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If you are at all interested in how the Internet's Domain Name System (DNS) works, then one of the most rewarding meetings that is dedicated to this topic is the DNS OARC workshops. I attended the spring workshop in Amsterdam in early May, and the following are my impressions from the presentations and discussion. What makes these meetings unique in the context of DNS is the way it combines operations and research, bringing together researchers, builders and maintainers of DNS software systems, and operators of DNS infrastructure services into a single room and a broad and insightful conversation. more»
I am excited to announce the recent release of the industry first Best Common Practices document for Cloud and Hosting providers for addressing abuse issues that was created by M3AAWG and the i2Coalition. M3AAWG has been collaborating with the Best Practices Working Group of the i2Coalition over the past 2 years to discuss ways to solve malicious activity within hosting and cloud ecosystems. more»
Arthur C. Clarke said any sufficiently advanced technology is indistinguishable from magic. Milton Friedman said there's no such thing as a free lunch. The validity of the former statement does not invalidate the later. From this we can see that even magic has a price. Hence, its application is subject to cost-benefit analysis. There are many developing technologies that may eventually qualify as magic. more»
In those circles where Internet prognostications abound and policy makers flock to hear grand visions of the future, we often hear about the boundless future represented by "The Internet of Things". This phrase encompasses some decades of the computing industry's transition from computers as esoteric piece of engineering affordable only by nations, to mainframes, desktops, laptops, handhelds, and now wrist computers. Where next? more»
As security breaches increasingly make headlines, thousands of Internet security companies are chasing tens of billions of dollars in potential revenue. While we, the authors, are employees of Internet security companies and are happy for the opportunity to sell more products and services, we are alarmed at the kind of subversive untruths that vendor "spin doctors" are using to draw well-intentioned customers to their doors. Constructive criticism is sometimes necessarily harsh, and some might find the following just that, harsh. But we think it's important that organizations take a "buyers beware" approach to securing their business. more»
The movement for upping the minimum wage in the US is gathering momentum. Protests and placard waving are on the increase, and the quest for $15 per hour is well underway. There are plenty of arguments as to why such a hike in minimum wage is necessary, and what the consequences could be to those businesses dependent upon the cheapest hourly labor. But, for the information security industry, upping the minimum wage will likely yield only good news. more»
The cybersecurity debate can be highly confusing at times. There is perhaps an analogy to be made between "Cybersecurity" and "The Economy". We all want to fix the economy but making progress is not an easy task. As soon as you are beyond that statement you notice that there is a lot of nuance. Issues like trust, influence, actors, and affectivity all come to play when you want to fix the Economy. The cybersecurity discourse has similar features. more»
Denial of service attacks have been around since the Internet was commercialized and some of the largest attacks ever launched relied on DNS, making headlines. But every day a barrage of smaller DNS-based attacks take down targets and severely stress the DNS ecosystem. Although DNS servers are not usually the target of attacks they are often disrupted so attention from operation teams is required. There is no indication the problem is going away and attackers continue to innovate. more»
You have just a couple of days to either complete a survey or submit a paper to join the "Coordinating Attack Response at Internet Scale (CARIS)" Workshop happening on June 19, 2015, in Berlin, Germany... If you are interested in helping improve the overall security and resilience of the Internet through increased communication between the groups responding to the large-scale attacks happening on the Internet every day, I would strongly encourage you to apply! more»
Everyone has heard of the cyber security attacks on Target (2013), Home Depot (2014), Neiman Marcus (2014), Sony Pictures (2014), and the United States' second-largest health insurer, Anthem (reported February 2015), but have you heard of the security breaches for Aaron Brothers, Evernote (denial of service attack), P.F. Chang's China Bistro, Community Health Services, Goodwill Industries, SuperValu, Bartell Hotels, Dairy Queen, U.S. Transportation Command contractors, and more. more»
How do we help coordinate responses to attacks against Internet infrastructure and users? Internet technology has to scale or it won't survive for long as the network of networks grows ever larger. But it's not just the technology, it's also the people, processes and organisations involved in developing, operating and evolving the Internet that need ways to scale up to the challenges that a growing global network can create. more»
The most interesting feature of the newly-described "Equation Group" attacks has been the ability to hide malware in disk drive firmware. The threat is ghastly: you can wipe the disk and reinstall the operating system, but the modified firmware in the disk controller can reinstall nasties. A common response has been to suggest that firmware shouldn't be modifiable, unless a physical switch is activated. more»
