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OSADL: Realtime Linux for the Atom Processor
OSADL, a consortium of Linux users in the automation industry, has released a realtime Linux kernel for Intel's Atom processor.
The Open Source Automation Development Lab (OSADL) thereby continued its work on its existing RT-Preempt realtime patch for Kernel 2.6.24 that it released in July. The developers have tried to stay as close as possible to Linus Torvalds's mainline Kernel.
The resulting Kernel 18.104.22.168-rt20-atom was successfully tested on Intel's Crown Beach evaluation board and Kontron's microETCpress SP module, with other board testing to follow.
An OSADL webpage provides an overview of the supported features and the available download of the patch.
Issue 180/2015
Buy this issue as a PDF
Dyreza Malware is Back
Powerful man-in-the-middle attack is now targeting online shopping.
Kernel Developer Matthew Garrett Forks the Linux Kernel
Another high-profile coder says the kernel team needs a kinder, gentler culture.
Bugzilla Bug
Bug database has a bug of its own that could allow an intruder to create an unauthorized account.
US Launches New Strategy for Broadband Access
Report focuses federal resources on achieving universal Internet access.
RC4 Finally Gets the Fork
Leading browser makers say "no" to porous encryption algorithm
IBM Says TOR Network a Vehicle for Ransomware
Report from the X-Force group says attackers are using TOR to hide their crimes
Mozilla Announces new Firefox API
Future Firefox extensions will be compatible with Chrome.
Weird Security Flaw Affected Intel Chips for 16 Years
Better read this if you bought your computer before 2011
OpenSSH 7.0 Secure Shell Arrives
Users should upgrade to the new version as soon as possible
Qemu Flaw Lets the Guest Escape
Xen project announces a privilege escalation problem for Qemu host systems


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Small town mourns tragic loss of 30-year-old Katie Hamilton, daughter of volunteer firefighter.
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subset: commoncrawl


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BI (Business Intelligence) Consultant
- Chicago, IL
- Full-time
Company Description
GoodData is on a mission to help global companies monetize big data. We deliver the leading cloud-based analytics platform and business mashups,enabling companies of any size to convert data into business advantage. We attract the best and brightest in our industry who are dedicated to the possibilities of technology innovation. Headquartered in San Francisco, GoodData is privately held and backed by leading venture capital firms, including Andreessen Horowitz, General Catalyst Partners, Fidelity Growth Partners, Next World Capital, Tenaya Capital and Windcrest Partners.
You will join the Account Management team that works with our customers to ensure their success. We are motivated by long-term customer relationship and helping customers maximize the business value receivedfrom GoodData solutions. Our pro-active and strategic approach to account management enables win-win long-term customer relationships. The Account Manager owns the primary responsibility for customer relationship management and success..
Job Description
Our implementation consulting team is multi-skilled, passionate, consultative problem solvers dedicated to making our customers amazingly successful while building a world-class company together!
We are growing and are looking for Consultants who will lead, develop and execute an implementation plan that will make our customers amazingly successful with GoodData's platform solutions.
As a key member of our Consulting team, you will be an expert and advocate that help guide our customers through their implementation. Working directly with customers to understand requirements, you will help customers develop powerful dashboards and data visualizations, and provide best practice advice and training on GoodData.
The right candidate for this position are customer oriented with strong communication and analytical skills, project management expertise, passion for data visualization, reporting and functional BI knowledge. You must be able to articulate value, inspire and sell the future and potential of GoodData and be comfortable consulting our customers.
This role is perfect for someone who has 5+ years of work experience, has a quantitatively-oriented degree (of any variety) from a top-tier school, and who thrives in extremely analytical, technically-oriented, consultative role.
- Serves as project lead and primary interface for customer during GoodData
- Owns all of the functional aspects of customer projects including project management, requirements design and wireframes, data visualization and report creation, and mentoring customers on using GoodData
- Coordinates and works closely with the other members of the GoodData consulting team
- Develop a trusted advisor relationship with customer executive sponsors such that all GoodData activities are closely aligned with the customer's business case and business strategy, allowing the full potential of their GoodData solution to be realized
- Serve as a customer advocate in driving industry best practices and the evolution of the GoodData
- Possess a comprehensive understanding of the GoodData products and services
- Assist the continued development of the GoodData's Consulting team by mentoring other consultants, contributing to our knowledge base and training content, and improving the GoodData development methodologies
& Requirements
- Superior listening, speaking, presentation and writing skills
- Self-directed with the ability to manage simultaneous engagements with minimal
- Ability to set and manage customer expectations and to negotiate appropriate resolutions to issues
- Exceptional project management skills, PMP certification a plus but does not replace relevant work experience
- Expertise in large cloud / SaaS platforms (e.g. Salesforce.com, Marketo, Netsuite, Workday, Google, etc), advertising platforms, and/or social platforms (e.g. Facebook, Twitter, etc) preferred
- Experience working with business intelligence and visualization tools is preferred
- 5+ years relevant work
- Strong consulting skills and experience
- Enthusiastic and data visualization and information design
- BA/BS degree, ideally in a quantitatively-oriented degree (of any variety) from a top-tier school
Are you this person? Do you have the DNA to work in a fast moving, dynamic and exciting, well-funded hot start-up? If so, we want to hear from you.
Additional Information
GoodData is headquartered in San Francisco and located in the cloud at http://www.gooddata.com.
Agency Recruiters Take Note:
Please do not spam us with unsolicited candidate resumes.
Unless you have a fully executed agreement with us, we will consider them a gift.
Do not send resumes to any of the executives.
All your information will be kept confidential according to EEO guidelines.


text:
PDF Password Remover
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text:
Amicus: The Machinery of DeathThe Supreme Court is taking up capital punishment in a big way this term, against a backdrop of continuing foul-ups with lethal injections by states.
Pope Francis' Message Isn't Echoed at Red MassA reminder that the only faith that should matter at the Supreme Court is faith in the Constitution.
How the Supreme Court Justices Spent Their Summer VacationAs the new term begins, can they ever forgive one another?
Jesus TownAn Arizona city council votes for Christian-only prayers, citing the Constitution.
Amicus: 2015 Term PreviewLiberals are bracing for a rightward swing in some of the most important cases on the docket this fall.
Who Feeds the Supreme Court?The judges who send law clerks—the future leaders of the legal profession—are still white men.
Zero Tolerance for ChildhoodA reminder as the school year opens: The juvenile justice system eats kids for breakfast.
All the Wisdom of the AgesMemorizing poems sucks at the time, but it will make your life richer.
Amicus: Sock the VoteOn the 50th anniversary of the Voting Rights Act, efforts to gut the landmark law are as robust as ever.
Should Colorado Execute James Holmes?A jury will soon sentence the mentally ill mass murderer.
