text:
We regularly get together at noon
on the third Saturday or Sunday of the month.
Join us if you're in town!
Urban Sketchers Chicago Facebook: https://www.facebook.com/groups/UrbanSketchersChicago/
Twitter: @USk_Chicago
Instagram: @USKCHICAGO
Pinterest: Urban Sketchers Chicago
Hashtags: #uskchicago #urbansketchers #usk
Instagram: @USKCHICAGO
Pinterest: Urban Sketchers Chicago
Hashtags: #uskchicago #urbansketchers #usk
Chicago Athletic Association Hotel
Saturday April 21, 12 p.m. - 3 p.m.
|Sketch by: Brian Wright
Chicago Athletic Association HotelDetails:
Dating back to 1893 and the Chicago World's Fair, this restored architectural gem is a treasure on Michigan Avenue. Designed by Architect Henry Ives Cobb, this Venetian-Gothic landmark originally served as an elite men's club reserved for the privileged, and remained so for 122 years. Re-opened as a boutique hotel in 2015, doors are now open to welcome all at the downtown destination and historic monument. Learn more about this living, breathing ode to Chicago's rich history. https://www.chicagoathletichotel.com/about
Great News! Lisa Wronski, an architect and member, has agreed to do a short Perspective demo on how to tackle Skyline building and the 8th story view. This is voluntary participation if you would like to learn more about Perspective. She is a fantastic artist and has many Skyline sketches on her Instagram account - check out her work @lisawronski
This event is free and open to the public. Attendees must bring all personal sketching materials- no art materials will be provided. Some chairs will be available with Sketching space first come , first served. Upon arrival , please take the elevator to the 8th floor and meet at 12pm in the White City Ballroom.
Public Transportation: CTA bus #146- Michigan ave bus
CTA lines :closest stations are Adams/Wabash, Monroe/Red line , Randolph /Wabash and Monroe/Blue (warning - none of these stations are handicap accessible)
Milennium Station on the Metric and South Shore lines is a short walk
Buses from Ogilvie train station - please consult map
If driving - try SPOTHERO or PARKWHIZ app to find the best parking deals in the area
VALET PARKING is available at the hotel's 71 E Madison entrance , hourly rates as follows:
0-4 with restaurant validation $18
0-4 without restaurant $28
4-8 hrs $38
Questions: Please email email@example.com
Saturday, Feb. 17, 12 p.m. - 3 p.m.
Union Station
225 S Canal St, Chicago 60661
Originally designed by famed architect Daniel Burnham ("make no small plans, they have no magic to stir men's blood") and completed in 1925 by the Graham, Anderson, Probst and White Firm, the Great Hall is considered to be one of the greatest indoor spaces in the United States.
The rich history and beauty of the Great Hall has made it a popular location for feature films including The Untouchables, My Best Friend's Wedding, Flags of Our Fathers and many others. Within the Great Hall lies the newly-restored Burlington Room. This room was originally constructed as the Women's Lounge where ladies traveling could wait for the train and enjoy the services of the adjacent beauty parlor and other facilities. Now it is a flexible event space that will charm any guest. With the perfect touch of history and elegance it is ideal for gatherings of any kind.
Urban Sketchers Chicago has met here many times and continue to be impressed by its presentation of architecture and variety of visitors. It has all of the features we look for in an urban setting and most especially warmth and easy access.
For questions: If you have any questions or if you are having trouble locating the group on the day of the event, call your host Wes Douglas, wed317@gmail or 630-776-2393 mobile.
Architectural Artifacts, Inc.
Navy Pier600 E Grand Ave, Chicago
INITIAL LOCATION: We'll meet in front of the main entrance. If it is nice weather we'll meet outside (shown in the sketch) and if the weather is cold or rainy, let's meet inside, downstairs in front of the Crystal Garden.
PARKING: Parking available at Navy Pier
If you have any questions or if you are having trouble locating the group on the day of the event, call your host Wes Douglas, 630-776-2393
4325 North Ravenswood Avenue
Chicago, IL 60613
HAPPY 5th ANNIVERSARY TO URBAN SKETCHERS OF CHICAGO!
Host: are Alex Zonis and Mary Jo Ernst (847-431-3422)
Sunday March 19, 2017, 12:00pm – 3:00pm
Fermi National Accelerator Laboratory
Garfield Park Conservatory
300 N Central Park Ave.
Chicago, IL 60624
INITIAL LOCATION: We'll meet at the front entrance at noon.
PUBLIC TRANSPORTATION: Easily accessible by the Green line L. Look for the massive greenhouse building just North of Lake St. on the West side of the street.
PARKING: Free parking in the Visitor's Parking Lot just South of the Conservatory entrance.
Garfield Park Conservatory's wesite:
Host: If needed you can text or call Paul at 773-819-0566.
Saturday, October 29, 11:30 a.m. - 3 p.m.
Sketching in the Loop, Dick Blick's
Saturday, October 22, 2 p.m. - 7 p.m.
Halloween Parade, Chicago Cultural Mile
Exciting and busy day at the Halloween Gathering sponsored by the Chicago Cultural Mile!
Sunday, September 18, 12 p.m. - 3 p.m.
Saturday, August, 12 p.m. - 3 p.m.
Unity Church
Host: Alex Zonis and Daniel Young, 773.203.0380
Saturday, June 18, 12 p.m. - 3 p.m.
Jackson Park Yacht Club
Jackson Park Yacht Club is a private club, so we will sketch on the docks, park, and areas near the building, but not inside.
Situated in South Shore, along the southern shores of Lake Michigan in Chicago, Jackson Park Yacht Club is one of the oldest yacht clubs in Chicago. Founded in 1896, shortly after the Chicago World's Fair of 1893, the club was built in the outer harbor across from the old U.S. Coast Guard Station. Primarily, a habor for sailboats, there are powerboats that are harbored here as well. http://www.jacksonparkyachtclub.org/
HOST: Joel Berman
PARKING: There is plenty of free parking to the west and north of the the yacht club, in addition to spaces near La Rabida Childrens' Hospital.
ACCESS BY BICYCLE: Take the Lakefront path south to the end, http://www.cityofchicago.org/cityinfo/cdot/bikemap/usemap/19-R.html
PUBLIC TRANSPORTATION: Take the Red Line to 69th Street, then take the 67 bus east bound. At the end of the line, walk to Promontory Drive.
CELL: On June 18, this phone number will forward to the host's cell: 773.275.5968 x103
Sunday, March, 20, 12 p.m. - 3 p.m.
Architectural Artifacts, Inc.
Architectural Artifacts is a sprawling multilevel warehouse of upscale architectural salvage & furnishings curated from around the world. From planes and cars to crystal chandeliers to typesetting plates and everything else that you cannot even imagine. If you have not been there before, prepare to be amazed!
4325 North Ravenswood Avenue
Chicago, IL 60613
PARKING: There is a lot of parking available on Ravenswood Ave.
PUBLIC TRANSPORT: Montrose station on CTA Brown line is the closest.
We will meet right inside the glass entrance in Architectural Artifacts at 12 noon and sketch until 3 p.m. We will gather again at 3 p.m. for a group photo and show of sketches.
AFTER SKETCHING: Snacks, drinks, and community -
O'Shaughnessy's Pub
4557 North Ravenswood Avenue
Chicago, IL 60640
Saturday, February, 20, 12 p.m. - 3 p.m.
The Field Museum
HOSTS: Joan Stachnik and Rebecca Banasiak
INITIAL LOCATION: We will meet at 12noon by Sue, the T. rex in Stanley Field Hall at the Field Museum of Natural History (1400 S Lake Shore Drive; www.fieldmuseum.org).
We will re-group at 2:45 pm for a group photo and to share sketches.
General admission to the museum is free that day for Illinois residents, so be sure to have an ID for free admission.
SPECIAL FEATURE for USk Chicago: For those interested, Rebecca Banasiak, our member, artist and Field Museum researcher, has kindly offered to arrange for a tour of the lower level research area. IF YOU ARE INTERESTED IN THE TOUR, PLEASE RESPOND TO THE EVENT NO LATER THAN SUNDAY FEBRUARY 7th to provide Rebecca with enough time to organize the tour. The tour should last about 20 to 30 minutes. The meeting time for the tour (or tours if needed) will be announced at the initial meeting time.
PARKING: Parking is available in the Solder Field North Garage, the East Museum Lot (this tends to fill up early in the day), and the lot adjacent to the Adler Planetarium. Rates range from $19 to $22.
PUBLIC TRANSPORTATION: The #146 bus—Inner Drive/Michigan Express/Museum Campus—stops directly in front of the museum. If arriving via the L, exit at the Roosevelt Red Line Stop – servicing Red, Orange and Green Lines. Connection to the #146 bus is located at State Street and Roosevelt Road.
HOSTS: Joan Stachnik 773-213-0006 (If you are running late or need additional info, please call or text)
Rebecca Banasiak
Alex Zonis 773 203 0380
Drawing of Sue on the banner is (c) Dave Wittekind
Saturday, November 14, 12 p.m. - 3 p.m.
Rockefeller Memorial Chapel
Host: Allana Coxwell Pierce
INITIAL LOCATION: We met at the main entrance of Rockefeller Memorial Chapel (5850 S Woodlawn Ave, Chicago, IL 60637). There are also several nice outdoor sketching areas nearby.
SKETCH NOTES: All sketch materials are welcome in this space.
Sunday, October 18, 12 p.m. - 3 p.m.
Chicago Architecture Foundation (CAF) Open House
Host: Wes Douglas
- McCormick Bridgehouse,
- The Oriental Theater and
- The Rookery.


text:
GoDaddy Rs. 99 Domain Name – 86% OFF [Working]
GoDaddy offers best discounts on Domain, Web Hosting, Virtual Private Servers, Bulk Domain Registrations, SSL Certificates, Email Marketing and more.
Why GoDaddy?
- World's largest domain name registrar.
- They have 13+ million customers.
- 14 facilities, including Ariz, Calif, Asia, Europe, India & more.
- More than 4,500 employees working hard.
- Manages more than 61 million Domains.
- 24/7/365, round-the-clock customer care support.
We have published 16 working GoDaddy Coupon Codes to SAVE your money on Domain and Hosting purchases.
Godaddy Domain Coupon Codes:
cjc99r – IN Rupees - Rs 99 .COM Domain! (Revised Price - Rs. 125)
cjc99com – .COM Domains at the price $0.99
cjcclub – Get 1 year .CLUB domain for $14.99 and get the 2 year for FREE!
cjcdom001t – Bulk Domain Registrations from GoDaddy!
cjcmsc002t – Backorder Domains from GoDaddy.com
cjctdnamt1 – GoDaddy Auctions: Find, buy & sell Domain
Godaddy Web Hosting Coupon Codes:
cjch99r – Web Hosting for Just Rs .99 / month!
cjcwph1 – $1.00/mo WordPress Hosting!
cjc1hos5 – $1.00/mo Web Hosting + Free domain!
Godaddy Big Discount Coupon Codes:
cjc2off30 – 30% off! Big Savings For Your Small Business.
cjcssl1t – 25% off Standard SSL Certificates from GoDaddy!
Godaddy Special Deals:
cjcEEM411 – Email Marketing from GoDaddy!
cjcwsb16 – Website builder, domain and email starting at $1/ mo!
Godaddy Renewal Coupon Codes:
ACOSKS65 – Save $10 off for your order of $50 or more. You can use this coupon to renew All products (Domain names, SSL, Web Hosting…) for multiple years.
cjc30c – Save $15 when you spend over $75 or more. You can use this coupon to renew All products (Domain names, SSL, Web Hosting…) for multiple years.
CJCRMN33 – Save 33%* OFF Discount Domain Club!
We regularly update GoDaddy Coupon Codes to SAVE your money on Domain and Hosting purchases. If any of the above code is not working, please let us know in comment section. We will find some other working coupons for you.
Subscribe to get FREE tech advice, tips and in-depth articles directly in your Inbox.
You May also Like to Read:
5 Responses to GoDaddy Rs. 99 Domain Name – 86% OFF [Working]
I purchase domain only Rs 125
Nice Site
Thanks for sharing those coupon codes. It helps me much. Thanks again.
nice i purchase from here in 125 rs . thanks site admin
thanks i purchase from here a domain in Rs. 99
Nice site i purchased domian from here in 99 rs. thanks site admin

subset: commoncrawl


text:
Lands End Shipwreck
Lands End Shipwreck
The most recent Lands End Shipwreck was the 1,846-tonne RMS Mulheim was making passage from Cork to Lubeck, Germany, when she ran aground between Land's End and Sennen at 0500 GMT on Saturday 22nd March 2003The six-strong Polish crew were airlifted to safety by a rescue helicopter from RNAS Culdrose.
They were taken to the Sennen Cove Lifeboat Station, where they were treated for shock.There was a Major salvage operation that involved a crane and a pulley system on the cliffs immediately above the wreck.
The RMS Mulheim was loaded with more than 2,200 tonnes of car scrap metal. We have more information and another video of the RMS Mulheim wreck.