Here we are, half-way through this list of the top 10 IPv6 security myths! Welcome to myth #6. Since IPv6 is just now being deployed at any real scale on true production networks, some may think that the attackers have yet to catch up. As we learned in Myth #2, IPv6 was actually designed starting 15-20 years ago. While it didn't see widespread commercial adoption until the last several years, there has been plenty of time to develop at least a couple suites of test/attack tools. more»
Lastly, and certainly not the least, part four of my security predictions takes a deeper dive into mobile threats and what companies and consumer can do to protect themselves. If there is one particular threat category that has been repeatedly singled out for the next great wave of threats, it has to be the mobile platform -- in particular, smartphones... The general consensus of prediction was that we're (once again) on the cusp of a pandemic threat. more»
A number of security predictions have been doing the rounds over the last few weeks, so I decided to put pen to paper and write a list of my own. However, I have a quite a few predictions so I have listed them over several blog posts. After all, I didn't want to bombard you with too much information in one go! Part three examines the threats associated with data breaches. more»
Chris Grundemann Internet Technologist, Author, and Speaker. All opinions are his and his alone 19 Blogs / 10 Comments


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Book Review: A Memory of Light (Final Book, Wheel of Time Series) by Robert Jordan and Brandon Sanderson
Quite a while ago, I wrote a blog post about the book that caused me to become a bibliophile and eventually brought me to pursue my dreams as an author. I told of how my dad found a random fantasy book and tossed it on my lap one day. I also told of how I had never read fantasy and thought it was somewhat dorky… well, it is dorky, but I'm okay with that. You can read that blog post here.
If you haven't guessed by now, the first book in the Wheel of Time series, The Eye of the World by Robert Jordan was the catalyst. I began this journey almost fifteen years ago and now that journey has come to an end… or has it? I mentioned in an earlier post that my wife asked how I would feel when I finished the final book in the series. While I stared at the ceiling for a good half-hour once finished, I quickly straightened up and pulled out my notebook to jot down my own fantasy genre ideas.
I've moved on in multiple ways. The fantasy will never end for me because I am an author. I'll just write more! And of course, there will always be other fantasies out there to keep my interest, though it will take one heck of a story to compare to my first love.
On to the review!
A quick note about the description below. It's not as much of a description for the book as it is a description of the entire series and the culmination of the entire project.
Book Description via Amazon
Since 1990, when Robert Jordan's The Wheel of Time® burst on the world with its first book, The Eye of the World, readers have been anticipating the final scenes of this extraordinary saga, which has sold over forty million copies in over thirty languages.
When Robert Jordan died in 2007, all feared that these concluding scenes would never be written. But working from notes and partials left by Jordan, established fantasy writer Brandon Sanderson stepped in to complete the masterwork. With The Gathering Storm (Book 12) and Towers of Midnight (Book 13) behind him, both of which were # 1 New York Times hardcover bestsellers, Sanderson now re-creates the vision that Robert Jordan left behind.
Edited by Jordan's widow, who edited all of Jordan's books, A Memory of Light will delight, enthrall, and deeply satisfy all of Jordan's legions of readers.
The Wheel of Time turns, and Ages come and pass.
What was, what will be, and what is,
may yet fall under the Shadow.
Let the Dragon ride again on the winds of time.
Book Review
If that last line isn't one of the coolest lines I've ever read, I don't know what is.
A reminder here that I try for no spoilers, but don't read on if you're worried I may give something away. I haven't had any complaints yet, but I know I'm the type of reader who wants to know virtually nothing about the storyline of the next book in a series I'm reading. There's your fair warning.
Sanderson had hundreds of main characters, and hundreds of story arcs, to cover in this one. A close friend and I have discussed how this series would end for years and neither of us could comprehend how Sanderson could put the finishing touches on this one. While there were a couple small areas we complained about after finishing the series, the rest of the story was amazing enough that it didn't change the way we felt one bit. The Wheel of Time series is, and will continue to be, one of the greatest epic fantasies ever told.
I didn't think it was possible, but Sanderson kept an unbelievable pace throughout the entire novel. The chapters and sections were shorter than in previous WoT (Wheel of Time) books, but not only did it work well, I believe it was needed in this case. The reader is all over the map at first as we're brought into the state of mind of all the characters and reminded where we're headed: Tarmon Gai'don, the final battle, possibly the end of all things.
The final battle isn't just one battle, but many. Some on a larger scale and some that are small yet lead to larger fights that may or may not be known by the other characters. Matrim, Perrin, Rand, Egwene, Nynaeve, Lan, Tam, and I could go on, all have their part to play, and they're each significant.