The "Term in Review" TranscriptHow will history remember this Supreme Court term? Read what Dahlia Lithwick talked about on her latest episode of Amicus.
Fates Worse Than Death?Justice Kennedy's own logic shows why he should make the Supreme Court abolish capital punishment.
The "Storm Arrives" TranscriptHistoric decisions on same-sex marriage and Obamacare. Read what Dahlia Lithwick talked about on her latest episode of Amicus.
Jerry Brown's Humble, Humane DecisionAll lawmakers should follow his example, especially when thinking about life and death.
The Supreme Court's Most Important Cases of the New TermJust how bad will things be for abortion, affirmative action, unions, and voting rights?
Carly Fiorina's Big LieEven if you take your facts on some sort of sliding scale, her fibs about Planned Parenthood are appalling.
Just Ignore Them?Unfortunately, that won't make publicity-hungry, unserious, outrageous people go away.
The Supreme Court Has Nothing to Hide, So Don't LookStephen Breyer swears the justices are just the nicest people.
Amicus: Sandra and RuthThe author of a new book about the first two female justices describes what it took for them to get to the high court.
Sonia Sotomayor, OutsiderIn a remarkable conversation, the justice talks about why she doesn't feel like she belongs on the Supreme Court.
Make This Death Not in VainHow did a mentally ill man charged with a trivial crime waste away in jail for months?
A Regrettable DecisionThis astonishing, anti-science, states'-rights court decision begs for a ban on abortion.
When Prosecutors Believe the UnbelievableA man is finally freed, but that doesn't mean the system worked.
Official ActsFormer Virginia Gov. Bob McDonnell just lost his best chance of staying out of prison.
The Planned Parenthood Hearings Aren't About Those Videos They are group therapy sessions for pro-lifers.
Dyrehavsbakken: The World's Oldest Amusement Park First Opened in 1583 and You Can Still Go There Today
Slate Plus
Introducing the Best of Slate Podcasts Only have time for one podcast this week? Make it this one.
Future Tense
Good News for Kanye West: California Bans Paparazzi Use of Drones to Spy on Celeb Homes


text:
On the Middle East with Ian Black + Israel
In the year since the 2014 war ended little has happened to change the conditions that will inevitably lead to another one, warns a bleak new report
Arab-Jewish relations in the Holy Land never enjoyed a golden age, but lives now scarred by unending conflict were once lived in common, argues an original new study
New book tackles intriguing question of whether the leader of the notorious Stern Gang was really shot 'while trying to escape', Ian Black writes
Leaked details of US secretary of state's framework proposals do not boost already low expectations that a deal can be done
Bashar al-Assad looks like the least bad option as fears mount of growing strength of jihadi groups, writes Ian Black
Ian Black writes: Declassified documents reveal how US intelligence helped Jimmy Carter strike a Middle Eastern bargain
Ian Black writes: The 30-year-old 'axis of resistance' based on Iran is still alive and supporting Bashar al-Assad
Ian Black: European statesmen express 'strong concern about the dying chances of a settlement based on two separate, sovereign and peaceful states of Israel and Palestine.'
Ian Black: Arab League determination to tackle regional crises masks doubts about whether it can make a difference
Ian Black: Centrist Israeli politician to lead future negotiations with Palestinians. But substantive talks still look unlikely.
Ian Black: 'Cooperation' with Israel over West Bank water supplies helped consolidate illegal settlements and undermine the two-state solution, a new study shows.
Ian Black: The Palestinian issue looks like being sidelined amidst apathy, despair and uncertainty elsewhere in the region
Newly-declassified secret British government documents shed light on a key moment in the Middle East and the elusive search for an Israeli-Palestinian peace settlement
Ian Black: The hit drama show is generally well researched and, on the whole, plausible. But cognoscenti might well ask why there are posters in Hebrew in what is supposed to be Lebanon
Rare auction of Arab world currencies provides rich pictorial display of region's turbulent modern history, Ian Black writes


text:
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text:
It's been a while since we've gotten anything new from one of my favorite workout hotties Julianne Hough, but then I stumbled across this old workout video of hers on YouTube, and trust me, it'll definitely get your heart rate pumping. And since it would probably take a couple days to watch the full hour video 30-45 seconds at a time, my favorite part is at 13:11. That's definitely going to be playing on a loop in Tuna headquarters for the rest of the week. So enjoy. And just remember to stretch first.
CELEBRITIES > Julianne Hough
Julianne Hough's Workout Video Will Make You Pump It
Julianne Hough Needs A Pair Of Daisy Dukes
I know it's been a while since we've gotten a good leg show from Julianne Hough and I'm sorry to disappoint you perverts, but it looks like we're going to have to wait a little longer. Because the baggy jean shorts on Julianne here definitely aren't doing it for me. What happened to a nice skintight pair of leggings? I know Julianne probably doesn't want to take fashion advice from a guy who thinks sweatpants count as formal wear, but trust me, with a booty like hers, spandex is the perfect choice for any occasion. It goes with everything.
Photos: PacificCoastNews
Julianne Hough Works It Good For Shape Magazine
Because these days, even fitness mags seem to be putting out hotter photoshoots than the so-called men's magazines, here's Julianne Hough in the December issue of Shape with a shoot so good this thing should probably come in a brown paper bag. I've always said that Julianne is one of my favorite workout hotties, but forget the leggings, if she worked out in outfits like this, I think she could probably even take the top spot someday.
Julianne Hough Reveals Some Awesome Boobage
So Julianne Hough showed up to do a segment on Extra the other day, and normally, I'd complain about how now that Maria Menounos and her perfect Greek booty are gone, it robbed us of the chance for one hell of a lesbian fantasy. But forget all that, because thanks to this outfit Julianne's got on and all that cleavage of hers, she's currently giving me more than enough for a fantasy all on her own. Like the one where the two of us are stranded on a desert island and our only hope is to motorboat our way back home.
Photos: PacificCoastNews, Fameflynet
Julianne Hough Wears Short Shorts
It's been at least a couple months since we've gotten a good Julianne Hough leg show, and even longer since we've seen my favorite workout hottie in a pair of leggings. It's like she doesn't even want to be perved on by a bunch of dudes over the Internet or something. I know, weird, right? But good news, because we're in luck today thanks to these shots of Julianne in short shorts. Phew, and just in time too. Because any longer and I would've had to file a missing person's report for that perfect booty of hers. I was starting to get worried about it.
Photos: WENN.com
Just In! Julianne Hough Takes Out The Trash
I've posted a lot of hotties doing a lot of weird things over the years, but I think this might be the first time I've ever done a post on one taking out the trash. I know, what's taken me so long, right? Anyway, for the rest of you pervs out there who don't have a garbage fetish, you'll probably be way more interested in the peek Julianne Hough's giving us at her bra here. See, now this is why I always knew Julianne would become a big star someday. She even knows how to make garbage day hot. Well done.
