon long-term culture (Fig. S4B). Notably, also CpGs in close neighborhood to the CpG represented on the microarray revealed very similar methylation patterns (Fig. 2C,D; Fig. S4C,D). This demonstrates that methylation changes are not restricted to individual CpG sites but rather affect whole CpG islands.
We have previously shown that long-term culture and aging have similar effects on the gene expression profile of MSC (Wagner et al., 2008, 2009). Therefore, we hypothesized that these two processes might also reveal an association in DNA methylation changes. Only 13 CpGs were more than 20% differentially methylated in both comparisons and therefore we have chosen a less stringent cutoff of 15% differential methylation resulting in 80 CpGs (Fig. 3). These demonstrated a correlation in long-term culture and aging (R = 0.61) and chi-square analysis revealed a statistical significance of 4.6 × 10−9. Hence, it might be speculated that senescence-associated changes are more pronounced in samples from younger donors. Indeed, the number of more than 20% differentially methylated CpG sites upon long-term culture was 24% higher in younger vs. elderly donors (248 CpG sites vs. 189 CpG sites). These data indicate that long-term culture and aging resemble two processes with related epigenetic modifications.
DNA methylation profiles were integrated with gene expression data to determine the impact of epigenetic modifications on mRNA expression. For each CpG on the HumanMethylation27 BeadChip the corresponding gene was matched to the Affymetrix mRNA gene expression data by gene symbols (Wagner et al., 2009). Signal intensity in gene expression data was used as an indicator for gene expression levels. As expected, nonmethylated genes revealed higher gene expression levels than methylated genes (Fig. 4A). Subsequently, we tested if differential methylation was also reflected in differential gene expression. Overall the two microarray platforms did not reveal a clear correlation between differential CpG methylation and differential mRNA expression in long-term culture or aging (Fig. 4B,C). This might be due to the different sensitivity of the two techniques: 20% differential methylation can be detected, whereas 20% differential gene expression can not be reliably discerned. We have selectively addressed gene expression changes in genes that have been analyzed by pyrosequencing and that revealed higher methylation changes. mRNA expression of these genes (DLX5, HOXA5, CDKN2B, S100A4, RUNX3, and C10orf27) was analyzed by quantitative RT-PCR. For these selected genes hypermethylation coincided with lower gene expression and vice versa (Fig. 4D,E).
Epigenetic modifications play a central role in differentiation and development. This study indicates that modification of DNA methylation is also involved in replicative senescence upon long-term culture of MSC. Furthermore, a similar pattern of differential methylation was observed between MSC from young and elderly donors. These results support the perception that replicative senescence and aging are not only due to stochastic accumulation of cellular defects but rather represent a developmental program. On the other hand, the process is overlaid by stress-induced senescence changes e.g. due to high oxygen content or the artificial environment. Furthermore, the cellular aging of MSC might be accompanied by a different composition of heterogeneous subpopulations and loss of self-renewal potential under in vitro conditions. Hence, senescence of MSC has many facets that need to be further elucidated (Wagner et al., review submitted).
Methylation was analyzed by using the novel HumanMethylation27 BeadChip representing 27 578 CpGs that are associated with promoter regions of more than 13 500 annotated genes. This array was designed to cover CpG dinucleotides in the promoter region of genes and therefore is clearly biased toward the assessment of gene-specific methylation. Differential CpG methylation was highly reproducible in different donor samples and was verified by pyrosequencing for all sequences tested. Thus, this technique facilitates reliable profiling of global methylation patterns.
Other authors have reported that global DNA methylation levels decrease upon long-term culture (Wilson & Jones, 1983; Young & Smith, 2001). Therefore, it has been speculated that maintenance of DNA methylation by DNA methyltransferase 1 (DNMT1) is incomplete resulting in a gradual decrease of DNA methylation with repeated cell cycles (Nilsson et al., 2005). However, our results did not reveal an overall decrease in DNA methylation levels. In fact, methylation remained constant over the whole culture period at most CpG sites represented on the BeadChip. To our knowledge this study demonstrates for the first time that only specific CpG islands become differentially methylated upon long-term culture. DNA methylation changes upon aging may also depend on individual differences although differential methylation was verified by pyrosequencing in independent donor samples. It is also likely, that age-related changes are influenced by the different cellular composition upon aging as well as by the different tissue and isolation technique.