Best Aspects of the Book
Sanderson keeps to the storyline and to Jordan's vision of an epic story while continuing to focus on the individuals and their actions that brings us to the final climax.
Again, THE PACING! I swear my heart was racing through almost every page of this darn book. Not just the battle scenes, but with every decision the characters made bringing us closer to the end of the world, and the book.
The epic battle scenes. Sorry for using the word "epic" again, but it's the only word that encompasses the breadth of this story. The tactics, the blood, the soldiers, the individuals; they all play significant roles in the final battle. Another reviewer said something similar, but the way the scenes played out it was as if Sanderson had first hand knowledge of that battle, as if he were there copying down what he saw.
Many may not see this as a positive, but it made the book more realistic in my opinion. Characters are dying all over the place in this one. It makes sense considering it was the final battle. If we'd had this grand battle to only have a few characters die, not only would it not have been as realistic, but the reader wouldn't be on the edge of their seat wondering when their other favorite characters were going to "wake up from the dream," as the Aiel would say.
Sanderson completed so many story arcs in one this one book that it was simply amazing. In a recent conversation with the friend I mentioned above, we discussed how Sanderson could have easily written another four or five books in the series with each of those story arcs being a story of their own.
The Negative Aspects
With how much I loved this story, it's hard for me to say anything negative. However, I try to keep an unbiased tone when I write a review, though that's pretty much impossible in this situation. While I say some of these things are negative, it's only my opinion and other readers may disagree.
Where was Thom Merrilin? No, he wasn't removed from the story, and he played an important role, but I loved his character and I wanted more. With the how big of a part of the story he was throughout, I thought there would be more.
There were some parts that were 100% Sanderson, particular lines especially. He sticks to a Jordanesque style throughout the previous two books he wrote, yet in this one, I felt there were times when the things he wrote were something Jordan probably would have done so differently. Of course, we'll never know, and I've no doubt that Sanderson did the better than any other author could have in his position. My argument against this is that I'm not sure Jordan could have completed this series the way Sanderson did, and that's saying a lot, because I love Jordan's writing. Before this book, I wasn't completely sold that Sanderson could finish the WoT series the way it should have been. Now, I can see that Robert Jordan and his wife picked the perfect author to finish this epic story.
This part goes with one of the positive aspects. I mentioned that Sanderson completed so many story arcs above, but there were some I wish he'd spent more time on. "Wish" is the key word there. We all wish we could read more of our favorite book or series, but that's just not possible. I won't mention which one, but there was one point that had been alluded to almost since the first book, and then when we reach the actual moment, it's over in less than two pages. I can't complain too much, the story arc was completed, even if not in the way that I had imagined.
The actual ending. I could probably put this in both the positive and the negative. In my mind, it fit perfectly. So why does it go in the negative section? Because, in my mind, there were just enough questions left that the ending read like the final line was a precursor to the next book in the series, which of course we won't have. I think my brain is still working on what exactly I think about everything.
I give A Memory of Light by Robert Jordan and Brandon Sanderson five of five stars, and the series itself, five of five stars. If you're looking for a true epic fantasy with an epic story line, an epic cast, and now an epic ending, the Wheel of Time is for you.
I doubt Brandon Sanderson or Harriet, Robert Jordan's wife, will ever see this, but I want to take this moment to say thanks to all three of you for sharing this amazing story with the world. Thanks for the time and effort you put in, despite the untimely passing of Mr. Jordan. Thanks for showing me what it's like to get lost in a book, to care for characters as if they're my family, and for giving me this pipe dream to chase.
Grab a copy of A Memory of Light here on Amazon US, here on Amazon UK, or here on Barnes and Noble.

subset: commoncrawl


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List all words ending with geezer
Only the word geezer was found, nothing longer.
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A Touring Taste of Dance Salad Festival 2007
Just in time for Thanksgiving, A Touring Taste of Dance Salad Festival 2007 reminds us of the advantage of leftovers: You can pick out the best parts. The film features the tastiest bits from the festival, including the now world-renowned performance by Russias Bolshoi Ballets star dancer, Natalia Osipova, which Londons International Herald Tribune said sent the usually staid British audiences into a standing, shouting ovation and created ecstasy among the sometimes finicky critics. Osipovas solo will be featured alongside clips from other tippy-toed talent, including Italys Compagnia Aterballetto, The Netherlands Kylián Foundation and Koreas Kim Eun Hee Dance Company. Those who missed out can see what they, well, missed, and others can feed their anticipation for next years festival in March with plenty of past pliés, pirouettes, promenades and pointy toes. 6:30 p.m. Museum of Fine Arts, Houston, 1001 Bissonnet. For information, call 7133559011 or visit www.dancesalad.org. Free.