Photos: WENN.com
Julianne Hough, Nikki Reed And Cara Santana Work It Good In Leggings
You know, I've been at this blogging game a long time, and if I've learned anything over the years, it's that the only thing better than one hottie in spandex is four of them. I'm pretty sure that's just a proven scientific fact. So anyway, here's Julianne Hough, Nikki Reed, Cara Santana and their hot nobody friend all sweating it out and going for a walk together. And after that, they hit the showers together too. At least according to
my sources the fantasy playing out in my head right now.
Photos: WENN.com
Julianne Hough Reminds Me Of Her Brother
So in case you've been wondering why I haven't put up any new workout pictures from Julianne Hough lately, here you go: she cut all her hair off a while back and now she looks like her brother. Or even worse, like Anne Hathaway. I guess it's not all bad though, because her body still looks as good as ever in those leggings and that tight tank top. Man, I'm so confused. Does this mean I'm gay?
Photos: WENN.com
Julianne Hough Pictures Are A Real Workout
Now that it's January, gyms everywhere are packed with a bunch of people convinced that they're actually going to follow through with their New Year's Resolutions this year. And by February, they're going to be empty again. Which is why I try to make my yearly goals way more manageable. For instance, this year instead of going to the gym myself, I resolved to look at more pictures of Julianne Hough going to her gym. So far, so good. And trust me, twenty minutes with these leggings pictures is a much better cardio workout than any treadmill or elliptical.
Photos: Fameflynet, PacificCoastNews
Julianne Hough and Cara Santana Make A Great Pair
Here's BFF Julianne Hough and Cara Santana working the streets the other day in LA. I'm a little disappointed that Julianne didn't wear her trademark leggings, but luckily Cara made up for it by sporting an awesome pair of Daisy Dukes. I want to see these two go at it in a leg wrestling match. I think Julianne being the dancer would win, but we'd be the big winners.
Photos: WENN.com
Julianne Hough's Delicious Cheeks In Leggings!
Over the last few weeks Julianne Hough tried to change up her outfits and ditch the leggings. However, none of her choices seemed to work. I guess when you were born to wear a specific article of clothing you stick with it. Here she is leaving the gym after a workout wearing a pair of leggings which showcase her awesome little booty. All I know I'd love to sink my teeth into that cheek. Delicious!
Photos: WENN.com
Julianne Hough's Booty Shot Needs A Little Work
Now that Julianne Hough has expanded her wardrobe to include outfits other than skintight leggings, her booty posing could use some work. See, she hasn't properly adjusted for all that added fabric. Because here she is attempting to model at the gas pump like all the other young hotties, but it's not quite working. Luckily, the Tuna is here to help. First thing Julianne needs to do is get better jeans, and by better, I mean tighter. Then she needs to arch her back more. And finally, get that phone out of her back pocket. It's my patented three-step method for perfect booty shots, and it works every time. Now it's just up to Julianne to put these lessons to good use.
Photos: Fameflynet
Breaking News! Julianne Hough Is Not Wearing Leggings!
Breaking news, everybody! Julianne Hough went out in public again wearing something other than leggings. So you might want to keep an eye out for flying pigs, just in case. Anyway, luckily Julianne went from one of my favorite hottie outfits to the other, but as much as I love Daisy Dukes, these are a bit too baggy. It's still a nice leg show, and I figure Julianne will get the hang of this new look in a couple days. You can't expect her to be an expert right out of the gate.
Photos: Fameflynet


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My Nonprofit Reviews
Review for Last Chance Animal Rescue Inc, Waldorf, MD, USA
It has been three years since my dog died at this clinic without having a neuter surgery, on April 28, 2008 They used Xylazine 100mg/ml labeled for cattle & horses. The drug manufacturer stated NOT recommended for cats & dogs, yet they used it anyway. My dog died at this clinic . I do not recommend this clinic. My life has been changed by this and my heart forever remais broken. I had to pick a star as a rating but they don't deserve not even one star.
Review for Last Chance Animal Rescue Inc, Waldorf, MD, USA
Waldorf Well Pet Clinic is where I took my dog to have a neuter surgery not knowing that this clinic was operated by last chance rescue. My dog was given a drug 43 times with a higher risk factor than other comparable ,safer drugs and he died before any surgery occurred.(XYLAZINE)T,his is a drug that is over 30 years old now their are safer drugs.I was charged for a surgery that NEVER happened. My dog should not have died. My life will never be the same. My dog was only 2 years 10 months old and he was found to be a normal dog on a necropsy report. I DO NOT RECOMMEND THIS FACILITY TO ANYONE.
More Feedback
What I've enjoyed the most about my experience with this nonprofit is...
The kinds of staff and volunteers that I met were...
I had a HORRIBLE EXPERIENCE HERE.
If this organization had 10 million bucks, it could...
In my opinion I am scared for the animals.
Ways to make it better...
My dog had not died using a drug off label without my informed consent and if I had been made aware that this clinic was operated by a rescue group to begin with. I was NOT informed.
In my opinion, the biggest challenges facing this organization are...
The way they treat people when their dog dies at their facility and not informing clients that they are affillated with a rescue group. Using drugs not recommended by the drug Manufacturer.
One thing I'd also say is that...
How frequently have you been involved with the organization?
One time
When was your last experience with this nonprofit?


text:
Search: open-mindSearch profiles »
MasseurForFemaleMatch % Unknown Join to find out!
An Intelligent Man will open Your Mind. A Handsome Man Will Open Your Eyes. And A Gentleman Will Open Your Heart. If you love me I'll always be in your heart, if you hate me I'll always be in your mind.
mystic_writrMatch % Unknown Join to find out!
always meantand whatever a sun will always sing is youhere is the deepest secret nobody knows(here is the root of the root and the bud of the budand the sky of the sky of a tree called life;which growshigher than soul can hope or mind can hide)and this is the wonder that's keeping the stars aparti
birdsofbrooklynMatch % Unknown Join to find out!
wall stare wrack dish invent invert per form filth songz in mind in itroat inside outlowdoh luvly littl ditty doo ditty y dontchew caws
ColorsWolfMatch % Unknown Join to find out!
If you are always open to all possibilities, an honest person, love to laugh, never separate parts of yourself like your emotions, and don't believe in the concept of shame.
- See More Profiles »
The Sexual Scenarios Test
This is the sexual scenarios test. Since I know that ALOT of people masturbate while on OKCupid I decided to write this test for those of you who like...
The how open is your mind Test
"A mind is like a parachute: it works better when it's open." so what do you think about that? or don't you care?
The Open mind Test
This is my first test. What I'm looking for is the open mindedness in people. I am a very openminded individual, just very opinionated. Well, enjoy!
This test is hidden based on your maturity setting. Change your maturity setting
The OPEN MINDED Test
This is my test to see exactly how open minded u can be with ur sexuality and how open minded u can be in your relationship.