text:
loading map...
- Summary
For anyone looking to mix ancient history with enough adventure to keep things interesting, a trip to the ancient city of Caral, north of Lima, might be right up your alley. Constructed in 2,900 BCE, this UNESCO New World Heritage Site has the official distinction of being the oldest known city in the Americas. Though the adventurous elements of this excursion might be slight, be prepared for a 3 hour ride over paved and gravel roads, as well as a brief flat-terrain hike before reaching the entrance to the site itself. Once there, you'll have the opportunity to explore the site's large, 5,000 year old stone pyramids and examine its wealth of artifacts (musical instruments, textiles, etc.) that many believe influenced the development of later Andean cultures such as the Incas. As long as you come prepared for the heat (bring water and a hat) and the tour group (almost unavoidable), you'll leave this magnificent, isolated and ancient city duly enlightened and impressed.
Location:170 km (106 miles) north of Lima, Peru
Loading comments...
Been here? Add a comment to get the ball rolling.


text:
Dress: Sosolo
Cardigan: New Look
Necklace: Cheap Frills
This dress has been worn so much, I can't believe I haven't featured it on here sooner! I was given it by Sosolo, and as soon as I opened the parcel I knew it would be something I would get a lot of wear out of. In the warmer weather it was great with bare legs, and now it's colder, I've been wearing it with different cardigans and tights - so easy to just throw on and instantly look cute! Isn't the collar amazing?!
I'm also wearing a super cute necklace which was sent to me from Cheap Frills - check out their site if you're wanting some little presents for friends for Christmas. I'm definitely going to!
Had a bit of a bad hair day, so decided to throw it into a loose plait. I still haven't mastered the fishtail plait, so had to settle for the good old way of plaiting! I'm seriously considering cutting my hair off, I'm wanting it quite a bit shorter, but like I've said on here before, I'm pretty sure I would regret it straight away! I also want to dye it dark brown again... hmm what to do?!
If you haven't entered my Miners Cosmetics giveaway yet, you've still got time. Just leave a comment here :)
6 comments:
So so pretty! And that necklace is adorable :).ReplyDelete
That's such a cute dress, it really suits you :) xReplyDelete
Love the collar on that dress, its amazing! xReplyDelete
Love the look!ReplyDelete
you are too cute!
You look beautiful, that dress is so darling! xxReplyDelete
looking super cute hereReplyDelete
Thank you for taking the time to read my blog.
I'd love to hear your thoughts... Leave a comment below!


text:
- Encrypted offsite backups on auto-mounted media with Bacula & vchanger
- Preparing the Drives (securely wiping)
- Preparing the Drives (encryption)
- Preparing the Drives (filesystem)
- Preparing the Drives (testing)
- Automating the Process (udev)
- Automating the Process (autofs)
- Configuring vchanger
- Configuring Bacula
- Credits
Encrypted offsite backups on auto-mounted media with Bacula & vchanger
Configuring Bacula:
In the Bacula configuration files listed below, only the parts relevent to this tutorial are included. On Gentoo, the default location for Bacula's configuration files is /etc/bacula, but we keep ours one directory below in /etc/bacula/include so that we can allow Gentoo's dispatch-conf (configuration file updating tool) to keep the default Bacula config files up-to-date for reference while not touching our modified files.
Configuring Bacula's Director Daemon
To keep our configuration managable, each of the configuration sections have been pulled out of bacula-dir.conf and are stored in separate files which are pulled into the bacula-dir.conf file via the file include option:
/etc/bacula/include/bacula-dir.conf Director { Name = backup-dir DIR port = 9101 QueryFile = /usr/libexec/bacula/query.sql WorkingDirectory = /var/lib/bacula PidDirectory = /var/run Password = "verysecurepassword" Maximum Concurrent Jobs = 6 Messages = Daemon } --[snip]-- # Include Storage @/etc/bacula/include/Storage # Include Pools @/etc/bacula/include/Pools # Include Jobs @/etc/bacula/include/Jobs --[snip]--
In the Storage file, a Storage definition block is configured. The Name = c0 is the same as the changer_name in our vchanger configuration.
The Device = eSATA-Changer is our changer device that will be configured later in the Autochanger definition block of the Bacula storage daemon confgiuration file.
The Autochanger = yes line must be present for this Storage resource, and the Media Type = Offsite-File needs to match the Pool defined next.
/etc/bacula/include/Storage Storage { Name = c0 Address = storagedaemon.example.com Password = "verysecurepassword" Device = eSATA-Changer Autochanger = yes Media Type = Offsite-File }
In the Pools file a Pool resource named Offsite-eSATA is created with pretty basic options. The Storage = c0 line defines that this pool will use the c0 Storage resource defined above.
/etc/bacula/include/Pools Pool { Name = Offsite-eSATA Storage = c0 Pool Type = Backup Recycle = yes AutoPrune = yes Volume Retention = 4 weeks Recycle Oldest Volume = yes Volume Use Duration = 2 hours Maximum Volume Bytes = 10737418240 # Small (10GB) easy to move/transfer volume sizes }
NOTE: The Volume Use Duration = 2 hours line should be enough to prevent Bacula from trying to use an already-written-to volume that has a status of "append" when/if a magazine is changed, while allowing enough time for consecutively run jobs to complete writing to the relatively small 10GB volume. For more details on this issue, see the vchanger docs, section 12.3, paragraph 3.
Since the vchanger "magazines" (the encrypted drives with multiple 10GB Bacula volumes) will be swapped in and out, we need a way for Bacula to know which volumes are available for use. That is, which 10GB volumes are on the drive(s) currently plugged into the system.
In the bconsole command line interface to Bacula, the command update slots instructs bacula to poll a specified tape library to see which volumes are available and which slots they are in. Bacula then updates the InChanger and Slot columns of the Media table in the database with this information. Now Bacula knows which volumes are currently available to it and in which slot of the library each of them is.
To automate this process so that no one needs to manually load bconsole and run the "update slots" command each time a magazine is plugged in or unplugged, a custom script that uses bconsole will be used. It will be scheduled to run via a special Bacula job type of Admin.
In the Jobs file, a job called UpdateSlots with a type of Admin is created. This purpose of this job is to run a script via Bacula's RunsScript definition. The script will call the bconsole program and run the "update slots" command. This job will use the same schedule as the other nightly jobs, but will have a higher priority (lower number) so that it will be the first job to run every night. This way, the magazine(s) may be swapped out each morning when all the backup jobs have finished. No other manual steps are required.
/etc/bacula/include/Jobs Job { Name = UpDateSlots Client = None Type = Admin FileSet = None Storage = c0 Schedule = WeeklyToOffsiteDisk Messages = Standard Priority = 8 # Other nightly jobs have a priority of 10 SpoolData = no Pool = Default RunScript { RunsWhen = Before RunsOnClient = no Fail Job On Error = no Command = "/etc/bacula/include/runbefore-updateslots.sh" } }
Here is the simple bash shell script that uses bconsole to run the "update slots" command:
/etc/bacula/include/runbefore-updateslots.sh #!/bin/bash BC="/usr/sbin/bconsole" CONF="/etc/bacula/include/bconsole.conf" STORAGE="c0" DRIVE="0" # If multiple drives are defined in the Changer # device bconsole will ask for a drive number $BC -c $CONF << WAAEOF update slots storage=$STORAGE drive=$DRIVE quit WAAEOF
Configuring Bacula's Storage Daemon
In the Bacula storage daemon configuration file an Autochanger called eSATA-Changer is first defined. Per the vchanger documentation, the Changer Command points to the vchanger binary. vchanger is configured to run as the user bacula and group of bacula. Your configuration may differ, but the user configured here must have read/write access to the vchnager directories created during the vchanger installation as well as read/write access to the Bacula "file" volumes on the encrypted drives.
The rest of the parameters are the substitution variables that will be passed to the vchanger program and are defined in Bacula's documentation as follows:
- %c - Changer Device Name
- %o - Command (Loaded, Load or Unload)
- %S - Autochanger slot to act on (base 1)
- %a - Archive Device Name
- %d - Changer drive index (base 0)
/etc/bacula/include/bacula-sd.conf Autochanger { Name = eSATA-Changer Device = eSATA-1 Changer Command = "/usr/local/bin/vchanger -u bacula -g bacula %c %o %S %a %d" Changer Device = /etc/bacula/include/vchanger.conf }
Per the vchanger documentation, the Changer Device points to /etc/bacula/include/vchanger.conf, the vchanger configuration file.
The Device in the Autochanger definition block points to the name of a tape drive, or a filetape drive which needs to be defined in the badcula-sd.conf file as well:
Next in the Bacula storage daemon configuration file the eSATA-1 referenced in the eSATA-Changer Autochanger definition is configured:
/etc/bacula/include/bacula-sd.conf(cont) Device { Name = eSATA-1 DriveIndex = 0 Autochanger = yes Device Type = File Media Type = Offsite-File Label Media = no Random Access = yes Removable Media = yes Automatic Mount = yes Archive Device = /var/lib/bacula/vchanger/c0/0/drive0 }
Few Modifications
A few things I ran into running this on current versions of cryptsetup.
1. You can create the encrypted drive WITH key in one command now;
cryptsetup -v luksFormat /dev/sdb --key-file /etc/bacula/include/Bacula_Key_File
2. There is a new format for the arguments? for key-file. For example;
cryptsetup -v luksOpen --key-file /etc/bacula/include/Bacula_Key_File /dev/sdb tempcontainer
3. I had to install some requirements in my ubuntu server 12.04 x64.
sudo apt-get install libblkid-dev
sudo apt-get install uuid-dev
4. I had a lot of trouble with the Client = None and Fileset = None. I thought they were built in keywords, wasn't until I read http://blog.serverfault.com/2011/01/10/some-notes-on-setting-up-backups-... that I realized they were just dummy ones created.
Very informative ,well written.
Thank you, this tutorial helped a huge amount.I've been struggling to automate the decryption and mounting/unmounting. This tutorial enabled me to accomplish exactly what we needed.
Great job!
Hi! Great job with this howto!
I'm using Bacula since 2.4 releases and it's the first time I found a solution to encrypt all the Bacula volumes and get the 'perfect' OUT-OF-OFFICE solution.
Thanks so much for this!
Incredibly thorough. As a recent Bacula convert I've found it really useful.