Wed., Nov. 28, 6:30 p.m., 2007
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|By PR Newswire
Article Rating:
|March 12, 2014 07:09 AM EDT
DUBLIN, Mar. 12, 2014 /PRNewswire/ --Research and Markets (http://www.researchandmarkets.com/research/6ns9jw/rapid_medical) has announced the addition of the "Rapid Medical Diagnostic Kits Market - Forecasts to 2019" report to their offering.
(Logo: http://photos.prnewswire.com/prnh/20130307/600769)
Rapid medical diagnostics are tools for detection of various infectious diseases caused by virus and bacteria. Since the last decade remarkable prevalence of infectious diseases has been observed worldwide and its morbidity rate is rapidly growing. Increased detection of new pathogens-causing infections and increasing rate of weak immune system resulting from aging of population worldwide are some of the major factors leading to increase in the infectious diseases due to which the rapid medical diagnostic kits market is showing massive growth globally. Some other factors contributing to the growth of the rapid medical diagnostic kits market include cheaper cost of the rapid medical diagnostic kits, and increasing awareness about the available technologies for diagnosis of the infectious diseases caused by virus and bacteria.
Rapid medical diagnostic kits market is mainly categorized on the basis of applications namely, clinical testing, home-use, infectious disease and veterinary and on the basis of technology used to develop rapid medical diagnostic platform namely, lateral flow technology, agglutination assays, flow-through and solid phase assays. The home-use rapid medical diagnostic application segment held the major market share of rapid medical diagnostic kits market in 2012 on account of the highly dominated blood glucose testing market. While on the technology side, lateral flow technology led the technology segment in the global rapid medical diagnostic kits market owing to presence of large number of approved rapid medical diagnostic kits with this technology. Moreover, its ease of use and quick turnaround time has been beneficial in malaria and pregnancy testing at home.
The global rapid medical diagnostic kits market is highly fragmented in nature with many companies competing in the market. Well established market players dominate this market with their proprietary products and predatory growth strategies. Patent repository of the established market players is restraining growth and entry of the new market players entering into the market. Companies such as Millipore Corporation and Alere, Inc. are seeking patent approvals to secure their market share in the rapid medical diagnostic kits market.
Geographically, North America dominates the global rapid medical diagnostic kits market and is followed by Europe. North America is expected to maintain its leading position till 2019. Asia Pacific is expected to show highest growth rate in the rapid medical diagnostic testing kits market owing to factors such as, increasing infectious disease prevalence, increasing awareness about the rapid medical diagnostic tools and increasing disposable income of the population in this region. Highest infectious disease rates in developing countries such as African countries will drive the growth of rapid medical diagnostic kits market in these countries.
Key Topics Covered:
Chapter 1 Introduction
Chapter 2 Executive Summary
Chapter 3 Rapid Medical Diagnostic Kits Market Overview
Chapter 4 Global Rapid Medical Diagnostic Kits Market, by Applications, 2011 - 2019 (USD Million)
Chapter 5 Global Rapid Medical Diagnostic Kits Market, by Technology, 2011 - 2019 (USD Million)
Chapter 6 Global Rapid Medical Diagnostic Kits Market, by Geography, 2011 - 2019, (USD Million)
Chapter 7 Recommendations
Chapter 8 Competitive Landscape
Chapter 9 Company Profiles
Companies Mentioned:
- Abaxis
- Abbott Laboratories
- Alere
- Becton, Dickinson & Company
- Bio-Rad Laboratories
- Danaher Corporation
- MegaCor Diagnostik
- F. Hoffmann-La Roche
- Trinity Biotech
- Zoetis
For more information visit http://www.researchandmarkets.com/research/6ns9jw/rapid_medical
Media Contact: Laura Wood , +353-1-481-1716, [email protected]
The Internet of Things is not only adding billions of sensors and billions of terabytes to the Internet. It is also forcing a fundamental change in the way we envision Information Technology. For the first time, more data is being created by devices at the edge of the Internet rather than from centralized systems. What does this mean for today's IT professional? In this Power Panel at @ThingsExpo, moderated by Conference Chair Roger Strukhoff, panelists will addresses this very serious issue of profound change in the industry.