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text:
Best of News
Canadian singer Garou performs during the 26th Telethon, France's... News PhotoAnnual Event,Biggest,Biological Process,Event,France,Global Communications,Live Event,Paris - France,Performance,Saint-Denis,Seine-St-Denis,Social Issues,Telethon,VerticalPhotographer AFPCollection: AFP 2012 AFPCanadian singer Garou performs during the 26th Telethon, France's biggest annual fund-raising event during 30 hours of live television transmission, on December 8, 2012 in Saint-Denis, north of Paris. The event aims at collecting funds for research on genetic diseases such as myopathy, a neuromuscular disease. This year's edition raised 81,065,239 Euros. AFP PHOTO THOMAS SAMSON (Photo credit should read THOMAS SAMSON/AFP/Getty Images)


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Doc McStuffins Musical Light Up Microphone
Item No.1710103328
Item No.1710103328
70% Off - Save a total of £5.25
Dottie 'Doc' McStuffins, a six year old girl, 'fixes' toys, with a little help from her stuffed animal friends, Stuffy, Hallie, Lambie and Chilly.This cool Doc Mcstuffin Microphone lets girls take the stage like a superstar! This Doc Mcstuffin's Microphone makes singing fantastic fun. Sing-along to "Time for your check-up" song from the Doc McStuffins show with this musical microphone and also see cool lights flash up as they sing to their heart's content. Portable and attractive, this pink & purple item features Doc Mcstuffin on the handle with flashing lights! For ages 3+
UK Home Delivery Free (UK orders over £30) or £3.49
Delivered within 5 working days
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Sunday to Friday - order by 9pm - £3.99
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Collect from store the next day - FREE
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Subject to exceptions, Debenhams is happy to exchange or refund your purchase within 28 days of delivery. Please check here for more details on our returns policy and exceptions.
From 1st July 2007, all retailers of electrical goods must comply with the Waste Electrical and Electronic Equipment (WEEE) legislation.
This is a scheme to prevent all such waste being disposed of in general landfill sites and to promote recycling of components and materials. The legislation aims to minimise the impact of electrical and electronic equipment on the environment by creating products with recycling in mind and encouraging reuse, recycling, and recovery of WEEE.
How do I know what product is "WEEE"? The definition of a WEEE product is one whose primary function is dependent on electrical currents to carry out the main function, e.g. a toaster. However, some product, such as a child's soft toy with an electric circuit, is not classed as WEEE as its primary function is a toy. For further details on product definitions, please click here.
You may also take the item(s) to your local authority Civic Amenity Site if this is more convenient.


text:
Bon Secours Health Jobs in Richmond, VA
Hiring? Post a Job
Show: All Results Last 7 Days
No jobs found – change your filters above for more results
Bon Secours Health Reviews
Recommend to a friend
Approve of CEO
Rich Statuto
36 Ratings
"hospital employee - Memorial Regional "
RecommendsPositive OutlookNo opinion of CEO
- Work/Life Balance
- Culture & Values
- Career Opportunities
- Comp & Benefits
- Senior Management
I have been working at Bon Secours Health (More than 5 years)
It's corporate - if you know how to play the game, you're in. The benefits are tremendous.
the talk is not always aligned with the walk. Always needing to be cautious as you never know how someone is interpreting what you are doing erroneously.
Advice to Management
Be more authentic with your employees - and show appreciation and praise !
Work at Bon Secours Health? Share Your Experiences
Bon Secours Health
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Don't Miss Out On a Job You Love
Upload a resume to easily apply to jobs from anywhere. It's simple to set up.


text:
RIP3 (Receptor-interacting protein 3) pathway was mainly described as the molecular mechanism of necroptosis (programmed necrosis). But recently, non-RIP3 pathways were found to mediate necroptosis. We deliberate to investigate the effect of calpain, a molecule to induce necroptosis as reported (Cell Death Differ 19:245–256, 2012), in RGC-5 following elevated hydrostatic pressure.
First, we identified the existence of necroptosis of RGC-5 after insult by using necrostatin-1 (Nec-1, necroptosis inhibitor) detected by flow cytometry. Immunofluorescence staining and western blot were used to detect the expression of calpain. Western blot analysis was carried out to describe the truncated AIF (tAIF) expression with or without pretreatment of ALLN (calpain activity inhibitor). Following elevated hydrostatic pressure, necroptotic cells pretreated with or without ALLN was stained by Annexin V/PI, The activity of calpain was also examined to confirm the inhibition effect of ALLN. The results showed that after cell injury there was an upregulation of calpain expression. Upon adding ALLN, the calpain activity was inhibited, and tAIF production was reduced upon injury along with the decreased number of necroptosis cells.
Our study found that calpain may induce necroptosis via tAIF-modulation in RGC-5 following elevated hydrostatic pressure.
Keywords:Retinal ganglion cells-5; Calpain; Elevated hydrostatic pressure; tAIF; Necroptosis
Calpains are calcium-activated neutral protease, which belongs to the family of cytosolic cysteine proteinases. They form heterodimers which are composed of a large 80 kDa catalytic subunit and a common 30 kDa regulatory subunit [1]. The calpains are widely distributed in most of the mammal tissues. The calpains are also implicated in physiological and pathophysiological processes, such as cytoskeletal reorganization, signal transduction pathways, cell cycle regulation and certain apoptosis pathways. The dysfunction of calpains is related to certain diseases like cataract, Parkinson's disease and Alzheimer's disease [2-5].
During cerebral hypoxic-ischemia, there's an overload of intracellular calcium which activates calpains, as a result neuronal apoptosis is triggered via caspase-3 activated pathway [6]. Recent studies show that calpains, caspase-3, caspase-8 and caspase-9 are all up-regulated in experimental retinal detachment, which suggests calpains are involved in caspase-dependent photoreceptor death [7]. Pharmacological inhibition of phosphodiesterase 6 (PDE6) induces retinal degeneration in rod and cone-enriched retinal explants with activation of caspase-3, calpain and poly (ADP-ribose) accumulation, which suggests a potential connection between calpain activation and apoptosis [8]. However, besides its role in apoptosis, a new feature of calpains has been found recently. Cellular necrosis, which is mediated by recombinant clostridium perfringens b-toxin (rCPB) occurs upon the activation of host cell calpains [9]. Another study reported that calpains may be involved in necroptosis as well [10]. Calcium-dependent calpain is activated by increasing calcium concentration in cytoplasm in N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG)-treated cells. The activated calpains cleaves BID (BH3 interacting domain death agonist) to trucked BID sequent; tBID redistributes from the cytosol to mitochondria where it regulates BAX (Bcl-2-associated X protein) activation. Once activated, BAX provoked mitochondrial tAIF release and resulted in necroptosis [10,11].