text:
About the Author
In March, 2011, Baen Books will release Overkill, Robert's sixth novel, and first in his Orphan's Legacy series, and in July, 2011 his seventh, Undercurrents.
Born in 1947 on Manhattan Island, he graduated with Honors in Geology from the College of Wooster in 1969, and received his J.D. from the University of Cincinnati in 1973. He served as a U.S. Army intelligence officer, a Director of the Southwestern Legal Foundation, and was a National Science Foundation Fellow in Paleontology. He is a member of the Heinlein Society and the Science Fiction and Fantasy Writers Association.
As attorney of record in more than three thousand cases, he practiced in the U.S. federal courts, before courts and administrative tribunals in no fewer than thirteen states, and in five foreign countries. Six, if you count Louisiana.


text: It is always a pleasure photographing this family! Especially since this family of three became a family of four late last year! As I am posting this, I realize that I never blogged Mr. S's newborn portraits, I will have to remedy that sometime soon! 🙂 It is an honor to be able to be a part of documenting a family's history and watching they grow. Thank you, guys! I look forward to next time! 🙂


text:
#ForteLive: Trina DasGupta
Thursday, May 17, 2018
Facebook Live
As a kid, Trina DasGupta learned how to be an American from '80s TV. Now, as founder and CEO of Single Palm Tree Productions, she's on a mission to make media more diverse and help clients tell stories that are funny, inclusive, and reflect people like her.
Join us for a conversation with Trina on Facebook Live, hosted by Morra Aarons-Mele. They'll talk about how to put your communication skills to work in the corporate world, what it's like to work in multiple verticals and then take a career break, and how to use humor and entertainment to change culture for the better.
Got questions for Trina, or insights to share? Drop us a line on Facebook or Twitter with #ForteLive. We can't wait for you to join the conversation!


text:
Butiran syarikat
Golden Mouth Advertising (H.K)Co.,Ltd. ( Jie Da Advertisement Co.,Ltd)
We provide digital printing service on banners, flags , stickers ect
Digital printing processing has gone from proofing to small and medium-sized production. Digital printing equipment has gone through the product introduction period and entered the growth and...
silk screen printed banners and flags
Manual silk screen banner, pennants Proceed as follows: First, prepare the screen (easy approach): (requires 2 people to cooperate) 1. Use a 5070mm wood to make two rectangular boxes of 3000mm*600mm...


text:
When building systems using OpenKantu you will notice that it is rather easy to find strategies that generate high R² results on symbols like the EUR/USD and the USD/JPY while it is generally significantly hard to get positive results on symbols like the AUD/USD. I have done a lot of research about why this is the case – you can read more about that here – but finding systems on symbols where it has been traditionally difficult for me to find any has always been an interesting goal. Today I want to show you how you can actually use the OpenKantu software to find some highly historically stable strategies on the AUD/USD, we will discuss why this is the case and the potential problems that are present when this is done.
When you use OpenKantu to mine trading strategies on the 1D charts and you choose a simple stop-loss as the only way to control position closes what you have is that most systems found by OpenKantu behave somewhat like trend followers. This is due to the fact that the generated systems have their SL updated whenever a signal is generated in the same direction as an open trade – this is done to avoid trade chain dependency – and a trade is only closed either when the SL is touched or when a signal in the opposite direction is triggered. What happens is that the SL tends to behave like a "trailing stop" within the profitable systems that are generated and the systems end up benefiting from long term momentum, in the end you end up with systems that are in some senses trend followers.
When you mine systems like this on the EUR/USD, USD/JPY, USD/CHF or GBP/USD you tend to find a large number of strategies but on other symbols, especially a symbol like the AUD/USD, the landscape is usually terribly barren. This is because on these first symbols there is an abundance of strong momentum through their history while in the AUD/USD there does not seem to be enough of this to generate strategies of this type at all. The solution to avoid generating this type of system and instead find some highly linear strategies is basically to simply enable the use of the take-profit and use a space where the max shift is restricted to be lower than 100.
Doing the above allows you to find systems with R²>0.95 with a frequency of more than 10 trades per year using daily data from 1987 to 2016. The produced strategies are alike the strategy showed above where historical results are quite consistent through the entire period. Although as you can see in the options the SL and TP are allowed to vary between 0.5 and 5 in 0.1 steps we actually do not find very extreme SL to TP ratios but as a matter of fact – as showed below for a sample of mined systems – there are both SL>TP and TP<SL cases but most actually have R:R ratios between 0.8 and 1.3. There is only one extreme case where the R:R is 0.56 where the TP is 1.3 and the SL is 2.7 (the most extreme ratio is the samples I mined).
Of course the introduction of the TP implies the introduction of an additional degree of freedom which implies that the mining bias of the process has been increased. It is possible that all of the generated results are therefore the result of pure mining bias – rather than the presence of true historical inefficiencies – reason why it's important to repeat the exact same mining process on data generated using bootstrapping with replacement to ensure that the mining bias of the process is low and that the systems generated have a low probability to come from random chance.
The above results also support some of my previous observations regarding reward to risk ratios and how establishing different reward to risk ratios forces the creation of trading systems that are very fundamentally different in nature (see here). Not only do the reward to risk ratio can affect the resulting data mining bias of the process – because the number of systems that can be found just by random chance chances – but it can also increase the number of systems that are in fact found on the real data. Not all pairs might be suited for all reward to risk ratios and finding systems on pairs where it has been traditionally harder might only be a matter of changing the system character we're asking for. If you would like to learn more about trading systems and how you too can learn to design and trade your own strategies please consider joining Asirikuy.com, a website filled with educational videos, trading systems, development and a sound, honest and transparent approach towards automated trading.strategies.


text:
Amazon | Barnes & Noble | Goodreads
Title: Counter Clockwise (Clockwise #5)
Author: Elle Strauss
Publication Date: August 18, 2015
Synopsis: High school senior Casey Donovan is in trouble. Again. If only she had trusted in Nate's loyalty when he traveled to Spain with his college basketball team – despite the accompaniment of the cheerleading squad and Fiona the Floozy who'd made it super clear she wanted Nate for herself. Then she wouldn't have made that impulsive trip to Hollywood and let Aiden do that stupid thing that threatened her relationship with Nate, and triggered a trip into the past.
Only something is wildly wrong. She's not in the 1860s like she should be. It's 1929. And she didn't come alone.
Review: I really enjoyed the premise behind this book - a time-traveling girl who ends up in the wrong time? Yes please! I was a little bit concerned about reading this book, since it's book 5 in the series, but one of the first things after the title page is a note that you can start with this book and be alright, so I decided to just go for it.
I was a little confused for a while, since there's not really a lot of explanation about Casey and her abilities or her relationship with Nate, so I spent a lot of time trying to figure things out. I mean, it wasn't terrible, there was definitely enough information there to understand what was going on and why it was such a big deal that Casey ends up in 1929 instead of 1860, but I just feel like reading the rest of the series first would have made this book more enjoyable.
Other than not knowing all of the information though, I actually really did enjoy this story. Casey is very immature, and it was kind of annoying how little she trusts Nate, but she usually did have a pretty good reason, so I couldn't hold it against her that much. It was fun to see her adapt to a new time period and try to figure everything out. I was happy for her that she didn't have to go about learning everything by herself, because I could see how that might be terrifying and awful.
This book is well written and I found myself not wanting to put it down! Luckily, it was relatively short, so I could read the whole thing in one setting. I was left wanting more, so I picked up the rest of the series as soon as I finished reading, and I can't wait to get a chance to read them! I'll definitely be looking for more from Elle Strauss, and would give this book a 3.5/5 (although I bet it will be higher once I read the other books and can pick up the subtle references!).


text:
FILM CLIPS / Opening today
Drama. With Maggie Cheung and Nick Nolte. Directed by Olivier Assayas. (R. 111 minutes. In English, French and Cantonese, with subtitles. At the Lumiere and the Shattuck in Berkeley.)
The divine Maggie Cheung may look as if she got too close to an electrical outlet in "Clean" -- is that her real hair or a Chia Pet? -- but her appearance perfectly reflects the mess of her character's inner life. Cheung plays a fading rock singer whose heroin addiction is fueling her self-destruction.
"Clean" is an unflinching look at the ravages of substance abuse, and it's also a sobering redemptive tale. Yet despite Cheung's efforts (she won the best actress award at Cannes in 2004) and a strong and subtle supporting performance by Nick Nolte, the story could have used a good dose of adrenaline to keep it from often seeming lifeless.
"Clean" is the second collaboration between Cheung, a Hong Kong star ("2046," "Hero," "In the Mood for Love") and French director Olivier Assayas. (The two became an item while making "Irma Vep," released in 1996, and separated during the "Clean" shoot. The story goes that Cheung signed her divorce papers on the set.)
Things don't get off to a good start for Cheung's character, Emily. The film begins with her and her rocker husband playing end-of-the-road gigs somewhere in Canada. After he dies of an overdose, Emily is sent to prison for possession of drugs. As a result, she loses custody of her young son, Jay (played with remarkable maturity by James Dennis).
Once out of jail, Emily tries to follow the straight and narrow. She moves to Paris, where she waits tables and works at a department store in an attempt to fit into society. She does this because she desperately wants to be reunited with her child, who is being raised by her late husband's parents (Nolte and the equally understated Martha Henry).
Cheung isn't entirely credible or engaging as a long-suffering parent, and she isn't aided by Assayas' plodding direction. It's Nolte, as a caring grandfather and father-in-law, who gives "Clean" its true clarity and soul.
-- Advisory: Adult language, drug use.
-- John McMurtrie
'Guys and Balls'
Comedy. Directed by Sherry Hormann. In German with English subtitles. (R. 106 minutes. At the Roxie.)
Germany is not exactly a prudish country. For next month's World Cup, the country is expected to be invaded by 60,000 extra prostitutes, and the city of Dortmund is even installing temporary "sex huts." Makers of sex toys are expected to do brisk business selling World Cup-themed erotica.
Yet the last public vanguard of discrimination, as in America, is the sports locker room. So gay people are no big deal -- but gay soccer players are. "Guys and Balls" may not be the ideal way to warm up for the World Cup, but it's surefire entertainment: loopy and predictable, but tremendously likable.
It stars Maximilian Bruckner as Ecki, a goalkeeper for a small, working-class soccer club, who is dropped from the team after he blows the championship by missing a penalty kick and is outed as gay on the same day. He vows revenge by traveling to nearby Dortmund to gather a ragtag team of all-gay players and challenging his ex-club to a game.
If there were an American remake, the sport would be football, and the working-class town would be Green Bay, Wis.
Naturally, Ecki's parents have issues with his orientation, so in Dortmund he enlists the support of his sister, Susanne (Lisa Maria Potthoff), a nurse. She has a crush on a fellow hospital worker, Sven (David Rott), but when Sven meets Ecki, it's love at first sight -- and he's a hell of a goal scorer, too.
Ecki and Susanne round out the squad by trolling the bars and nightclubs of Dortmund's gay districts. Then it's only four weeks of practice to train for the big match and -- well, I'll bet you can guess what happens.
Director Sherry Hormann plays it very safe -- not much edge here, or issues explored -- but has slickly packaged a crowd-pleaser that has won several awards on the gay and lesbian film festival circuit. Nice ensemble cast, too.
-- Advisory: This film contains nudity, some foul language and risque sexual situations.
-- G. Allen Johnson
'Mongolian Ping-Pong'
Drama. Co-written and directed by Ning Hao. In Mongolian with English subtitles. (Not rated. 102 minutes. At the Balboa.)
I wonder, in this era of globalization, what Zhuang Zedong and Glenn Cowen would think of the movie "Mongolian Ping-Pong." Zedong and Cowen were the members of the Chinese and United States table tennis teams, respectively, whose instant friendship led the U.S. team to spontaneously visit China in 1971, a historic event that helped thaw Chinese-American relations and led to President Nixon's trip to China.
Thus came the catchphrase "pingpong diplomacy," but I doubt that Ning Hao's new film will help Outer Mongolia join the global culture -- mainly because the people of the steppes, who live without electricity or running water, have enough trouble joining mainstream Mongolian culture. Would they feel at home in the modern capital city of Ulaanbaatar, for instance?
"Mongolian Ping-Pong" is about a boy, Bilike, who finds a pingpong ball and doesn't know what it is. Because it is so white and round and perfect, he and his two friends -- all of them children of nomadic peoples -- think it's something pretty special.
His grandmother calls it "glowing pearl." The boys trek to a Buddhist monastery, but the monks there cannot help identify the mysterious orb. Eventually, a traveling film projectionist identifies it as a pingpong ball, and the kids find out it is the national sport of China, and thus the pingpong ball is the national ball of China. They decide to go to Beijing to return the national ball, but the Gobi desert proves too rough.
It's hard to say what Hao, who is Chinese, thinks of the nomadic people, who make up a quarter of the nation's population of 2.8 million and whose lifestyle has remained largely unchanged since the days of Genghis Khan. Is it unfortunate that they have been left behind as the world moves ahead? Or do they enjoy a purity of life very few experience? The children seem happy enough -- perhaps happier than, say, the average American child.
Hao doesn't seem to have a point of view. "Mongolian Ping-Pong" is episodic and meandering, with several tedious stretches. Although the children are charming, they are clearly nonactors, and that becomes a distraction as they try to carry a 102-minute movie. The best thing Hao does is show us a snapshot of the lifestyle of Mongolian nomads. For that, his film has value.
-- G. Allen Johnson