May. 26, 2015 11:00 AM EDT Reads: 331
An entirely new security model is needed for the Internet of Things, or is it? Can we save some old and tested controls for this new and different environment? In his session at @ThingsExpo, New York's at the Javits Center, Davi Ottenheimer, EMC Senior Director of Trust, reviewed hands-on lessons with IoT devices and reveal a new risk balance you might not expect. Davi Ottenheimer, EMC Senior Director of Trust, has more than nineteen years' experience managing global security operations and assessments, including a decade of leading incident response and digital forensics. He is co-author of t...
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The Internet of Things is a misnomer. That implies that everything is on the Internet, and that simply should not be - especially for things that are blurring the line between medical devices that stimulate like a pacemaker and quantified self-sensors like a pedometer or pulse tracker. The mesh of things that we manage must be segmented into zones of trust for sensing data, transmitting data, receiving command and control administrative changes, and peer-to-peer mesh messaging. In his session at @ThingsExpo, Ryan Bagnulo, Solution Architect / Software Engineer at SOA Software, focused on desi...
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SYS-CON Events announced today that MetraTech, now part of Ericsson, has been named "Silver Sponsor" of SYS-CON's 16th International Cloud Expo®, which will take place on June 9–11, 2015, at the Javits Center in New York, NY. Ericsson is the driving force behind the Networked Society- a world leader in communications infrastructure, software and services. Some 40% of the world's mobile traffic runs through networks Ericsson has supplied, serving more than 2.5 billion subscribers.
May. 26, 2015 10:15 AM EDT Reads: 1,102
While great strides have been made relative to the video aspects of remote collaboration, audio technology has basically stagnated. Typically all audio is mixed to a single monaural stream and emanates from a single point, such as a speakerphone or a speaker associated with a video monitor. This leads to confusion and lack of understanding among participants especially regarding who is actually speaking. Spatial teleconferencing introduces the concept of acoustic spatial separation between conference participants in three dimensional space. This has been shown to significantly improve comprehe...
May. 26, 2015 10:00 AM EDT Reads: 3,008
Buzzword alert: Microservices and IoT at a DevOps conference? What could possibly go wrong? In this Power Panel at DevOps Summit, moderated by Jason Bloomberg, the leading expert on architecting agility for the enterprise and president of Intellyx, panelists will peel away the buzz and discuss the important architectural principles behind implementing IoT solutions for the enterprise. As remote IoT devices and sensors become increasingly intelligent, they become part of our distributed cloud environment, and we must architect and code accordingly. At the very least, you'll have no problem fil...
May. 26, 2015 10:00 AM EDT Reads: 1,748
The 4th International Internet of @ThingsExpo, co-located with the 17th International Cloud Expo - to be held November 3-5, 2015, at the Santa Clara Convention Center in Santa Clara, CA - announces that its Call for Papers is open. The Internet of Things (IoT) is the biggest idea since the creation of the Worldwide Web more than 20 years ago.
May. 26, 2015 09:00 AM EDT Reads: 1,538
The Domain Name Service (DNS) is one of the most important components in networking infrastructure, enabling users and services to access applications by translating URLs (names) into IP addresses (numbers). Because every icon and URL and all embedded content on a website requires a DNS lookup loading complex sites necessitates hundreds of DNS queries. In addition, as more internet-enabled 'Things' get connected, people will rely on DNS to name and find their fridges, toasters and toilets. According to a recent IDG Research Services Survey this rate of traffic will only grow. What's driving t...
May. 26, 2015 09:00 AM EDT Reads: 5,080
The Internet of Things promises to transform businesses (and lives), but navigating the business and technical path to success can be difficult to understand. In his session at @ThingsExpo, Sean Lorenz, Technical Product Manager for Xively at LogMeIn, demonstrated how to approach creating broadly successful connected customer solutions using real world business transformation studies including New England BioLabs and more.
May. 26, 2015 08:00 AM EDT Reads: 5,863
Today's enterprise is being driven by disruptive competitive and human capital requirements to provide enterprise application access through not only desktops, but also mobile devices. To retrofit existing programs across all these devices using traditional programming methods is very costly and time consuming – often prohibitively so. In his session at @ThingsExpo, Jesse Shiah, CEO, President, and Co-Founder of AgilePoint Inc., discussed how you can create applications that run on all mobile devices as well as laptops and desktops using a visual drag-and-drop application – and eForms-buildi...
May. 26, 2015 08:00 AM EDT Reads: 5,370
The recent trends like cloud computing, social, mobile and Internet of Things are forcing enterprises to modernize in order to compete in the competitive globalized markets. However, enterprises are approaching newer technologies with a more silo-ed way, gaining only sub optimal benefits. The Modern Enterprise model is presented as a newer way to think of enterprise IT, which takes a more holistic approach to embracing modern technologies.