High intra-ocular pressure (HIOP) is identified as one of the characteristics of glaucoma and it is the main factor that causes visual functional damage [12]. Related studies have been confirmed that elevation, volatility and continuous rise of intraocular pressure (IOP) could cause the death of retinal ganglion cells (RGCs), retinal pigment epithelium cells, etc. and eventually lead to vision loss [13,14]. Rapid elevation of IOP is a critical susceptible factor that causes acute glaucoma. The study indicated that RGCs necroptosis could also exist at the early stage of aHIOP (acute high intra-ocular pressure) [15]. Our further investigation suggested that up-regulation of receptor-interacting protein 3 (RIP3) might be involved in cellular mechanism of RGCs necroptosis [16]. As reported previously, RIPs is not the only cellular pathway which modulates early neuronal necroptosis, other cellular pathways may also participate in this process [17-19]. As mentioned earlier, the potential role of calpain involved in necroptosis in RGC after aHIOP is still unknown and further studies need to be done to evaluate it. Moreover, the various types of complex pathophysiological mechanisms in aHIOP-induced retinal injury, including retinal hypoxic-ischemia, accumulation of excitatory amino acid and inflammatory molecules, etc. needs to be considered [20,21]. But little is currently known about whether there is necroptosis in RGC under elevated hydrostatic condition, which is an initial factor in aHIOP-induced retinal injury in vivo, hence, we investigated the existence of necroptosis in elevated hydrostatic condition states by PI-staining and flow cytometry, and then evaluated the effect of calpain in necroptosis of RGC under elevated hydrostatic condition. We expect that the results will lead to a better understanding of the cellular mechanism of early RGC necroptosis and looking for rational interventional targeted therapy in the future.
Cell culture
The mouse retinal ganglion cell line (RGC-5) was contributed by Department of Ophthalmology, Second Hospital of Ji Lin University in China [22]. RGC-5 cells grew in Dulbecco's Modified Eagle Medium (DMEM) (HyClone Laboratories, Inc. UT) and supplemented with 10% fetal bovine serum (FBS, HyClone Laboratories, Inc. UT), 100 U/ml of penicillin and 100 μg/ml of streptomycin (HyClone Laboratories, Inc. UT). The RGC-5 cells used in the experiment were within 2-3 passages post-thawed to minimize the variability in the assays based on our observations. The density of RGC-5 cells was around 80% in 6 ml culture media in 50 ml flask before EHP (elevated hydrostatic pressure).
PCR analysis
The cells cultured in the flasks were harvested and RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA). cDNA was synthesized using Thermoscript (Invitrogen, Carlsbad, CA) from 1 μg of total RNA. Each primer pair (β-actin: forward primer
reverse primer 5′-ACTTTTATTGGTCTCAAGTCAGTGTACAG-3′.
NGF: forward primer 5′-AGAACCGTACACAGATAGCAA-3′
reverse primer 5′-TTAATGTTCACCTCGCCCAG-3′.
BDNF: forward primer 5′-AAAACCATAAGGACGCGGACTT-3′
reverse primer 5′-AAAGAGCAGAGGAGGCTCCAA-3′.
GFAP: forward primer 5′-ACGCAATTCCATTTTACCTG-3′
reverse primer 5′-AGGCCATAACTCATGCAAC-3′) was designed to cross at least one intron and was specific to the gene of interest. Negative control was used diluted water for instead. Oligonucleotide primers that recognized the housekeeping gene β-actin were used as normalized control. PCR program consisted of 94°C for 3 min, 36 cycles of 94°C for 45 s, 55°C for 50 s, and 72°C for 2 min, with a final extension at 72°C for 10 min.
Immunofluorescence staining
The coverslips with fixed cells were washed in 0.01 M PBS for 3 min, incubated in 5% BSA and then reacted with anti-rabbit calpain antibody (Abcam, ab39170, USA, 1:500) or anti-rabbit Brn3a (Boster, BA1773, China, 1:200) and anti-mouse THY1.1 (Abcam, ab8872, USA, 1:500) overnight. Then coverslips were reacted with Cy2 or Cy3-conjugated donkey anti-rabbit or anti-mouse secondary antibodies at 1:200 (Invitrogen, USA). Counterstaining with DAPI, then covered with an anti-fading mounting medium before microscopic examination. Control RGC-5 incubated simultaneously in a conventional incubator at 37°C.
Cell injury and ALLN or Nec-1 usage
A pressurized incubator was designed to expose the cells to an elevated hydrostatic pressure as described in Ju's study [23]. After 2 hr exposure in this pressure system, the pressure present in three different values: 100 mmHg, 60 mmHg and 30 mmHg, cells were then moved to conventional culture incubator to recover at each recovery time point (6 hr, 12 hr and 24 hr). ALLN (Merck, Germany) and Nec-1 (Sigma-Aldrich, USA) were dissolved in Dimethyl sulfoxide (DMSO) for storage in 10 mmol/L and 1 mg/mL, which were pretreated in 10 μmol/L for 24 hr before cell injury.
PI staining
At each recovery time point (6 hr, 12 hr and 24 hr), the coverslips were washed in 0.01 M PBS for 3 min, and incubated in 10 μg/ml PI-dye solution at 37°C. After that, cells were fixed in 4% PF (Paraformaldehyde) and washed in PBS counterstaining with DAPI, and then covered with anti-fading mounting medium before microscopic examination. Control RGC-5 was incubated simultaneously in a conventional incubator at 37°C. Quantitative analyses were conducted using approximately 20 merged images (magnification = 40×) to estimate the frequency of cell necrosis.
Western blot
At each survival time point, cells were digested by sonication on ice in a digestion buffer [150 mM NaCl, 25 mM Tris–HCl (pH 7.4), 2 mM EDTA, 1.0% Triton X-100, 1.0% sodium deoxycholate, 0.1% SDS] containing a cocktail of protease inhibitors (Sigma, USA). Cell lysates were centrifuged at 10,000 × g for 20 min at 4°C. The supernatants were collected, and protein concentration was determined by Bicinnchoninic acid (BCA) assay (Pierce, USA). A total of 100 μg of protein in 62.5 mM Tris loading buffer (pH 6.8, containing 25% glycerol, 2% SDS, 0.01% bromophenol blue and 5% β-mercaptoethanol, Bio-Rad, USA) was boiled for 10 min, and loaded into each lane of 4-20% linear gradient Tris–HCl ready gel (Bio-Rad, USA). The polypeptides were electrotransferred to Trans-Blot pure nitrocellulose membrane (Bio-Rad, USA). Non-specific binding was blocked with PBS containing 5% nonfat milk (Bio-Rad, USA) and 3% bovine serum albumin (Sigma, USA). Membranes were incubated with tAIF (Santa Cruz biotechnology Inc, SC-113116, USA, 1: 200), β-tubulin (Abcam, ab6046, USA, 1:1000), calpain (Abcam, ab39170, USA, 1:500) or actin (Abcam, ab3280, USA, 1:500) antibodies overnight, and then in HRP-conjugated secondary antibodies (1:20000, Bio-Rad, USA) for 1 hr. Immunoblotting products were visualized with an ECL Plus™ Western Blotting Detection kit according to manufacturer's instruction (GE Healthcare Life Sci., USA), and images were captured in a Molecular Dynamics Phosphor imager (Nucleo Tech Inc., USA). Western blot bands were measured with Image J (National Institutes of Health, USA) to analyze the integrated density value (IDV). The average IDV values of tAIF or calpain with β-tubulin and actin were compared, and the average relative value was obtained.