text:
If you or a loved one has suffered complications as a result of taking Risperdal, our dangerous drug lawyers at Buckfire & Buckfire, P.C. are an excellent choice to handle your lawsuit. Our firm has the experience, skill, and resources that you need on your side to acheieve a settlement against the large pharmaceutical companies. We have been named to Super Lawyers, have a perfect 10 rating on AVVO, and have achieved an "AV" rating which is the highest possible rating for skill and integrity from the prestigious Martindale-Hubbell service.
At Buckfire & Buckfire, P.C., our attorneys have extensive experience filing lawsuits against large drug companies. We are certified to practice throughout the entire United States and have an excellent track record. We can help you recover the maximum possible settlement for your complications. For your claim against Risperdal, we can help you decide if you should join a class action lawsuit, or file an individual case. We will explain your best course of action and be with you every step of the way.
Our No Fee Promise on Risperdal Cases
You can afford to have our great team of lawyers on your side. When you choose us, it literally costs nothing to get started. We promise you in writing:
- No money to get started
- We pay all case costs and expenses
- No legal fees whatsoever unless you receive a settlement
- Phone calls are always free.
Start Your Risperdal Claim
Our Risperdal lawyers will help you file your lawsuit. To get started, you can:
- Submit the Free Case Review Box on this page, or
- Call (866) 280-4722 any time of day to tell us about your case.
We will listen to your story and answer your questions. If you have claim, we will start immediately.
WARNING: There are strict time deadlines for filing Risperdal lawsuit claims