May. 25, 2015 11:00 PM EDT Reads: 5,939
The true value of the Internet of Things (IoT) lies not just in the data, but through the services that protect the data, perform the analysis and present findings in a usable way. With many IoT elements rooted in traditional IT components, Big Data and IoT isn't just a play for enterprise. In fact, the IoT presents SMBs with the prospect of launching entirely new activities and exploring innovative areas. CompTIA research identifies several areas where IoT is expected to have the greatest impact.
May. 25, 2015 09:00 PM EDT Reads: 4,787
Every day we read jaw-dropping stats on the explosion of data. We allocate significant resources to harness and better understand it. We build businesses around it. But we've only just begun. For big payoffs in Big Data, CIOs are turning to cognitive computing. Cognitive computing's ability to securely extract insights, understand natural language, and get smarter each time it's used is the next, logical step for Big Data.
May. 25, 2015 08:00 PM EDT Reads: 1,878
There's no doubt that the Internet of Things is driving the next wave of innovation. Google has spent billions over the past few months vacuuming up companies that specialize in smart appliances and machine learning. Already, Philips light bulbs, Audi automobiles, and Samsung washers and dryers can communicate with and be controlled from mobile devices. To take advantage of the opportunities the Internet of Things brings to your business, you'll want to start preparing now.
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With major technology companies and startups seriously embracing IoT strategies, now is the perfect time to attend @ThingsExpo in Silicon Valley. Learn what is going on, contribute to the discussions, and ensure that your enterprise is as "IoT-Ready" as it can be! Internet of @ThingsExpo, taking place Nov 3-5, 2015, at the Santa Clara Convention Center in Santa Clara, CA, is co-located with 17th Cloud Expo and will feature technical sessions from a rock star conference faculty and the leading industry players in the world. The Internet of Things (IoT) is the most profound change in personal an...
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P2P RTC will impact the landscape of communications, shifting from traditional telephony style communications models to OTT (Over-The-Top) cloud assisted & PaaS (Platform as a Service) communication services. The P2P shift will impact many areas of our lives, from mobile communication, human interactive web services, RTC and telephony infrastructure, user federation, security and privacy implications, business costs, and scalability. In his session at @ThingsExpo, Robin Raymond, Chief Architect at Hookflash, will walk through the shifting landscape of traditional telephone and voice services ...
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The 17th International Cloud Expo has announced that its Call for Papers is open. 17th International Cloud Expo, to be held November 3-5, 2015, at the Santa Clara Convention Center in Santa Clara, CA, brings together Cloud Computing, APM, APIs, Microservices, Security, Big Data, Internet of Things, DevOps and WebRTC to one location. With cloud computing driving a higher percentage of enterprise IT budgets every year, it becomes increasingly important to plant your flag in this fast-expanding business opportunity. Submit your speaking proposal today!
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Explosive growth in connected devices. Enormous amounts of data for collection and analysis. Critical use of data for split-second decision making and actionable information. All three are factors in making the Internet of Things a reality. Yet, any one factor would have an IT organization pondering its infrastructure strategy. How should your organization enhance its IT framework to enable an Internet of Things implementation? In his session at Internet of @ThingsExpo, James Kirkland, Chief Architect for the Internet of Things and Intelligent Systems at Red Hat, described how to revolutioniz...
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All major researchers estimate there will be tens of billions devices - computers, smartphones, tablets, and sensors - connected to the Internet by 2020. This number will continue to grow at a rapid pace for the next several decades. With major technology companies and startups seriously embracing IoT strategies, now is the perfect time to attend @ThingsExpo, June 9-11, 2015, at the Javits Center in New York City. Learn what is going on, contribute to the discussions, and ensure that your enterprise is as "IoT-Ready" as it can be
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The security devil is always in the details of the attack: the ones you've endured, the ones you prepare yourself to fend off, and the ones that, you fear, will catch you completely unaware and defenseless. The Internet of Things (IoT) is nothing if not an endless proliferation of details. It's the vision of a world in which continuous Internet connectivity and addressability is embedded into a growing range of human artifacts, into the natural world, and even into our smartphones, appliances, and physical persons. In the IoT vision, every new "thing" - sensor, actuator, data source, data con...
May. 25, 2015 01:00 PM EDT Reads: 6,164


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History Lectures 2013
Published 02/01/2013 | 09:35
THE Kilmacanogue History Societies are already up and running for 2013 the spring programme is already announced.