Flow cytometry
The cells attached to the flasks were trypsinized followed by a gentle wash. Resuspending the cells in 200 μl of 1× binding buffer, and then added 5 μl of 20 μg/ml Annexin V and 10 μl of 50 mg/ml PI, incubated at RT for 15 min in the dark. After the cells were washed and analyzed by FACS Calibur (Becton, Dickinson Company, USA). The percentages of cells in each quadrant were analyzed using ModFit software (Verity Software House Topsham, USA). Statistical results of flow cytometry were conducted by calculating the PI+ cells numbers. All the results were repeated for three times.
Calpain activity assay
Calpain activity was determined by cleavage of the substrate Ac-LLY-AFC (Abcam, USA). The attached cell were digested, and then centrifuged for 1 min in a microcentrifuge (10,000 × g) then the supernatant was transferred to a fresh tube, a part of the supernatant were used to measure the protein concentration. The fluorescence was measured after 60 min incubation at 37°C along with the substrate in the reaction buffer. Fluorescence was recorded in a Fluoroskan Ascent Fluorimeter (Labsystems, Eragny-Parc, France), and the final results were expressed as Relative Fluorescent Unit (RFU) per milligram protein of each sample.
Data analysis
Figure panels were assembled by using Photoshop CC. The data were analyzed by using SPSS 19.0 (SPSS, USA). One-way analysis of variance (one-way ANOVA) was performed to test differences in average value between groups. All results were presented as mean ± SD. A value of p < 0.05 was considered statistically significant.
RGC-5 has the same features with retinal ganglion cells
First we verified that the RGC-5 cells in our culture conditions expressed BDNF and NGF, but not GFAP (Figure 1A). As shown in Figure 1B, the cells expressed RGC marker Brn3a and Thy1.1. The transcription factor Brn3a was expressed and appears to localize at the nuclei of RGC-5 cells. On the other hand, the expression of Thy 1.1 was detected in the cytoplasm of RGC-5 cells. Negative control immunostaining (second antibody only) showed no positive signal (data not shown), confirming the specificity of the antibodies. Therefore, expression of RGC marker genes and protein indicated that the cells cultured were RGCs.
Figure 1. Identification of RGC-5 cell line. Panel A: RT-PCR show retinal ganglion cell marker gene expressed in our cultured cell, Panel B: Two other types of protein marker also detected with immunostaining, scale bar = 50 μm.
Necroptosis happened at early stage of EHP
Propidium iodide (PI) is one of the dyes that emit red fluorescent under the excitation of 535 nm with the combination of DNA double strain through necrosis cell's membrane, but it does not penetrate into live cells. Thus, PI dye can be used to distinguish the necrosis cell from normal ones [24-27]. DAPI is one of the dyes that emit blue fluorescent under the excitation of 340 nm with the combination of DNA double strain through 4% PF fixation. It can identify PI-staining to be a realistic cell but not false-positive dying [28]. The double labeling of PI and DAPI showed that there was no significant PI staining in 30 mmHg insults (data not shown). With the counterstaining of DAPI, a few PI-positive cells were observed in the condition of 60 mmHg (Figure 2). In our observation time point, the number of PI-positive cells gradually increased to 13.45% in 12 hr and decreased to 4.32% afterward in 24 hr (Figure 3B). In the condition of high pressure in 100 mmHg, necrosis also occurs at 6 hr (Figure 3A). The number of PI-positive cells reached 15.35% at 12 hr, and gradually decreased at 24 hr (Figure 3B). These results indicated that within our observation time point, there was a small number of necrosis in RGC-5 cell line under 60 mmHg and the degree of cell necrosis increased following the pressure elevation (Figure 3B). During the same time point, we observed that as the pressure level elevated, the number of necrosis cells increased too. This result indicates that RGC-5 necrosis is affected in a pressure-dependent manner. Correspondingly, it is suggested that under 100 mmHg there is more necrosis cells in RGC-5. In order to be consistent with the conditions in vivo[29], we believed that 100 mmHg insult condition was suitable for the next step of our experiment for necroptosis detection.
Figure 2. Quantitative analysis of necrosis cells in low pressure. PI/DAPI staining in 60 mmHg, scale bar = 100 μm.
Figure 3. Quantitative analysis of necrosis cells in high pressure. Panel A: PI/DAPI staining in 100 mmHg, scale bar = 100 μm; Panel B: The statistical analysis of RGC-5 necrosis.
It's difficult to precisely identify between necroptosis, apoptosis or necrosis in various methods until recently [30]. Nec-1 is the specific inhibitor of necroptosis, as reported in other studies the cell number of necrosis decreased when using it [31,32]. In order to analyze necroptosis, we detected the PI-positive cells to analyze cellular necroptosis by flow cytometry with PI/Annexin V double staining using Nec-1. In our model of cell injury in 24 hr in 100 mmHg, we found that the ratio of necrosis cells (PI+) was about 13% (Figure 4B), but the percentage decreased to nearly 5% (Figure 4C) within the use of Nec-1 which have significant difference in statistical analysis (Figure 4D). These results indicate that with the exposure of Nec-1, necrosis is inhibited, thus necroptosis occurred after EHP.
Figure 4. Quantitation of necrotic cells under hydrostatic pressure and Nec-1 usage. Panel A: Normal control cells; Panel B: RGC-5 cell lines necrosis under elevate hydrostatic pressure; Panel C: RGC-5 cell lines were treated with necrostatin-1 (10 μmol/L) to block necroptosis for 24 hr in cell density of 0.6 × 105/ml before elevate hydrostatic pressure and analysis of necroptotic cells. Cells were stained with Annexin FITC and PI, and analyzed by FACS using FL1 (Annexin) and FL3 (PI) channels. Panel D: The statistical analysis of RGC-5 necrosis, * vs # p < 0.05.