text:
- Research article
- Open Access
Human neural progenitors express functional lysophospholipid receptors that regulate cell growth and morphology
© Hurst et al; licensee BioMed Central Ltd. 2008
- Received: 07 July 2008
- Accepted: 11 December 2008
- Published: 11 December 2008
Lysophospholipids regulate the morphology and growth of neurons, neural cell lines, and neural progenitors. A stable human neural progenitor cell line is not currently available in which to study the role of lysophospholipids in human neural development. We recently established a stable, adherent human embryonic stem cell-derived neuroepithelial (hES-NEP) cell line which recapitulates morphological and phenotypic features of neural progenitor cells isolated from fetal tissue. The goal of this study was to determine if hES-NEP cells express functional lysophospholipid receptors, and if activation of these receptors mediates cellular responses critical for neural development.
Our results demonstrate that Lysophosphatidic Acid (LPA) and Sphingosine-1-phosphate (S1P) receptors are functionally expressed in hES-NEP cells and are coupled to multiple cellular signaling pathways. We have shown that transcript levels for S1P1 receptor increased significantly in the transition from embryonic stem cell to hES-NEP. hES-NEP cells express LPA and S1P receptors coupled to Gi/o G-proteins that inhibit adenylyl cyclase and to Gq-like phospholipase C activity. LPA and S1P also induce p44/42 ERK MAP kinase phosphorylation in these cells and stimulate cell proliferation via Gi/o coupled receptors in an Epidermal Growth Factor Receptor (EGFR)- and ERK-dependent pathway. In contrast, LPA and S1P stimulate transient cell rounding and aggregation that is independent of EGFR and ERK, but dependent on the Rho effector p160 ROCK.
Thus, lysophospholipids regulate neural progenitor growth and morphology through distinct mechanisms. These findings establish human ES cell-derived NEP cells as a model system for studying the role of lysophospholipids in neural progenitors.
- Epidermal Growth Factor Receptor
- Neural Progenitor
- Neural Progenitor Cell
- Neurite Retraction
- Human Neural Progenitor Cell
We have previously generated a stable neuroepithelial (NEP) cell line derived from human embryonic stem (hES) cells (hES-NEP) that is grown under adherent conditions, is self-renewing, and stably maintains capacity for neuronal or glial differentiation. These hES-NEP cells recapitulate morphological and phenotypic features of neural progenitor cells isolated from fetal tissue [1]. Such a cell line has potential both as a source for specific neuronal lineages to be used in hES cell neural therapy and as an in vitro model system in which to study human NEP cell function and its regulation by signaling mediators such as lysophospholipids. The lysophospholipid signaling mediators Lysophosphatidic Acid (LPA) and Sphingosine 1-phosphate (S1P) are critical regulators of neural development, modulating neural growth, morphogenesis, and differentiation.
Lysophospholipid signaling has been implicated in mediating diverse physiological and pathological responses, including cancer progression, wound healing, angiogenesis, cardiovascular development, and, more recently, neural development (Reviews: [2–5]). There is strong evidence that both LPA and S1P are critical in early neural development, as mouse embryos that lack enzymes for S1P or LPA synthesis exhibit severe neural tube defects. Specifically, mice with genetic deletion of Sphingosine kinases required for production of S1P developed cranial neural tube defects as a result of increased apoptosis, decreased mitosis and subsequent thinning of the neuroepithelial progenitor cell layer [6]. These data suggest that S1P mediates anti-apoptotic and pro-growth signaling in normal neuroepithelial development. Similarly, genetic deletion of Autotaxin, the enzyme responsible for production of LPA in the brain, yields embryonically lethal mice with neural tube defects. In these embryos, the neural tube fails to close completely and is kinked [7]. Further, embryos lacking LPA exhibited asymmetric neural headfold, reflecting large effusions with high levels of apoptotic cells [8]. These studies demonstrate critical and distinct roles of S1P and LPA in early neural development.
LPA and S1P receptors are expressed in neural progenitors, neurons, and oligodendrocytes in the developing and adult brain, and both LPA and S1P are generated by neurons [9–11]. The biological consequences of lysophospholipid signaling in the nervous system are incompletely defined, but evidence for several roles in neural progenitors is emerging. As discussed above, there are clear roles for S1P and LPA in early neural tube development. Further, LPA appears to regulate cortical neurogenesis by promoting morphological changes, survival, and differentiation [12, 13]. Finally, S1P activity is implicated in mediating migration of neural progenitor cells toward sites of spinal injury [10]. Thus, LPA and S1P regulate critical responses in neural progenitor cells that may be exploited to manipulate these cells in traditional pharmacological or cell-based therapeutics.
LPA and S1P bind and activate cell surface G-protein coupled receptors (GPCRs) to regulate cell proliferation, differentiation, and morphological changes, all of which may contribute to their roles in regulating neural progenitor cell function. There are at least five distinct LPA receptors (LPA1-LPA5) and five S1P receptors (S1P1-S1P5) [14]. LPA and S1P receptors couple to multiple G-protein pathways to regulate ion channel activity, adenylyl cyclase mediated cyclic AMP (cAMP) production, phospholipase C (PLC) mediated inositol phosphate production and calcium release, activation of the small GTPase Rho, and transactivation of receptor tyrosine kinase receptors (Review: [15]).
Regulation of cell growth and morphology are common effects of lysophospholipids. LPA and S1P have potent proliferative effects in multiple neural cell lines [16–18]. For example, LPA induces proliferation in neurospheres isolated from rat embryonic cortex [19], and application of S1P to neural progenitor cells from embryonic rat hippocampus has been shown to stimulate Gi/o pathways which activate Mitogen-Activated Protein (MAP) kinases and DNA synthesis [20]. The latter observation is consistent with the mechanism for lysophospholipid stimulated proliferation in many cancer cells, in which LPA receptors transactivate the epidermal growth factor receptor (EGFR) pathway, resulting in MAP kinase activation and subsequent proliferation [16–18].
LPA and S1P also stimulate specific cytoskeletal rearrangements, likely contributing to their roles in axonal pathfinding and migration. Neural cell lines such as NIE-115 cells and PC12 cells undergo rapid and transient neurite retraction in response to LPA and S1P [21, 22]. LPA induces neurite retraction within minutes, and neurons re-extend neurites after LPA is removed; thus, the retraction is dynamic and may fine tune neurite growth [23, 24]. Similar neurite retraction and growth cone collapse occur in response to LPA in differentiating cortical neurons [23]. Morphological changes also occur in neural progenitor cells, which lack distinct neurites. Both LPA and S1P cause transient aggregation of rat hippocampal neural progenitor cells [20], and LPA stimulates cluster contraction, lamellipodia retraction and migration toward the center of the cluster in mouse cortical neuroblasts [11]. LPA stimulates cell rounding of cortical neural progenitors, important in cortical neurogenesis [11]. The mechanisms for these effects is incompletely understood, but in most cases LPA and S1P induced morphological changes can be partially or completely blocked by pretreatment with inhibitors of the small GTPase Rho or its primary effector in neurons, p160 Rho kinase (ROCK) [23, 24].
The goal of the current study was to define functional lysophospholipid receptor signaling pathways in hES-NEP cells. We have determined that functional LPA and S1P receptors are expressed in hES-NEPs and regulate second messenger pathways, MAP kinase-dependent cell proliferation, and Rho-dependent morphology changes. These results contribute to the molecular characterization of hES-NEP cells, and establish for the first time a human, multipotent, renewable model cell system in which to define the role of LPA and S1P in neural progenitor cell function.
LPA and S1P receptor mRNA transcript expression changes during the transition from ES cells to hES-NEP cells
NEP cells express functional LPA and S1P receptors
LPA and S1P promote growth of hES-NEP cells via Ptx-sensitive G-proteins, EGF receptors, and MAP kinases
LPA and S1P induce reversible morphological changes in hES-NEP cells
Additional file 1: Continuous video microscopy of LPA treated hES-NEP cells. hES-NEP cells were incubated with 1 μM LPA for 18 hours and subjected to continuous video microscopy and condensed to the attached 2 minute time-lapse movie file. (WMV 5 MB)
Additional file 2: Continuous video microscopy of S1P treated hES-NEP cells. hES-NEP cells were incubated with 100 nM S1P for 18 hours and subjected to continuous video microscopy and condensed to the attached 2 minute time-lapse movie file. (WMV 3 MB)
Lysophospholipids are hypothesized to be critical regulators of neuronal differentiation, proliferation, and migration during development and following injury. While rodent neural progenitor cells and human transformed cell lines have been used to establish these roles and investigate the pathways responsible, the effects of lysophospholipids in human neural progenitor cells has not been established until now. This study establishes our recently characterized human embryonic neural epithelial progenitor cell line as a valid model system to define the role of LPA and S1P in neural progenitors during human neural development, differentiation, and wound healing.
Our results demonstrate that hES-NEP cells express functional LPA and S1P receptors coupled to Gi/o mediated inhibition of adenylyl cyclase and to a pertussis toxin-insensitive PLC pathway, likely mediated by Gq. hES-NEP cells do not express functional Gs coupled receptors for either LPA or S1P. Like the cAMP inhibitory response, the proliferative response was also completely inhibited by Pertussis toxin (Ptx) and is therefore also mediated by Gi/o coupled receptor subtypes. In contrast, the morphological response was not inhibited by Ptx, and so is not mediated by Gi/o coupled receptors. Our data suggest that LPA and S1P morphological responses may be mediated by G12 coupled GPCRs, consistent with the observed Rho-dependency, although we cannot rule out a Gq-mediated mechanism. All LPA and S1P receptors except LPA3 and S1P4 were detected in hES-NEP cells. Studies including additional pharmacologically selective drugs are required to determine the molecular identity of the receptors mediating the observed responses in hES-NEP cells.
Both LPA and S1P stimulate proliferation of many cell types. Studies in multiple cell lines [16–18, 39, 40] suggest that LPA receptors coupled to Gi/o stimulate cell growth via EGF receptor transactivation and subsequent MAP kinase activation, which directly leads to cell proliferation. While we observed a strong effect of lysophospholipids on cell growth, our data do not distinguish between effects on proliferation versus survival pathways. Future work should directly address the effect of LPA and S1P on apoptosis in these cells. Indeed, LPA does function as a survival factor in many cancer cell types via activation of the PI3 Kinase pathway. Nonetheless, our data are consistent with the proliferative EGF receptor transactivation mechanism described above. The growth responses to LPA and S1P in these cells were completely inhibited by Ptx and inhibitors of EGF receptors and ERK Map kinases, but not by inhibitors of p160 ROCK. Notably, the basal growth of hES-NEP cells was also inhibited by EGFR and MAP kinase inhibitors but not p160 ROCK inhibitor, suggesting that basal growth is mediated by a similar pathway, although not necessarily initiated by LPA or S1P. This also suggests a basal level of ERK MAP kinase activity. Although the data shown in Figure 6 do not show basal ERK phosphorylation due to the short exposure times required to avoid saturation of peak bands for quantification, in longer exposures basal ERK phosphorylation was apparent (data not shown).
The proliferative effect of LPA has been directly demonstrated in rat embryonic neural stem cells [19]. Cui et al. report a bell-shaped LPA dose response relationship in proliferation assays in which LPA increased thymidine incorporation at concentrations between 10 nanomolar and 1 micromolar, but inhibited proliferation at higher concentrations. This biphasic effect of LPA on proliferation is consistent with both our observation that LPA stimulates hES-NEP cell growth between 1 nM and 100 nM, and a recent report in which 10 micromolar LPA did not stimulate proliferation in human neurospheres [25]. Similarly, LPA stimulated production of inositol phosphates reached a maximal level at 1 μM and a reduced activation at higher concentrations.
LPA and S1P effects on morphology of either neurons or neural progenitors are mediated by effects on the actin cytoskeleton and/or microtubules, and effects are typically, but not always, dependent on the small GTPase protein Rho. Rho is known to regulate axonal growth, neuronal differentiation, and neuronal survival, primarily through its well-characterized neuronal effector p160 ROCK (Review:[41]). Rho activation occurs primarily via activation of Rho exchange factors by G proteins of the G12 subfamily, and leads to activation of p160 ROCK which mediates morphological changes by altering cytoskeletal structure. Specifically, p160 ROCK increases actin contractility and stress fiber formation via myosin-II regulatory light chain (MLC) [41] and decreases actin depolymerization via LIM kinases to regulate growth cone collapse [24]. Alternately, Gi/o pathways can also alter the cytoskeleton through activation of Glycogen synthase kinase-3 (GSK-3) [42] or Rac, which promotes cell spreading [43, 44].
The effect of LPA on neural cell morphology varies with cell type and distinct morphology changes occur over different time scales. Typically, in neurons or neuronal cell lines that have neurites or growth cones, these retract and cells round in response to LPA within minutes. In NIE-115 and NG108-15 cells, and B103 cells expressing either LPA1 or LPA4, LPA causes a rapid, transient rounding which initiates at 5 minutes following LPA addition, and cells recover their flattened morphology after 20 minutes, even in the continued presence of LPA [21, 44]. Alternately, in rat hippocampal NP cells both LPA and S1P cause transient aggregation with a maximal response at 3 hours and a return to baseline at 18 hours [20, 44]. Similarly, in B103 cells expressing exogenous LPA4, but not LPA1, LPA stimulated a slow aggregation that peaked at three hours [44]. Like the rapid cell rounding, the slow cell aggregation response is dependent on the Rho effector p160 ROCK, as was the slow cell aggregation observed in this report. In contrast, the known activation time course of p160 Rho kinase is on a scale of minutes, and Rho activation occurs even faster. Thus, even though this response is dependent on Rho/Rho kinase activation, these are not the rate limiting factors in the response. In our experiments, LPA or S1P were added to the media and not washed out throughout the experiment. The long recovery time of shape changes may reflect time course of LPA stability in the media. Consistent with this explanation, when media was changed to remove S1P one hour after addition to cells, morphology changes immediately began to reverse.
Our data clearly implicate Rho-mediated activation of ROCK in mediating LPA and S1P stimulated rounding and aggregation in hES-NEP cells. Inhibition of p160 ROCK completely blocked LPA and S1P stimulated effects, while both phospholipids could still mediate cell aggregation and rounding following inactivation of EGFR, or ERK. Although LPA and S1P still clearly altered cell morphology following treatment with Ptx, Ptx treatment itself induced modest cell aggregation. This effect of Ptx may reflect inhibition of basal Gi/o mediated effects on GSK-3 or Rac as described above.
While the current study describes LPA and S1P effects on proliferation and morphological changes, hES-NEPs are also a promising model cell system in which to study LPA and S1P effects in multiple processes of neural development. There is growing evidence that S1P and LPA regulate neuronal differentiation; however, data from various models report contradictory effects [11, 19, 20, 25, 26]. For example, LPA is reported to increase neuronal differentiation of rat neural progenitors (NP) [19] and mouse neurosphere cultures [11], while more recently LPA was shown to inhibit neuronal differentiation of human ES cell-derived neurosphere cultures [25]. These contradictions may reflect bona fide differences in LPA signaling pathways in rodent versus human neural differentiation, or they may be a result of mixed cell populations and the various sources and developmental stages from which the neural stem cells were isolated. For example, significant differences in expression of FGF, wnt and LIF pathway genes are observed between human neural stem cells derived from hES cells and fetal neural stem cells [45]. Given these potential differences between neural stem cells from different cell sources, homogeneous multi-potent human ES cell-derived neuroepithelial (hES-NEP) cells may be a superior model system in which to elucidate the roles of LPA and S1P cell signaling pathways in neural progenitor cells. Future studies of LPA and S1P effects on differentiation in the homogenous hES-NEP cell system will serve to clarify the effect of lysophospholipids on human neural differentiation.
We have defined LPA and S1P signaling pathways in hES-NEP cells that promote cellular growth and morphological changes by distinct mechanisms. This cell system is superior to rodent and transformed cell systems in which LPA and S1P effects have been defined by virtue of its human origin, multi-potent status, and non-transformed state. Further, as a stable, homogeneous, adherent, renewable cell line, hES-NEP cells are a convenient model system for future studies defining the functional role of lysophospholipids in proliferation, differentiation, and migration in the developmentally important human neural progenitor cell type.
Carbachol, epinephrine, quinpirole, clonidine, bromocriptine, dopamine, and U0126 were purchased from Sigma-Aldrich (St. Louis, MO). Y27632 and AG1478 were purchased from Tocris Bioscience (Ellisville, Missouri). Pertussis toxin was purchased from List Biological Laboratories (Campbell, CA) and FR180204 from EMD Biosciences (La Jolla, CA). Oleoyl (18:1) LPA and D-erythro-sphingosine-1-phosphate were from Avanti Polar Lipids (Alabaster, AL).
Cell Culture
Commercially available stocks of hES-NEP cells [available as ENStem-A™ (Millipore, Temecula, CA)] were used. These cells were derived from WA09 human ES cells and maintained as described previously[1]. Briefly, cells were grown on poly-ornithine (20 μg/mL)/laminin (5 μg/mL) (Sigma-Aldrich St. Louis, MO) coated plates in ENStem-A™ Neural Expansion Medium with 2 mM L-Glutamine and 20 ng/mL b-FGF (all from Millipore, Temecula, CA). Cells were passaged approximately every 48 hours and split 1:2 following manual dissociation by trituration.
WA09 (WiCel) were cultured in Dulbecco's minimal essential medium/Ham's F12 medium (DMEM/F12), 2 mM L-glutamine, 0.1 mM minimal essential medium (MEM) nonessential amino acids, 50 U/ml penicillin, 50 μg/ml streptomycin (all from Invitrogen), 4 ng/ml basic fibroblast growth factor (bFGF; R&D Systems, Minneapolis, MN) and 20% KSR (Invitrogen). Cells were cultured on mitomycin-C (Sigma Chemical Co., St. Louis, MO) mitotically inactivated murine embryonic fibroblasts, manually dissociated, and passaged to new feeder layers every 4–5 days [46].