Published 02/01/2013 | 09:35
THE Kilmacanogue History Societies are already up and running for 2013 the spring programme is already announced.
She can be a little shy when you first meet her but this lady has a heart of gold and within moments of meeting you will see her loving and gentle nature shine through.
May Bray Cult Film Club will be screening John Hughes' classic 80s comedy caper Ferris Bueller's Day Off on Wednesday, May 20, at 8p.m. A card with 10 tickets costs €10 on the door. Tickets can be used for any future Cult Film Club screenings.
On Friday May 15th, a busy sports day itinerary was planned and implemented with precision by the P.E. department, ably assisted by other members of staff. Despite the grey conditions, the student body kitted out and participated in Track and Field events, Basketball, Volleyball and Soccer; the events rotating so that all students could take part.
Two German Shepard dogs spotted attacking sheep were shot dead by a farmer in Roundwood.News
A discretionary fund allocated to Bray Municipal District will be decided at local level, before going on to the full council in Wicklow for...News
In a notice of motion before Bray Municipal District, Cllr Joe Behan asked that Wicklow County Council undertake a traffic and...News
The council will examine the area surrounding the car...News
Some years of very hard work paid off for students of Colaiste Chraobh Abhann in Kilcoole last week.News
A ring, a necklace, brooch, a bracelet and a pair of earrings were all taken from the...News
A countywide parking survey is being carried out by Wicklow County Council.News
The painful death of a dog due to a horrific virus could have been prevented by treatment and earlier by vaccination.News
Residents of Elgin Wood, adjacent to Springfield Cemetery, are concerned about trees planted at the graveyard.News
Housing stock, both public and private, is badly needed in north County Wicklow was the message from councillors at last Tuesday's...Bray People


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Gary "Watchu talking about Willis" Coleman dead at age 42
Gary Coleman has died as the result of injuries he suffered earlier this week. We've learned Gary's wife, Shannon, made the decision to pull life support early this AM.
We're told Coleman died at 12:05 PM Mountain Standard Time. He died of a intracranial hemorrhage.
Family members and close friends were at Coleman's side when life support was pulled.
He was 42
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Leftovers: Gaming
Netflix in Steam OS
Android Leftovers
Fedora 22 Coming Next Tuesday and Converting Users
At the Fedora release Go/No-Go meeting last night it was determined that three bugs were serious enough to violate the release readiness criteria. As a result, the Final was blocked and a second Go/No-Go was scheduled for today. The results of that meeting are in! Elsewhere, Jack Germain said, "Simplicity Linux is easy to use and runs fast" and Swapnil Bhartiya shared his secret to converting users to Linux.


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There's a crop of girls who are making their mark on the fashion world, mostly for being, well, themselves. They're not typical models, though some do walk the runway, yet they're included on magazine's "best-dressed" lists and chosen for fashion ad campaigns. They're young, they're beautiful, and they're making us a little jealous. (Oh, and it probably helps that they all have fabulous parents.)
Is it a boy? A girl? A potato?
Bonus points for that brow cred.
Exceptions made for Josh Duggar, who need only write a note of apology to Jesus H. Christ.
Somebody call the wahmbulance.


text:
Please make Blood viable in Heroic FL. Please?
Pretty please?
Pretty please?
We're looking for feedback on your class as we work on changes and adjustments for a future game update. While we may be making some specific class changes in 4.3, what we're looking for in this thread is overall feeling on the class as a whole for more long term changes in the future.
Here is the kind of feedback that is NOT helpful in this particular thread:
- Concerns that your current damage, healing or tanking is not competitive. While this kind of feedback is often useful, it also is the kind of thing that can change from patch to patch, and so it isn't as useful for long term vision changes.
- Concerns that your feedback is being ignored or that we don't take these kind of posts seriously, or otherwise posting anything that is off-topic or not in the spirit of this thread's intent.
- Requests for responses to your suggestions or feedback. The purpose of this thread is to get information to us, not the reverse.
We hope you take these threads seriously, as we're going to be reading them very carefully. We ask that you take time to think and formulate a useful and objective reply, as this feedback could be used to alter your class in substantial ways.
We won't be replying here addressing your concerns. This is purely for us to read your answers to the below questions.
Please copy the below questions into your reply and answer each one.
- What type of content do you focus on? [PvE/PvP/Both]
PvE and PvP
- If PvE, what type of PvE? [Heroics/Raids/Other]
Heroics, Raids, so both.