Calpain is up-regulated following elevated hydrostatic pressure
Immunofluorescence staining results showed that calpain is mainly present in cytoplasm in normal control group in RGC-5 (Figure 5A); meanwhile, no difference in distribution was observed between injury group and normal controls. Generally, in contrast with the normal controls, significantly more distinct and heavier calpain immunoreactive was found with strong cellular labeling of calpain in 6 hr and 12 hr groups in high pressure models, but weaker labelling in 24 hr group. The western blot results showed that calpain exhibited mainly as a single 75 kDa band in all groups (Figure 5B). The bands in high pressure groups were apparently thicker and larger than those of normal control groups. The bands in 24 hr group were thinner and smaller than those in injury groups and tended to be normal. Statistical analysis of IDV indicated that high pressure up-regulated the expression of calpain in the early stage (Figure 5C), significantly more distinct calpain bands were shown in 12 hr injury group (p < 0.05). It demonstrated that calpain distribution have no difference between injury groups and normal controls, the protein expression levels increased at first and then decreased as time extend within 1 day, which reached the maximum detection in 12 hr group.
Figure 5. Calpain protein expression level detection. Panel A: Immunofluorescence staining of calpain; Panel B: Western blot of calpain after cell injury; Panel C: The statistical analysis of calpain western blot bands; *: 6 hr, 12 hr and 24 hr group vs CTL group: p < 0.05, scale bar = 100 μm in panel A.
AIF cleavage product decreased after calpain inhibition
At first, the bands of tAIF in high pressure groups of 6 hr, 12 hr and 24 hr increased and then decreased as time extended the maximum detection in 6 hr group (Figure 6A). The statistical analysis of IDV suggested that high pressure up-regulated the cleavage of tAIF in the early stage (Figure 6B), significantly more distinct tAIF band were shown in 12 hr injury group (p < 0.05). After ALLN addition, no significant changes of tAIF band in all four groups were detected (Figure 6C), the statistical analysis of IDV also confirmed about these results (Figure 6D), which indicated that there is no difference in protein expression levels in tAIF production in all groups.
Figure 6. tAIF protein expression level detection. Panel A: Western blot of tAIF after cell injury; Panel B: The statistical analysis of tAIF western blot bands after injury; Panel C: Western blot of tAIF after cell injury pretreated with ALLN; Panel D: The statistical analysis of tAIF western blot bands after calpain inhibition. *: 6 hr group vs other groups: p < 0.05.
Calpain activity assay
With both the inhibitor and injury groups preteated with ALLN for 24 hr, the calpain activity assay appeared to be higher in the inhibitor group (Figure 7). However, at 12 hr there were significant changes between the inhibitor and injury group (p < 0.05). It suggested that ALLN may effectively inhibit up-regulation of calpain activity following hydrostatic pressure treatment.
Figure 7. The statistical analysis of calpain activity assay. CTL: Normal control RGC-5; CTL-positive: Normal control RGC-5 cell lysate with active calpain addition according to the detection kit; CTL-inhibitor: Normal control RGC-5 cell lysate with Z-LLY-FMK addition according to the detection kit; 6 hr, 12 hr and 24 hr means the survival time point after cell injury; 6 h-ALLN, 12 h-ALLN and 24 h-ALLN means pretreated with ALLN for 24 hr before cell injury; *: 12 hr group vs 12 hr ALLN treatment group: p < 0.05.
ALLN may decrease the rate of RGC-5 necrosis
Under 100 mmHg insult, our study showed the occurrence of necroptosis in RGC-5 for 2 hr but recovery at 24 hr. Therefore, the cells were treated under this condition with the addition of ALLN. After that, we analyzed cellular necroptosis by using flow cytometry with PI/Annexin V double staining to detect whether it could decrease the rate of necrosis in RGC-5 under high pressure condition after calpain activity has been inhibited. The results showed that the ratio of necrosis cells is about 12% (Figure 8B), the percentage decreased to nearly 8% upon adding ALLN (Figure 8C) in the injury group in 24 hr. Similar with Nec-1 treatment, it demonstrated that the ratio of RGC-5 necrosis decreased when treated with ALLN under high pressure condition. Meanwhile, statistical analysis indicated that there were significant changes in PI-positive cells upon adding ALLN compared to normal control group and EHP group (Figure 8D), these results indicated that RGC-5 necroptosis in the early stage may be related to the up-regulated calpain activity.
Figure 8. Quantitation of necrotic cells under hydrostatic pressure and calpain inhibition. Panel A: Normal control cells; Panel B: RGC-5 cell lines necrosis under elevate hydrostatic pressure; Panel C: RGC-5 cell lines were pre-treated with ALLN to inhibit calpain activity for 24 hr before elevating hydrostatic pressure; Panel D: The statistical analysis of RGC-5 necrosis. Cells were stained with Annexin FITC and PI, and analyzed by FACS using FL1 (Annexin) and FL3 (PI) channels. *: 24 hr group vs CTL or 24 hr-ALLN treated group: p < 0.05.
HIOP is one of the main features and risk of visual impairment with ganglion cell death in glaucoma [33-35]. Some research demonstrated that acute increase or continuous arise of intra-ocular pressure (IOP) could lead to visual injury or cell death in retinal ganglion cells, pigment epithelial cells and finally resulting in vision lost. Particularly, retinal ganglion cell death is the most important behavior [36,37] in acute glaucoma. The pathological research model of acute glaucoma is divided into animal model and cell model [16,38,39]. Elevated hydrostatic pressure (EHP) in cultured cell line is commonly used in glaucoma cell model in vitro[40]. In this study, we used open cycling air pressure culture system to gain hydrostatic pressure, the system could set pressure values as required and may easily gain more information via high-throughput experiments [41].
In our study, we chose three pressure values (100 mmHg, 30 mmHg and 60 mmHg) to detect possible levels in necrosis after injury. Among these three pressure values, 100 mmHg represented acute glaucoma (high pressure), 30 mmHg represented low pressure in glaucoma [40] and 60 mmHg was a maximum pressure value in human IOP [42]. The results indicated that the number of PI positive cells increased first and then decreased followed by the time passage in 100 mmHg and 60 mmHg. This is consistent with our previous study on rat (110 mmHg for 1 hr) [16], but there was a small number of PI-positive cells in 30 mmHg (data not shown). It was inconsistent with the chronic glaucoma mouse model following 20-30 mmHg [43], one possibility may be that glaucoma is a neuronal degenerative disease; the symptom might take a longer time to exhibit. Therefore, the fact that having a few obvious necrosis cells in our acute insult for a short time seems to be reasonable. Joo's study have found that necrosis cells existed in ganglion cell layer in 160-180 mmHg insult for 90 min following HIOP at 4 hr, but a few necrotic cell was observed after 24 hr [44], but we found some necrosis cells existed in 24 hr, it might be due to the higher pressure and also cells being kept longer in their study. It may also be related to the complexity in the micro-environment (neighboring cells and the secretion of various factors in interstitial cells). Our experiment demonstrated that cell model after EHP could accurately reflect the injury degree of RGCs. Moreover, Nec-1 has become one of the widely recognized inhibitors in necroptosis [31]. Based on our data, the necroptosis can be found after high pressure insult (100 mmHg), and the proportion was reflected by the fact that the necrosis rate decreased to about 5% after adding Nec-1. Therefore, we speculated one signaling molecules that may induce necroptosis of RGC-5 after EHP.