Real Time Reverse Transcriptase PCR
RNA was extracted using Qiashredder and RNeasy kits (Qiagen, Valencia, CA) according to the manufacturer's instructions. The RNA quality and quantity was verified using a RNA 600 Nano Assay and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Total RNA was reverse-transcribed using the cDNA Archive Kit (Applied Biosystems Inc., Foster City, CA) according to manufacturer's protocols. Quantitative RT-PCR (Taqman) assays were chosen for the transcripts from a pre-validated library of human specific QPCR assays, and incorporated into a 384-well Micro-Fluidics Cards. Relative quantification was carried out on the ABI PRISM 7900 Sequence Detection System (Applied Biosystems Inc., Foster City, CA). Expression data for each LPA or S1P receptor was first normalized against endogenous 18S ribosomal RNA within each cDNA, and then the relative expression in hES-NEP was compared to hES cells using the ΔΔCT method of quantification in SDS software (Applied BIosystems Inc., Foster City, CA). Relative fold changes were determined as RQ values for positive changes and -1/RQ values for negative fold changes. ANOVA statistical analysis was performed using Tukey post-hoc analysis.
Inositol Phosphate Assay
Production of Inositol Phosphates (IP) was quantified using established protocols [47]. Briefly: To measure IP production by PLC activation, hES-NEP cells were plated in 24-well dishes at ~80% confluency. Cells were labeled with 1 μCi/well [3H] myo-inositol (American Radiolabeled Chemicals, St. Louis, MO) for 18 hours to label the cellular pool of phosphatidyl inositol. The cells were treated with Oleoyl (18:1) LPA (Avanti Polar Lipids, Alabaster, AL) or D-erythro-sphingosine-1-phosphate (Avanti Polar Lipids, Alabaster, AL) in the presence of 10 mM lithium chloride to inhibit degradation of inositol phosphates for 30 minutes at 37°C. Cells were then lysed in cold formic acid and neutralized with ammonium hydroxide, and the lysates were then loaded onto columns of AG 1-X8 anion exchange resin (Biorad, Hercules, California). The columns were washed with water and dilute ammonium formate to remove unhydrolyzed lipids. The [3H] IPs were then eluted with 1.2 M ammonium formate/0.1 M formic acid, and added to scintillation cocktail for counting. In some experiments, cells were treated with 100 ng/mL pertussis toxin (Ptx) for 18 hours prior to IP assay.
cAMP Assay
We used a modified version established protocols [48]. hES-NEP cells were plated in 12-well dishes and labeled with 0.6 μCi [3H]-adenine (Perkin Elmer, Waltham, MA) for three hours in the presence or absence of 200 ng/mL Ptx. Assay buffer containing 1 mM isobutylmethylxanthine (IBMX), 50 μM forskolin, and varying concentrations of LPA was added to the cells for 20 minutes at 37°C. Reactions were terminated by aspiration followed by the addition of stop solution containing 1.3 mM cAMP and 2% sodium dodecyl sulfate. [14C]-cAMP stock was added to each well to control for recovery of cAMP, followed by perchloric acid to lyse cells. Lysates were neutralized with KOH and cAMP was isolated using sequential column chromatography over Dowex AG-50-W4 cationic exchange resin (Bio-Rad, Hercules, CA) followed by neutral alumina columns. The resulting eluate was subjected to scintillation counting after the addition of scintillation cocktail.
Cellular Growth
hES-NEP cells were plated in 24-well plates at 50,000 cells per well and grown to reach 50% confluency (approximately 100,000 cells/well). In some experiments, cells were pre-treated with the indicated reagents for 18 hours, triturated to remove them from the plate, and counted using a hemacytometer to determine the number of cells per well. Cells were then treated with LPA, S1P, or vehicle for the indicated amount of time and counted again. Trypan blue exclusion was used to determine cell viability following drug treatment (0.4% (wt/vol) solution of Trypan Blue (Invitrogen, Carlsbad, CA)). Statistical significance of changes in growth was determined using an unpaired, two-tailed T-test.
p44/42 ERK MAP Kinase Phosphorylation
hES-NEP cells were plated in 24-well plates. Prior to the assay, cells were washed one time with ENStem-A™ Neural Expansion Media and allowed to incubate in 250 μL media for 15 minutes at 37°C. LPA or S1P was then applied to the cells for the indicated period of time. The reaction was terminated by aspirating the media and adding 100 μL protein sample buffer. Cells were harvested and lysed in protein sample buffer, separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted using a primary antibody targeted against phospho-ERK or total ERK (Cell Signaling Technology, Danvers, MA) and peroxidase-conjugated secondary antibodies (Bethyl Laboratories, Montgomery, TX). Bands were then visualized using SuperSignal Chemiluminescent substrate (Pierce, Rockford, IL). Densitometry analysis was performed using Total Lab 1D Gel Analysis software. Background bands were not subtracted out and all lanes and bandwidths were of equal size. Densitometry results for phospho-ERK were normalized to total ERK to control for loading, and then normalized to maximal ERK phosphorylation to compare between experiments. Statistical significance of increases in ERK phosphorylation over basal levels was determined using an unpaired, two-tailed T-test.
Cell Morphology Studies
Continuous video microscopy of hES-NEP cells was performed using the WaferGen Smart Slide System (WaferGen, Incorporated, Freemont, CA). hES-NEP cells were plated on a WaferGen Smart Slide 100 and maintained at 37°C, with the lid at 39°C to prevent condensation. CO2 was maintained at 5% over the course of the experiment, and negative flow was maintained through systemic purging every two minutes. Images were obtained using a Nikon Eclipse TE2000-S microscope, and captured every two minutes using a Retiga 2000R Fast 1394 camera (QImaging, Canada). Data were processed using Image Pro Plus5.1 version 220.127.116.11 (Media Cybernetics, Inc., Bethesda, MD).
To study the effects of pharmacological inhibitors on LPA and S1P stimulated changes in morphology, hES-NEP cells were plated in 6-well plates. Three areas with approximately equal cell densities were identified in each well and an image of each of these areas was captured with a Nikon AZ100 microscope mounted with a Nikon Digital Sight DS-QiMc camera set at 16× magnification. Cells were pre-treated with the indicated compounds for 18 hours. LPA or S1P was then applied for an additional 18 hours. Images of the cells were captured in triplicate after pre-treatment, approximately 5 hours after application of LPA or S1P, and then again 13 hours later.
The authors acknowledge Dr. Robert Arnold, Department of Pharmaceutical and Biomedical Sciences, University of Georgia, for technical assistance with microscopy studies. Funding was provided by the National Institutes of Health (NS053272, SLS), Aruna Biomedical, Inc. and the Georgia Tech/UGA Biomedical Research Initiative.
Authors' Affiliations
- Shin S, Mitalipova M, Noggle S, Tibbitts D, Venable A, Rao R, Stice SL: Long-term proliferation of human embryonic stem cell-derived neuroepithelial cells using defined adherent culture conditions. Stem cells (Dayton, Ohio). 2006, 24 (1): 125-138. 10.1634/stemcells.2004-0150.View ArticleGoogle Scholar
- Birgbauer E, Chun J: New developments in the biological functions of lysophospholipids. Cell Mol Life Sci. 2006, 63 (23): 2695-2701. 10.1007/s00018-006-6155-y.View ArticlePubMedGoogle Scholar
- Chun J: Lysophospholipids in the nervous system. Prostaglandins & other lipid mediators. 2005, 77 (1–4): 46-51. 10.1016/j.prostaglandins.2004.09.009.View ArticleGoogle Scholar
- Moolenaar WH, van Meeteren LA, Giepmans BN: The ins and outs of lysophosphatidic acid signaling. Bioessays. 2004, 26 (8): 870-881. 10.1002/bies.20081.View ArticlePubMedGoogle Scholar
- Mills GB, Moolenaar WH: The emerging role of lysophosphatidic acid in cancer. Nat Rev Cancer. 2003, 3 (8): 582-591. 10.1038/nrc1143.View ArticlePubMedGoogle Scholar
- Mizugishi K, Yamashita T, Olivera A, Miller GF, Spiegel S, Proia RL: Essential role for sphingosine kinases in neural and vascular development. Molecular and cellular biology. 2005, 25 (24): 11113-11121. 10.1128/MCB.25.24.11113-11121.2005.PubMed CentralView ArticlePubMedGoogle Scholar
- van Meeteren LA, Ruurs P, Stortelers C, Bouwman P, van Rooijen MA, Pradere JP, Pettit TR, Wakelam MJ, Saulnier-Blache JS, Mummery CL, Moolenaar WH, Jonkers J: Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development. Molecular and cellular biology. 2006, 26 (13): 5015-5022. 10.1128/MCB.02419-05.PubMed CentralView ArticlePubMedGoogle Scholar
- Bachner D, Ahrens M, Betat N, Schroder D, Gross G: Developmental expression analysis of murine autotaxin (ATX). Mechanisms of development. 1999, 84 (1–2): 121-125. 10.1016/S0925-4773(99)00048-9.View ArticlePubMedGoogle Scholar
- Birgbauer E, Chun J: New developments in the biological functions of lysophospholipids. Cell Mol Life Sci. 2006Google Scholar
- Kimura A, Ohmori T, Ohkawa R, Madoiwa S, Mimuro J, Murakami T, Kobayashi E, Hoshino Y, Yatomi Y, Sakata Y: Essential roles of sphingosine 1-phosphate/S1P1 receptor axis in the migration of neural stem cells toward a site of spinal cord injury. Stem cells (Dayton, Ohio). 2007, 25 (1): 115-124. 10.1634/stemcells.2006-0223.View ArticleGoogle Scholar
- Fukushima N, Weiner JA, Chun J: Lysophosphatidic acid (LPA) is a novel extracellular regulator of cortical neuroblast morphology. Developmental biology. 2000, 228 (1): 6-18. 10.1006/dbio.2000.9930.View ArticlePubMedGoogle Scholar
- Fukushima N, Ishii I, Habara Y, Allen CB, Chun J: Dual regulation of actin rearrangement through lysophosphatidic acid receptor in neuroblast cell lines: actin depolymerization by Ca(2+)-alpha-actinin and polymerization by rho. Molecular biology of the cell. 2002, 13 (8): 2692-2705. 10.1091/mbc.01-09-0465.PubMed CentralView ArticlePubMedGoogle Scholar
- Fukushima N, Shano S, Moriyama R, Chun J: Lysophosphatidic acid stimulates neuronal differentiation of cortical neuroblasts through the LPA1-G(i/o) pathway. Neurochem Int. 2007, 50 (2): 302-307. 10.1016/j.neuint.2006.09.008.View ArticlePubMedGoogle Scholar
- Anliker B, Chun J: Lysophospholipid G protein-coupled receptors. J Biol Chem. 2004, 279 (20): 20555-20558. 10.1074/jbc.R400013200.View ArticlePubMedGoogle Scholar
- Ishii I, Fukushima N, Ye X, Chun J: Lysophospholipid receptors: signaling and biology. Annual review of biochemistry. 2004, 73: 321-354. 10.1146/annurev.biochem.73.011303.073731.View ArticlePubMedGoogle Scholar
- Gschwind A, Prenzel N, Ullrich A: Lysophosphatidic acid-induced squamous cell carcinoma cell proliferation and motility involves epidermal growth factor receptor signal transactivation. Cancer research. 2002, 62 (21): 6329-6336.PubMedGoogle Scholar
- Daub H, Wallasch C, Lankenau A, Herrlich A, Ullrich A: Signal characteristics of G protein-transactivated EGF receptor. The EMBO journal. 1997, 16 (23): 7032-7044. 10.1093/emboj/16.23.7032.PubMed CentralView ArticlePubMedGoogle Scholar
- Kue PF, Taub JS, Harrington LB, Polakiewicz RD, Ullrich A, Daaka Y: Lysophosphatidic acid-regulated mitogenic ERK signaling in androgen-insensitive prostate cancer PC-3 cells. International journal of cancer. 2002, 102 (6): 572-579. 10.1002/ijc.10734.View ArticleGoogle Scholar
- Cui HL, Qiao JT: Promotive action of lysophosphatidic acid on proliferation of rat embryonic neural stem cells and their differentiation to cholinergic neurons in vitro. Sheng Li Xue Bao. 2006, 58 (6): 547-555.PubMedGoogle Scholar
- Harada J, Foley M, Moskowitz MA, Waeber C: Sphingosine-1-phosphate induces proliferation and morphological changes of neural progenitor cells. Journal of neurochemistry. 2004, 88 (4): 1026-1039.View ArticlePubMedGoogle Scholar
- Jalink K, Eichholtz T, Postma FR, van Corven EJ, Moolenaar WH: Lysophosphatidic acid induces neuronal shape changes via a novel, receptor-mediated signaling pathway: similarity to thrombin action. Cell Growth Differ. 1993, 4 (4): 247-255.PubMedGoogle Scholar
- Sato K, Tomura H, Igarashi Y, Ui M, Okajima F: Exogenous sphingosine 1-phosphate induces neurite retraction possibly through a cell surface receptor in PC12 cells. Biochemical and biophysical research communications. 1997, 240 (2): 329-334. 10.1006/bbrc.1997.7666.View ArticlePubMedGoogle Scholar
- Fukushima N, Weiner JA, Kaushal D, Contos JJ, Rehen SK, Kingsbury MA, Kim KY, Chun J: Lysophosphatidic acid influences the morphology and motility of young, postmitotic cortical neurons. Molecular and cellular neurosciences. 2002, 20 (2): 271-282. 10.1006/mcne.2002.1123.View ArticlePubMedGoogle Scholar
- Fukushima N: LPA in neural cell development. Journal of cellular biochemistry. 2004, 92 (5): 993-1003. 10.1002/jcb.20093.View ArticlePubMedGoogle Scholar
- Dottori M, Leung J, Turnley AM, Pebay A: Lysophosphatidic Acid Inhibits Neuronal Differentiation of Neural Stem/progenitor Cells Derived from Human Embryonic Stem Cells. Stem cells (Dayton, Ohio). 2008Google Scholar
- Pebay A, Wong RC, Pitson SM, Wolvetang EJ, Peh GS, Filipczyk A, Koh KL, Tellis I, Nguyen LT, Pera MF: Essential roles of sphingosine-1-phosphate and platelet-derived growth factor in the maintenance of human embryonic stem cells. Stem cells (Dayton, Ohio). 2005, 23 (10): 1541-1548. 10.1634/stemcells.2004-0338.View ArticleGoogle Scholar
- Learish RD, Bruss MD, Haak-Frendscho M: Inhibition of mitogen-activated protein kinase kinase blocks proliferation of neural progenitor cells. Brain research. 2000, 122 (1): 97-109. 10.1016/S0165-3806(00)00064-X.View ArticlePubMedGoogle Scholar
- Li BS, Ma W, Zhang L, Barker JL, Stenger DA, Pant HC: Activation of phosphatidylinositol-3 kinase (PI-3K) and extracellular regulated kinases (Erk1/2) is involved in muscarinic receptor-mediated DNA synthesis in neural progenitor cells. J Neurosci. 2001, 21 (5): 1569-1579.PubMedGoogle Scholar
- Deleyrolle L, Marchal-Victorion S, Dromard C, Fritz V, Saunier M, Sabourin JC, Tran Van Ba C, Privat A, Hugnot JP: Exogenous and fibroblast growth factor 2/epidermal growth factor-regulated endogenous cytokines regulate neural precursor cell growth and differentiation. Stem Cells. 2006, 24 (3): 748-762. 10.1634/stemcells.2005-0138.View ArticlePubMedGoogle Scholar
- Caverzasio J, Palmer G, Suzuki A, Bonjour JP: Evidence for the involvement of two pathways in activation of extracellular signal-regulated kinase (Erk) and cell proliferation by Gi and Gq protein-coupled receptors in osteoblast-like cells. J Bone Miner Res. 2000, 15 (9): 1697-1706. 10.1359/jbmr.2000.15.9.1697.View ArticlePubMedGoogle Scholar
- Cechin SR, Dunkley PR, Rodnight R: Signal transduction mechanisms involved in the proliferation of C6 glioma cells induced by lysophosphatidic acid. Neurochem Res. 2005, 30 (5): 603-611. 10.1007/s11064-005-2747-4.View ArticlePubMedGoogle Scholar
- Kim JH, Song WK, Chun JS: Sphingosine 1-phosphate activates Erk-1/-2 by transactivating epidermal growth factor receptor in rat-2 cells. IUBMB life. 2000, 50 (2): 119-124.View ArticlePubMedGoogle Scholar
- Kim MK, Lee HY, Park KS, Shin EH, Jo SH, Yun J, Lee SW, Yoo YH, Lee YS, Baek SH, Bae YS: Lysophosphatidic acid stimulates cell proliferation in rat chondrocytes. Biochemical pharmacology. 2005, 70 (12): 1764-1771. 10.1016/j.bcp.2005.09.015.View ArticlePubMedGoogle Scholar
- Mathieson FA, Nixon GF: Sphingolipids differentially regulate mitogen-activated protein kinases and intracellular Ca2+ in vascular smooth muscle: effects on CREB activation. Br J Pharmacol. 2006, 147 (4): 351-359. 10.1038/sj.bjp.0706600.PubMed CentralView ArticlePubMedGoogle Scholar
- Osinde M, Mullershausen F, Dev KK: Phosphorylated FTY720 stimulates ERK phosphorylation in astrocytes via S1P receptors. Neuropharmacology. 2007, 52 (5): 1210-1218. 10.1016/j.neuropharm.2006.11.010.View ArticlePubMedGoogle Scholar
- Sato K, Ishikawa K, Ui M, Okajima F: Sphingosine 1-phosphate induces expression of early growth response-1 and fibroblast growth factor-2 through mechanism involving extracellular signal-regulated kinase in astroglial cells. Brain Res Mol Brain Res. 1999, 74 (1–2): 182-189. 10.1016/S0169-328X(99)00279-X.View ArticlePubMedGoogle Scholar
- Van Brocklyn J, Letterle C, Snyder P, Prior T: Sphingosine-1-phosphate stimulates human glioma cell proliferation through Gi-coupled receptors: role of ERK MAP kinase and phosphatidylinositol 3-kinase beta. Cancer Lett. 2002, 181 (2): 195-204. 10.1016/S0304-3835(02)00050-2.View ArticlePubMedGoogle Scholar
- Darenfed H, Dayanandan B, Zhang T, Hsieh SH, Fournier AE, Mandato CA: Molecular characterization of the effects of Y-27632. Cell Motil Cytoskeleton. 2007, 64 (2): 97-109. 10.1002/cm.20168.View ArticlePubMedGoogle Scholar
- Shah BH, Baukal AJ, Shah FB, Catt KJ: Mechanisms of extracellularly regulated kinases 1/2 activation in adrenal glomerulosa cells by lysophosphatidic acid and epidermal growth factor. Mol Endocrinol. 2005, 19 (10): 2535-2548. 10.1210/me.2005-0082.View ArticlePubMedGoogle Scholar
- Zhao Y, He D, Saatian B, Watkins T, Spannhake EW, Pyne NJ, Natarajan V: Regulation of lysophosphatidic acid-induced epidermal growth factor receptor transactivation and interleukin-8 secretion in human bronchial epithelial cells by protein kinase Cdelta, Lyn kinase, and matrix metalloproteinases. J Biol Chem. 2006, 281 (28): 19501-19511. 10.1074/jbc.M511224200.PubMed CentralView ArticlePubMedGoogle Scholar
- Schmandke A, Schmandke A, Strittmatter SM: ROCK and Rho: biochemistry and neuronal functions of Rho-associated protein kinases. Neuroscientist. 2007, 13 (5): 454-469. 10.1177/1073858407303611.PubMed CentralView ArticlePubMedGoogle Scholar
- Sayas CL, Ariaens A, Ponsioen B, Moolenaar WH: GSK-3 is activated by the tyrosine kinase Pyk2 during LPA1-mediated neurite retraction. Molecular biology of the cell. 2006, 17 (4): 1834-1844. 10.1091/mbc.E05-07-0688.PubMed CentralView ArticlePubMedGoogle Scholar
- Hama K, Aoki J, Fukaya M, Kishi Y, Sakai T, Suzuki R, Ohta H, Yamori T, Watanabe M, Chun J, Arai H: Lysophosphatidic acid and autotaxin stimulate cell motility of neoplastic and non-neoplastic cells through LPA1. J Biol Chem. 2004, 279 (17): 17634-17639. 10.1074/jbc.M313927200.View ArticlePubMedGoogle Scholar
- Yanagida K, Ishii S, Hamano F, Noguchi K, Shimizu T: LPA4/p2y9/GPR23 mediates rho-dependent morphological changes in a rat neuronal cell line. J Biol Chem. 2007, 282 (8): 5814-5824. 10.1074/jbc.M610767200.View ArticlePubMedGoogle Scholar
- Shin S, Sun Y, Liu Y, Khaner H, Svant S, Cai J, Xu QX, Davidson BP, Stice SL, Smith AK, Goldman SA, Reubinoff BE, Zhan M, Rao MS, Chesnut JD: Whole genome analysis of human neural stem cells derived from embryonic stem cells and stem and progenitor cells isolated from fetal tissue. Stem cells (Dayton, Ohio). 2007, 25 (5): 1298-1306. 10.1634/stemcells.2006-0660.View ArticleGoogle Scholar
- Mitalipova M, Calhoun J, Shin S, Wininger D, Schulz T, Noggle S, Venable A, Lyons I, Robins A, Stice S: Human embryonic stem cell lines derived from discarded embryos. Stem cells (Dayton, Ohio). 2003, 21 (5): 521-526. 10.1634/stemcells.21-5-521.View ArticleGoogle Scholar
- Hepler JR, Nakahata N, Lovenberg TW, DiGuiseppi J, Herman B, Earp HS, Harden TK: Epidermal growth factor stimulates the rapid accumulation of inositol (1,4,5)-trisphosphate and a rise in cytosolic calcium mobilized from intracellular stores in A431 cells. The Journal of biological chemistry. 1987, 262 (7): 2951-2956.PubMedGoogle Scholar
- Hettinger-Smith BD, Leid M, Murray TF: Chronic exposure to adenosine receptor agonists and antagonists reciprocally regulates the A1 adenosine receptor-adenylyl cyclase system in cerebellar granule cells. Journal of neurochemistry. 1996, 67 (5): 1921-1930.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