Methylation changes in our study were often associated with homeobox genes. Expression of these genes is tightly regulated under temporal control during vertebrate development. It has been shown that chromatin modifications play an important role for regulation of the 'Hox clock' that coordinates body patterning (Illingworth et al., 2008; Soshnikova & Duboule, 2009). Furthermore, age-associated gene expression changes have been described in homeobox genes (Stelnicki et al., 1998; Wagner et al., 2009) and it is conceivable that they are involved in the timing of aging. Exemplarily, we have verified differential methylation for HOXA5 and DLX5 upon long-term culture and aging by pyrosequencing. The homeobox gene DLX5 is also involved in differentiation toward the osteoblastic lineage (Lee et al., 2005). Therefore, hypermethylation and decreased gene expression of DLX5 might be relevant for the impaired differentiation capacity of MSC upon long-term culture. It has been shown that methylation changes at specific CpG sites can affect differentiation of MSC (Noer et al., 2007; Zimmermann et al., 2008). In addition, age-related methylation changes have been described. Several tumor suppressor genes are hypermethylated upon aging with potential implications for cancer susceptibility in the elderly (So et al., 2006). Among these was RUNX3 that revealed age-associated changes in our analysis. Another differentially methylated gene was the cycline-dependent kinase inbibitor 2B (CDKN2B) that has been implicated in senescence in our previous work (Schallmoser et al., 2009).
Several genes such as RUNX3 and ALX4 were more than 20% hypermethylated at specific CpG sites and more than 20% hypomethylated in others. These antagonistic modifications within the same promoter region indicate that DNA modifications are restricted to relatively short and specific sequences. On the other hand, pyrosequencing revealed that methylation changes are not restricted to unique CpG sites but also include neighboring cytidines of the same CpG island. It has been suggested that senescence can also be induced in immortal cells by treatment with 5aza-2deoxycytidine (5AZA) (Vogt et al., 1998). However, it is difficult to discern toxic effects of 5AZA. The complex pattern of hypermethylation and hypomethylation observed in this study might explain why replicative senescence cannot be controlled by demethylating agents.
De novo methylation is facilitated by DNA methyltransferases DNMT3A and DNMT3B but it is mostly unknown how the extent of de novo methylation is regulated and where it takes place. Active DNA hypomethylation is also considered to occur but a specific enzyme that removes the methyl group from 5-methylcytosine has not yet been described. It has been suggested that DNA methyltransferases exhibit dual actions in CpG methylation and demethylation of 5mCpGs through deamination (Kangaspeska et al., 2008; Metivier et al., 2008). DNA methylation patterns may vary drastically within CpG islands and only methylation of a defined core-region silences gene expression (Gonzalgo et al., 1998; Deng et al., 1999; Ushijima & Okochi-Takada, 2005). We have shown that age-associated hypermethylated and hypomethylated genes are enriched in SP1 transcription factor binding sites and it has been proposed that binding of the SP1 transcription factor protects CpG islands from methylation (Brandeis et al., 1994; Macleod et al., 1994). 'Seeds of methylation' is another interesting mechanism that leads to increasing methylation of CpG islands. This hypothesis is based on the observation that little methylation is induced in an initially nonmethylated region whereas a high degree of methylation is induced in a sparsely methylated region (Song et al., 2002; Stirzaker et al., 2004). This mechanism might serve as a cell-cycle counter.
It is commonly accepted that DNA methylation silences gene expression. This is also reflected by the correlation of the two different levels investigated in our study: nonmethylated genes revealed higher signal intensity in mRNA expression profiling. However, differential DNA methylation at specific CpG sites does not necessarily correlate with observed gene expression changes. Relevant fold-changes in gene expression can only be expected if the CpG sites are predominantly methylated. It is also conceivable that selective modification of specific promoter regions either activates or inactivates binding of transcription regulators.