Previous studies have shown that beside calpain mediated apoptosis, calpain can also induce necroptosis through intracellular signal pathway. Calpain facilitates BAX activation and activated BAX favors the release of tAIF from mitochondria to the cytosol which could induce necroptosis [10,11]. Calpain may be one of the important regulatory molecules participating in necroptosis in the cells like fibroblast, nephrocyte, HeLa cell and vascular endothelial cells, etc. Some early necroptosis may be mediated by calpain [9-11,45]; however, the biological function of calpain-mediating early neuronal necroptosis in nervous system especially the visual nervous system is largely unveiled. Our western blot results showed that calpain protein levels appeared to decrease after an initial increase, and reached maximum at 12 hr following elevated hydrostatic pressure condition in RGC-5. In this study, the immunofluorescence staining results showed that calpain was mainly present in the cytoplasm of RGC-5. Moreover, in contrast with other groups, significantly more distinct and heavier calpain immunoreactive were shown in 12 hr group. Both of these results showed that the expression of calpain was significantly up-regulated in RGC-5 following elevated hydrostatic pressure condition accompanied with the phenotype of the increased ratio of necroptosis. This observation in regards to the expression profile of calpain consists of what we found in gene chip detection in RGC-5 following elevated hydrostatic pressure in our previous experiment (our previous unpublished data). In contrast, necroptosis was reduced to a certain extent when exposed to ALLN, a specific calpain inhibitor targeting the activity rather than the expression [46], measured by flow cytometry. Collecting all these results together, it suggested that calpain may play an important role in early RGC-5 necroptosis following elevated hydrostatic pressure condition.
In our experiment, with ALLN intervention the expression of calpain remained high while the activity was markedly inhibited in RGC-5 following elevated hydrostatic pressure condition. Moreover, tAIF (the cleaved form of AIF) didn't significantly increase, which resulted in less effect on RGC-5. Meanwhile, the number of necroptosis cells didn't significantly increase either. It further demonstrated that calpain in RGC-5 was mediated by the downstream molecule of tAIF to modulate necroptosis pathway. Overall, the necroptosis regulated by calpain mediated by tAIF may be a mode of RGCs death induced by elevated hydrostatic pressure. Regarding to another risk factor, cellular hypoxic exposure is not only the widely used nervous system injury model [47], but also one of the important pathophysiological mechanisms of aHIOP [48]. Therefore, it is worthwhile to further investigate whether necroptosis is mediated by calpain in hypoxic exposure. It will be helpful for comprehensive understanding about RGC-5 necroptosis in aHIOP. Taken all together, the result provided novel evidence for the molecular mechanism (non-RIP3 pathway) research of the early RGCs in aHIOP and new interventional target for reducing early RGCs necroptosis in aHIOP patients.
Our study found that calpain may induce necroptosis via tAIF-modulation in RGC-5 following elevated hydrostatic pressure.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
LS, KX and JFH designed the experiment, LS and KX performed the experiment, WD and SC analyzed the data, LS, RFM and KX drafted the manuscript, LXX participated in paper modification and revised the manuscript for English writing, all authors participated in critical revision of the manuscript and approved the final manuscript.
This study was supported by National Natural Science Foundation of China (No. 81371011, 81070729); Natural Science Foundation of Hunan Province (No. 11JJ2020); Young Teachers Training Program of University of Hunan Province. We would like to thank PhD. Anna Chun-Ling, Neuroprotection Research Laboratory, Massachusetts General Hospital, Boston, USA, who edited the manuscript for us.
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text:
The latest novel by Richard Powers, The Echo Maker, had its origin, as any good book probably should, in a visionary experience. "I'd taken a long cross-country trip down to Arizona and was heading back home to Illinois through Nebraska," he told me when I phoned him in Urbana, home to the university where he teaches. "I'd been driving for a very long time and suddenly saw this hallucinatory sight of birds along the Platte River." What Powers had stumbled upon was a breathtaking phase of the annual sandhill crane migration, in which an enormous congregation of these birds—half a million cranes, about four-fifths of the global population—use a seventy-five-mile stretch of the Platte and the surrounding locale as a staging area before heading north to their Arctic breeding grounds. "It really seemed like an
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From Our Editors
Butterfly Yoga's staff channels its experience and love of movement through the practice of yoga. Butterfly's courses encourage students of all experience levels to get fit, build self-awareness, and increase their ability to out-maneuver bendy straws. With an emphasis on a supportive environment and compassionate guidance, instructors seek to inspire self-exploration through movement and meditation. Additionally, Butterfly Yoga holds donations-based classes periodically each month to share yoga with more people in the community.
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Here in the village of Lisbane, close to the shores of Strangford Lough, we offer a unique dining and hospitality experience.
The Grade II listed building dates back to the 1840's and having lain derelict for many years, it has been lovingly restored and it now makes a charming home for Trevor and Alison Smylie's tea rooms and gift shop.
Trevor has years of experience as a baker and pastry chef in Australia and the U.K. and is now responsible for producing all the mouth watering scones, gateaux, pies and tarts which can be enjoyed in the three atmospheric tea rooms, each with its own cosy peat fire.
And, in fine weather, eating outside is always a treat, whether in the courtyard garden or by the lily pond where the gentle scent of the nearby herb garden wafts on the air.
As well as breakfast, teas, coffees and light snacks, wholesome lunch dishes such as salmon and asparagus quiche or chicken and broccoli bake with salad are available (with a big slice of chocolate tart to follow, perhaps).
Our Pantry offers a great range of take away options from home made scones and tray bakes to Quiche, Lasagne and a range of pies and salads.
Recent Customer comments
Recent Customer comments
"I have been to many afternoon teas across Northern Ireland and the food at the Chocolate Afternoon Tea was the best I've had. The service was also fantastic. Will be back soon!"
Feb 2015
"Afternoon tea is superb, a little different, delicious and served with a smile. In fact all the food served in the coffee shop and bought from the Pantry is superb, especially the chicken and broccoli bake. The gift shop, though small, has everything. Thank you for the candle and birthday rendition for my sister's birthday."
Feb 2015
Gift Vouchers
Gift Vouchers
Why not give a gift voucher for Christmas as a special gift?
Give us a call for details, or even order via the telephone 02897 343335
We also an Old Post Office Gift voucher available for either the Tea Rooms, Pantry or Gift Shop.


text:
Tutorial: Hibernate Logging using Log4jHibernate Logging using Log4j
Tutorial Details:
This section contains detail description of how you can use Log4j for logging configuration.
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