text:
Inside this area you could find properties in Navi Mumbai inside Hex Blox, Kharghar and Sai Nagar, Kalamboli. Actual estate may give returns in two methods- from capital beneficial properties as you sell property and/or through rental money flows. NRIs/PIOs can make fee for acquisition of immovable property by means of regular banking channels in India by means of inward remittance from anywhere outdoors India or by debit to his NRE/FCNR (B) account. To take advantage of your search Low-cost Flats Mumbai Thane you might strive the following key phrases Low-cost Flats Mumbai Thane, Condominium Mumbai, Home Mumbai.
Mickey Enclave is located on the Dominic-Colony Highway three, a major area in Malad West On supply are 1 BHK residences with a carpet space of 392 sq. ft, making it one of the main upcoming projects in Mumbai. Costs are down albeit barely and financial institution loans on housing are easily accessible with very low rates of interest. Before buying property from a company, it is necessary to confirm with the Registrar of Companies that the property should not be mortgaged or is just not getting used as a security towards a loan; otherwise it is not thought-about a freehold property. Areas like Goregaon (East), Chembur East, Vile Parle (East) & Bandra (East) have seen around 20% ROI in the final financial year.
Commonly known as the 'New York of India', Mumbai offers an opportunity to all classes to spend money on its property market. The property providers division of Tata Housing Growth Company Limited supplies a complete and big selection of companies to prospective tenants/landlords/investment firms searching for probably the most appropriate and price efficient premises. Actual Property Agencies Tata Pokhran Road Pre Launch obtainable in Mumbai with properties for sale are Ilesh Thakkar, Solanki Houses and Tulsi Estates. We respect the professional attendance offered by All Check Deals in aiding us to buy our property. Nevertheless, service-based mostly industries are allowed to operate from residential areas, on the condition that they are going to vacate the property if any complaint is acquired from other residential house owners.
Such NRI may switch any immovable property to an Indian Citizen Resident Outdoors India i.e. NRI or a PIO resident outdoors India. Mumbai Property Change focuses on dealing with NRI in Dubai, USA, UK, Singapore, Hongkong, South Africa and other components of the world that need to buy properties in Mumbai, Thane and Navi Mumbai and does full portfolio and property management and repatriation. A whole lot of startups and property broking companies have sprung up, however if you name their Gross sales guys after few years, they are lengthy gone. Unlike within the case of leasehold property, no annual lease expenses should be paid and the freehold property could be registered and / or transferred partly(s).
With upcoming residential projects, as well as ongoing housing and accomplished residential initiatives in Mumbai is main posh areas, Neo Infra Real Property Development Company is quick carving a niche for itself among the many most premium actual estate builders in Mumbai. There are a number of benefits: for those who convert the property to a freehold property, you change into a full-fledged proprietor by getting the sale deed and having it registered. On the website, one can find that a flat for lease in Andheri is are available from price starting from ₹ 10500. The final permission, however, covers only buy of residential and commercial property.