We have previously described age-related gene expression changes in human stem and progenitor cells and observed a moderate but significant concordance in the expression profiles upon aging in vivo and replicative senescence in vitro (Wagner et al., 2009). However, the proliferation rate was not higher in MSC from younger donors and they did not reach a higher number of cumulative population doublings. This might be attributed to the relatively low number of samples per group as other authors reported an inverse relationship between donor age and the replicative life span in vitro for fibroblasts or MSC (Schneider & Mitsui, 1976; Stenderup et al., 2003; Mareschi et al., 2006; Shibata et al., 2007). Also the doubling time of either freshly isolated marrow cells or MSC of early passage has been described to be significantly lower in samples from elderly people (Zhou et al., 2008) whereas this was not observed in our study. Analysis of age-specific changes might be affected by the different sources for MSC that were available for the two age groups. MSC were either isolated from human bone marrow from the iliac crest or from the femoral head. There were no differences in growth, cell morphology, immunophenotype or differentiation potential (Wagner et al., 2009). Thus, MSC from BM and HIP fulfilled the criteria that are commonly used for definition of MSC although these cell preparations are heterogeneous and may comprise different subpopulations (Dominici et al., 2006; Wagner & Ho, 2007). However, we can not exclude that some of the methylation changes are due to the different bone marrow regions. Notably, however, there was a relevant overlap of methylation changes upon long-term culture and aging of MSC. Furthermore, there is a significant concordance of our age-related changes in MSC and age-associated methylation changes in ovarian cancer samples (Teschendorff et al., manuscript in preparation). Further integrative analysis of methylation profiles of different cell types and tissues will help to discern the existence of a distinct methylation signature for aging.
Bone marrow is a complex tissue with various different cell types. Hence, it is apparent that aging and replicative senescence are not identical despite molecular similarities of the two processes. Long-term culture has been shown to impair differentiation potential of MSC, especially of adipogenic differentiation (Baxter et al., 2004; Bonab et al., 2006; Wagner et al., 2008). By contrast, other authors demonstrated that aging activates adipogenic differentiation and suppresses osteogenic differentiation (Moerman et al., 2004; Zhou et al., 2008). These authors also demonstrated that expression of the master transcription factor for osteoblast differentiation RUNX2 and DLX5 decreased upon aging and this is in line with higher methylation as observed in our data. It is conceivable, that the higher propensity for adipogenesis in BM of elderly people results in a lower number of osteogenic progenitor cells.
Aging is not an inevitable fate of all cells. Our germ line cells and their progeny are sustained through the generations and embryonic stem cells as well as induced pluripotent stem cells do not show signs of replicative senescence upon long-term culture (Zeng, 2007). It is therefore conceivable, that the loss of pluripotency is directly accompanied with limitation of the replicative live span (Marciniak-Czochra et al., 2009). Both mechanisms might be regulated by similar epigenetic modifications that govern differentiation and development. Our results demonstrate that specific CpG sites become either methylated or demethylated upon long-term culture. Analysis of these specific CpG sites might facilitate monitoring of the replicative lifespan of cells. It will be of central interest to further increase our understanding of the mechanisms regulating senescence-associated and age-associated epigenetic modifications.
Mesenchymal stromal cells were isolated from human bone marrow that was either taken from aspirates from healthy donors for allogeneic transplantation (BM) or from the caput femoris from elderly patients undergoing femoral head prosthesis (HIP) as described in our previous work (Wagner et al., 2009). All samples were taken after written consent using guidelines approved by the Ethic Committee on the Use of Human Subjects at the University of Heidelberg. The standardized culture conditions have been described in detail before (Reyes et al., 2001; Wagner et al., 2005).
Mesenchymal stromal cells were cultured for 2–3 month by serial passaging until the cells reached a senescent state and stopped proliferation. Culture conditions and sampling have been described in our previous work (Wagner et al., 2008, 2009). In brief, 7–10 days after the initial seeding of MNC the colonies were trypsinized and re-plated in a new culture flask (passage 1, P1). Upon sub-confluent growth (70%), cells were re-plated at a density of 104cells cm−2. Cells were counted at every passage by a Neubauer counting chamber and the cumulative population doublings were calculated (Cristofalo et al., 1998). As cell numbers were first determined at P1, the cumulative doubling number was first calculated for P2. From P2 onward, there were enough cells for simultaneous expansion of one fraction and harvesting another fraction for subsequent analyses: 106 cells were lysed in TRIzol for RNA isolation, 106 cells were cryopreserved for immunophenotyping and in vitro differentiation assays (Wagner et al., 2008, 2009) and 106 cells were pelleted and stored by −80 °C for subsequent DNA isolation.
Immunophenotypic characterization of MSC preparations was performed on a three-color FACScan (Becton Dickinson [BD], San Jose, CA, USA) with a five-color upgrade (Cytek Dev. Inc., Fremont, CA, USA) using the commonly used panel of antibodies as described before (Wagner et al., 2009): CD13-allophycocyanin (APC, clone WM15, BD), CD29-fluorescein isothiocyanate (FITC, MEM-101a, abcam, Cambridge, UK), CD31-FITC (BD), CD34-phycoerythrin (PE, 8G12, BD), CD44-PE (g44-26, BD), CD45-FITC (2D1, BD), CD73-PE (AD2, BD), CD90-PE (G7, BD), CD105-PE (MHCD10504, BD), CD146-PE (P1H12, BD), CD166-PE (3A6, BD), CD184-PE (12G5, BD). Osteogenic and adipogenic differentiation of MSC was induced as previously described (Wagner et al., 2009). After 21 days osteogenic differentiation was analyzed by Alizarin red staining and adipogenic differentiation by Oil Red-O staining.
Expression of pH-dependent senescence-associated beta-galactosidase (SA-beta-gal) activity was analyzed simultaneously in different passages of MSC of young and elderly donors using the SA-beta-gal staining kit (Cell Signaling Technology, Boston, MA, USA).
Genomic DNA of 106 MSC was isolated using the QIAGEN DNA Blood Midi-Kit. DNA quality was assessed with a NanoDrop ND-1000 spectrometer (NanoDrop Technologies, Wilmigton, DE, USA) and average fragment length was assessed by gel electrophoresis. Genomic DNA (500 ng) from each sample was bisulfite converted using the EZ-96 DNA Methylation Kit (Zymo research Corporation, Orange, CA, USA). This step leads to the deamination of nonmethylated cytosines to uracils, while methylated cytosines are refractory to the effects of bisulfite and remain cytosine. After bisulfate conversion each sample was whole genome amplified and enzymatically fragmented.
The HumanMethylation27 BeadChip (Illumina, San Diego, CA, USA) uses Infinium technology, previously described for SNP genotyping (Steemers et al., 2006), to perform genome-wide screening of DNA methylation patterns. In detail, the HumanMethylation27 panel targets 27 578 unique CpG sites located within the proximal promoter regions of transcription start sites of 14 475 consensus coding sequencing (CCDS) in the NCBI Database (Genome Build 36). In addition, 254 assays cover 110 miRNA promoters. On average, two assays were selected per CCDS gene and from 3 to 20 CpG sites for >200 cancer-related and imprinted genes. Further specifications of the platform can be found at http://www.illumina.com/products/infinium_humanmethylation27_beadchip_kits.ilmn. About 200 ng DNA was applied per BeadChip according to the manufacturer's instructions. During hybridization, the DNA molecules anneal to two different bead types with locus-specific DNA oligomers – one corresponds to the methylated (C) and the other to the unmethylated (T) state. Allele-specific primer annealing is followed by single-base extension using DNP- and Biotin-labeled ddNTPs. After extension, the array is fluorescently stained, scanned, and the intensities of the unmethylated and methylated bead types measured. Initial data analysis was performed with the BeadStudio Methylation Module. DNA methylation values, described as beta values, are recorded for each locus in each sample. Only few CpG sites did not reveal reliable signals, as determined by their detection P-value, and were therefore excluded from further analysis. Raw data were quantile normalized. Statistical analysis and clustering by Euclidian distance was performed using the MultiExperiment Viewer (MeV, TM4) (Saeed et al., 2003). For further analysis we have selected CpG sites with more than 20% differential methylation. These were further analyzed to calculate mean methylation, differential methylation and student's t-test. Furthermore, genes associated to the differentially methylated CpG sites were categorized by Gene Ontology analysis using GoMiner software (http://discover.nci.nih.gov/gominer/). Chromosomal distribution and transcription factor binding was assessed by Gene Set Enrichment Analysis (Subramanian et al., 2005). The complete CpG methylation values have been deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE17448.
Methylation changes of six selected CpG sites were validated by pyrosequencing. Independent bisulfite treatment and pyrosequencing assays were performed by Varionostic GmbH (Ulm, Germany). In brief, bisulfite converted DNA was subjected to PCR amplification. Primers are provided in Table S5. 40–45 μL of PCR product was immobilized to 3 μL Streptavidin SepharoseTM HP beads (GE Healthcare, Piscataway, NJ, USA) followed by annealing to 2 μL sequencing primer (5 μM) for 2′ at 80 °C. CpG analysis was done with Pyro Q-CpG software, and differential methylation was calculated as described above.
In this study we have combined DNA methylation data with gene expression profiles of our previous work (Wagner et al., 2008, 2009). Gene expression profiles have been analyzed by the U133_Plus_2.0 Affymetrix chip and two different data sets were used for this comparison.
Gene expression data was matched to HumanMethylation27 BeadChip by gene symbols (HUGO names). For comparative analysis we have only considered probe sets that were detected as present in more than 50% of the hybridizations in gene expression analysis.