text:
For a twelve month period, from Xmas 1985 to Xmas 1986, Elite were probably the best arcade conversion software house around - most of them down to the talents of Keith Burkhill - although he wasn't involved in the Bomb Jack conversion.
Bomb Jack is an absolute masterclass of a conversion - in fact I would rather play the Speccy version than the Tehkan/Tecmo coin-op - it's that good!
Overall it's a ridiculously playable game, with lovely colurful backgrounds, decent sound and although the graphics can become a bit of a mess when too many enemies congregate at the bottom of the screen it doesn't detract from a supremely playable game.
Mag Reviews: http://zxspectrumreviews.co.uk/Search.aspx?wosid=0000617
Not logged in [ log in | sign up | forgot password? ]


text:
Want to try yoga?
Or need a refresh?
Kick Start Yoga is for both complete newbies and those who want to brush up on the basics
Every Thursday lunch time from 24 March at 201 Borough
Join us in the gym to stretch out and find some calm in your day
6 Class Pass £65
Please select option 'Borough 6 Week Course' when booking.
Yoga mats, good music, friendly teachers and creative, alignment-based classes.
Please keep in mind: • Arrive at least 5 mins before class • Booking is advised • Drop in is cash only
Find Us: 201 Borough High Street London SE1 1JA


text:
Status: Open
Know more about this business than we do? Cool! Please submit any corrections or missing details you may have.Help us make it right
Be the first NOLA.com user to add photos or videos of Wadhwa Oberol
Oberol Wadhwa is located at 148 Woodland Dr, La Place, LA. This business specializes in Doctors & Clinics.


text:
About Cotecna
Cotecna Services
Cotecna Tools
Cotecna News & Media
Call or email us!
Verification of conformity is a service offered by Cotecna in the area of Certification & Training. This service, for which Cotecna is accredited, entails verification of product safety and manufacturer's conformity to the standards adopted by national bodies. It also includes the issuance of Product Certificates of Conformity.


text:
Exports still images into video format
Simple DVD converter into AVI, bin/cues, iso
Convert popular video and audio files to the ones supported by iPhone and iPod.
Easily decode files from OnlineTVRecorder and OnlineMusicRecorder
An useful application that lets you convert QuickTime files to another format
A open source/cross-platform solution for computer vision and machine sensing
Fixes radial wipe and OpenGL shadows in OS X 10.3
A easy to use VCD, MPEG, AVI video conversion and CD duplication tool
Capture continuous images on the screen as a Quicktime movie
Video player for your Mac
Chromakey Movie is an iMovie blue screening plug-in
Use your webcam to meet other people virtually
Drop MPEG here... X corrects those (*) MPEG files that cannot be read by QuickTime Player
Allows you to easily manage and edit your video files.
SDI-based video & audio routing switcher.
End-to-end system to explore streaming multimedia
A great plug-in collection for Final Cut Pro
Free and open source ffmpeg GUI
Opens up a world of cool chapter usage in QuickTime Player
Create a video from a sequence of images.
A free and Java based screencast tool for your Mac
Lets you do more stuff with your iSight camera.
Allows you to perform video and audio conversions
Cool Open GL 3D visualization plug in for iTunes which renders your favorite music!
Allows you to perform basing operations on video timings
An useful application that allows you to watch QuickTime movies in full screen
A useful and simple to use Mac application for movie playback
Create QuickTime movies from a series of images
Change your movie collection into iPod files
Easy to use video to iPod mp4 converter for Mac
An useful application that has a digital video catalog and exploration tools
The best DVD Copy software for Mac users.
QuickTime based application that retrieves save-disabled files
Lets you use your iSight to capture activity in front of your Mac's camera.
Movie organizer for your Mac
Blue screen, green screen, and compositing effects for iMovie
emphasize color - Accent a certain color (or color range) or reduce the prominence of a color...
DVD Ripper, Video Converter, DVD Copy and DVD creator for Mac.
Adds OS 10.2 and QuickTime 6 support.
Convert video and rip DVD to all sorts of video formats on Mac OS X.
A full-featured software-only model that offers a mobile and scalable editing environment
An useful application that makes one clip from several clips
An useful application that helps you play QuickTime DV or DV stream files using a media converter
An encoding software for DVD (and DV), managing telecine and interlaced
Convert video to MP4 formats on Mac OS X with high quality.
Shine is a fast light effect plug-in
QuickTime Effects - 8 video editing effects plug-ins
A program that lets you apply visual effects to your videos.
An Adobe AIR application that offers you an interactive Avatar's trailer experience allowing...
Capture and share videos and pics from iSight or other webcams via email


text:
Tacking on More News in Des Moines/
KCCI says they are going to add more news to their line-up.
The station is adding a 9PM newscast, but it will be on their smaller sister station.
The half hour-long "KCCI 8 News at 9" will air seven days a week on MeTV, the CBS affiliate's sister channel, and will be anchored by Shaina Humphries, who is currently the station's morning anchor.
The show will launch its first newscast on March 21.
The newscast will compete with WHO, which airs a newscast at 9 p.m. each night on KDSM.
"KCCI is committed to being Iowa's News Leader and we want to make our No. 1 coverage as available as possible to our viewers," KCCI-TV President and General Manager Brian Sather said in a statement. "We know people are looking for news all day long."
How they know that? We have no idea and we're fairly certain they don't either.


text:
Chief Constable Bill Skelly has released a statement to the people of Lincolnshire, after the UK threat level was raised from 'severe' to 'critical' following the Manchester terror attack.
Chief Constable Skelly said: "On Tuesday night, the UK threat level was raised from severe to critical, meaning a further attack could be imminent.
I understand people will be anxious and concerned, and I want to reassure everyone that we are well prepared.Chief Constable Bill Skelly
"Here in Lincolnshire, our 'daily business' does not change and we continue to police professionally and engagingly. We have made some changes in response to the escalation in the threat level.
"People in Lincolnshire can expect to see more officers on the streets, more routine checks and patrols will be taking place and, quite simply, you can expect that those officers will be around for you to approach if you have any concerns or if you need any information.
"Some of those officers will be armed. If you've ever been to a big event in the county or anything large scale and pre-planned, then you'll be used to seeing officers carrying firearms; this is a necessary response to the events of the last few days.
"At the moment you won't see soldiers on our streets. Military capability is there if we need it and, if we do, will be in accordance with a well-considered plan.
"I understand people will be anxious and concerned and I want to reassure everyone that we are well prepared. There are a number of events planned across the county coming up which people might be worried about. We are working with organisers to ensure those events are as safe and secure as possible and we hope that you are still able to go and enjoy them.
"This is an exceptional and difficult time. The key things to take away are that we are constantly reviewing and assessing and that you should be alert, not alarmed. If something seems out of place to you, talk to us. We'd much rather hear about something that comes to nothing than receive information too late to act.
"Please take note of safety advice and, in the very rare event you are caught up in any kind of weapons attack, please remember to follow the 'Run, Hide, Tell' advice.
"I'd also like to take this opportunity to remind people that any type of hate crime will not be tolerated. Emotive incidents like these can sometimes cause discord within communities. Over the coming days and weeks we will be reaching out to make sure those existing bonds and relationships stay strong. If you or anyone you know has been a victim of hate crime please report it straight away on 101 or via Crimestoppers on 0800 555 111.
Chief Constable Skelly added: "Terrorists seek to create fear and distrust. As the police, but more importantly as people, we will not let that happen. We stand with Manchester and we stand together as a country. A small amount of people carry out these heinous crimes and we will not let them win.
"Let us work closely together to unite against those who seek, through violence and extremism, to intimidate or cause fear."


text:
At the end of their time at Wilbury Primary School it is important that we celebrate our pupils' whole time here, their achievements and the friendships they have made. Our year 6 Graduation Assembly is a very special event where we celebrate in song, poem, and awards. Many parents attend and really value this final event for their children.
The Overall Achievement Award Winner


text:
RTA maintenance dollars at work on Westside
Pikes Peak Rural Transportation Authority (RTA) maintenance work on the Westside is scheduled to get started this week. The
work will be accomplished by crews from the City of Colorado Springs, in some cases combining city funding with the infusion
of tax money from the 1-cent RTA tax.
Scheduled projects this week are:
Citywide this year, RTA funds are slated as follows:
Westside Pioneer article


text:
Diergaarde Blijdorp /Rotterdam Zoo has become one of Europe's most beautiful zoos. Dive into the fully covered Oceanium and make a magical voyage over the bottom of the sea and along coastal regions, meeting many marine and coastal inhabitants such as fish (from herring to sharks), puffins, snakes and a large group of king penguins.
You wander through different parts of the world where you will see fascinating animals that feel perfectly at home in specially imitated natural landscapes. A walk through the fully covered Oceanium, the world of sharks, king penguins, sea lions and yellyfish will take an hour or more.
Discover Europe's largest and most beautiful butterfly paradise Amazonica (2013). Tropical temperatures, fragrant flowers, thousants of colourful South American butterflies, the awesome anaconda and greedy piranhas. Experience the Amazon!
In Africa you can end up by the Crocodile River. An exciting decking path takes you safely up past a group of large crocodiles! Once you're at the top you stand eye to eye with the giraffes and at a safe altitude you can admire the other inhabitants of the new savanna such as hyenas, kudus, ostriches and zebras. On the African Gorilla Island you will be delighted by Bokito's playful gorilla family with their little ones.
Okapis, a rare species of forest giraffe, are back once more in Rotterdam Zoo where they can be found in a new enclosure (2015) resembling the central African jungle. They share their new space with various species of birds and plants that are native to their natural habitat. Finish your African discovery at the African Vulture Rock next to the savanna.
Continue your voyage through 'Asia'. Strolling through the Asian marshes you will meet the elephant family in the indoor tropical forest 'Taman Indah' (mostly under cover).
Last but not least discover the Ice Cave and have a magnificient view on the polar bears under water in Artica!
Mostly under cover
Many sections are covered, such as the Oceanium, Taman Indah, the Asia House, Amazonica and the Riviera Hall.
Rotterdam Zoo supports a number of conservation projects in the wild. Blijdorp also actively participates in many breeding programmes for endangered species and is one of the international top ten of zoos in this field.