Quantification of mRNA expression for candidate genes was performed by real-time quantitative PCR (QRT-PCR) using the ABI PRISM® 7700HT Sequence Detection System Instrument (Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany). Total RNA was isolated and reverse transcribed as described in our previous work (Wagner et al., 2009). Primers were obtained from Biospring (Frankfurt, Germany) (Table S5). QRT-PCR reactions were performed with the power SYBR® green PCR master mix in a MicroAmp optical 96-well reaction plate with a ABI PRISM® 7700HT sequence detector (Applied Biosystems) according to the manufacturer's instructions. Gene expression levels were normalized to GAPDH expression, which was used as a housekeeping gene.
We want to thank Anke Diehlmann (Department of Medicine V, Heidelberg, Germany) for assistance in MSC expansion, Juliane Rieber (DKFZ, Heidelberg, Germany) for help with DNA isolation, Roger Fischer (DKFZ, Heidelberg, Germany) for excellent technical assistance in bisulfite treatment and array analysis, and Dr. Uwe Gerstenmaier (Varionostic GmbH, Ulm, Germany) for support on pyrosequencing. This work was supported by the German Ministry of Education and Research (BMBF) within the supporting program 'cell based regenerative medicine' (START-MSC and CB-HERMES), the German Research Foundation DFG (HO 914/7-1), the Network for Aging Research (NAR, Heidelberg), the Heidelberg Academy of Sciences (WIN-Kolleg) and the Stem Cell Network North Rhine Westphalia.
Additional supporting information may be found in the online version of this article:
Fig. S1 Immunophenotype and in vitro differentiation of mesenchymal stromal cells.
Fig. S2 Long-term culture of mesenchymal stromal cells.
Fig. S3 β-galactosidase staining and expression of p16 in early and late mesenchymal stromal cell passages.
Fig. S4 Methylation changes in independent donor samples and during long-term culture.
Table S1 Differentially methylated CpGs upon long-term culture.
Table S2 Gene ontology analysis of methylation changes in long-term culture.
Table S3 Differentially methylated CpGs upon aging.
Table S4 Gene ontology analysis of methylation changes upon aging.
Table S5 Primer sequences.
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Cargill Adds Second Dairy Farm Methane-to-Power Digester
An anaerobic digester on an Idaho dairy farm is helping convert manure from 6,000 cattle into about a million kilowatt hours of electricity every month, according to Environmental Protection.
This is Cargill's second such plant. The first has been operating since 2008 at another area dairy plant. Both of the dairies are operated in Idaho by Bettencourt Dairy.
The digester works by putting the manure into a massive, sealed, in-ground vessel that is free from oxygen. Bacteria in the mix anaerobically break down the manure into methane gas, which is captured and burned in generators.
Cargill sells the electricity to the grid, according to FinChannel.
Last year, Cargill registered and sold 400,000 tons of emission offsets from an Alberta beef operation through the Canadian Standards Association.
Those offsets come from a wastewater treatment system at Cargill's plant in High River, Alberta, which capture methane from the plant's wastewater lagoons for fuel. As a result, the system uses 25 percent less natural gas.
Similarly, Cargill plans to generate and sell emissions offsets from its Idaho dairy methane plants.
The first Idaho dairy plant is expected to produce about 1.3 million kWh of electricity a month, and Cargill is trying to sell 28,000 tons of emission offsets related to that project.
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Liberty Hill is located in Texas.
Liberty Hill, Texas has a population of
Liberty Hill is
family-centric than the surrounding county with
53.93% of the households
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The county average for households married with children is 42.4%.
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The median household income for the surrounding county is $70,849
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The median age of people living in Liberty Hill is
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The average high temperature in July is 96
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The average rainfall is approximately 33.7 inches
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Radiophysics and Quantum Electronics
Radiophysics and Quantum Electronics reports on topics such as: radio astronomy; plasma astrophysics; ionospheric, atmospheric and oceanic physics; radiowave propagation; quantum radiophysics; physics of oscillations and waves; physics of plasmas; statistical radiophysics; electrodynamics; vacuum and plasma electronics; acoustics; and solid-state electronics.
Radiophysics and Quantum Electronics is a translation of the peer-reviewed Russian journal Izvestiya VUZ. Radiofizika, published by the Radiophysical Research Institute and N.I. Lobachevsky State University at Nizhnii Novgorod, Russia.
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