- If PvP, what type of PvP? [Arenas, BGs, Rated BGs]
Arenas, and BGs.
- What are your biggest quality-of-life issues? For instance, no longer requiring ammo could be considered a quality-of-life improvement for hunters.
Rune cooldown being lower. :)
- What makes playing your class more fun?
Doing alot of dps, and being able to help the group.
- What makes playing your class less fun?
- How do you feel about your "rotation"? (Rotation is the accepted order in which abilities are used to maximum efficiency.)
I feel like im rune starved.
- What's on your wish list for your class?
Rune cooldown time decreased.
- What spells do you use the least?
Threats of violence. We take these seriously and will alert the proper authorities.
Posts containing personal information about other players. This includes physical addresses, e-mail addresses, phone numbers, and inappropriate photos and/or videos.
Harassing or discriminatory language. This will not be tolerated.


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Report from the annual Good Friday walk
mayo | environment | news report Saturday April 23, 2011 15:28 by Shell to Sea rossportsolidaritycamp at gmail dot com 085 114 1170
Shell to Sea!
The annual Good Friday walk went ahead yesterday despite the heavy rain. About 83 people walked the length of Shell's 9km proposed raw gas pipeline. The walk is a symbolic act to push Shell out of this beautiful area.
Despite over a decade of resistance to Shell, the community and campaign here in Erris is still going strong and people are determined to protect their families and land. The crosses of the 9 Ogoni people hung by the Nigerian Government and Shell in 1995 led the walk and were laid at the gates the Shell compound in Aughoose and the proposed compound in Glengad. The 'Justice' sign in the field over in Rossport, which has been maintained so well over the years, stood out clearly over the estuary. Let's hope people wake up and stop this project before it's too late.
Comments (7 of 7)
by Shell to Sea Sat Apr 23, 2011 15:39
More photos from yesterday's walk
by Shell to Sea Sat Apr 23, 2011 15:43
More photos from yesterday's walk
by C Sun Apr 24, 2011 10:37
Here is the photo that was supposed to go up as the first picture
by m.m.mccarron Mon Apr 25, 2011 13:39
Thanks for the Ogoni memorial in the Walk. We had a Nigerian lady staying in our house for Easter so I was able to read the account to her and show her the pictures. Dying on the gallows as the Ogoni 9 did is as near to a crucifixion as one can get as is dying in war or being executed as the 1916 leaders were. The cross remains a strong symbol of how we order society and gives us time for reflection.
by Be Corrected Mon Apr 25, 2011 14:21
Sorry you have not the full story above. The 614m is Shell Ireland's share - the full cost of developing the field - one or more wells? - is " expected to top 2.5 BILLION " which the Irish state has agreed to be written off tax on profit. So the Sate is actually underwriting costs of 2.5 BILLION to Shell on what is for now ONE gas well.
by Energy Policy Tue Apr 26, 2011 12:25
Piece in Irish Independent tody tells us that lessons have been learned from Corrib. We need to know them When will we hear? Before the next round? Contracts should not be signed dependent on consents. Communities to be fully informed, agree freely, well in advance ... the State needs to say what amount is being produced, what profit for the State...do we have a projection for profit on Corrib - energy supply is the weak part of the argument. Have we people currently and specifically in training to manage our resources if we are expecting licences to start producing. To start small - have we the An Bord Pleanala Project Moitoring Committee in place to oversee the Shell work taking place at Aughoose. What kind of expertise has Mayo County Council for such tasks? The An Bord Pleanala conditions are demanding of expertise.
by shelver Mon May 02, 2011 13:05
All the people walking past at Aghoos looks great - well done!


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One Hit Wonders of the 1980's
It happens all the time. You turn on the radio or throw your iPod on shuffle and hear a song you love but haven't heard in a while. After singing from the top of your lungs and embarrassing yourself in public you undoubtably ask yourself one question:
"What happened to (insert band name here)?"
We don't know why or how it happens, but sometimes an artist strikes gold and can never reclaim that magic again. Sadly, when that happens they get labeled as a "One Hit Wonder," doomed for the rest of eternity as segment pieces on VH1's Where Are They Now and Behind The Music.
The 1980's was filled with one hit wonders. Many of them fill the majority of Top 10 lists and were even some of the best songs of the decade. And if you're like us, they always put a huge, cheesy smile on your face.
Big Country "In A Big Country"
Frontman Stuart Adamson once said about their one hit wonder status, "If we're known for nothing more than just that one song, I'd be pretty happy with that."
Falco "Rock Me Amadeus"
Falco is extremely popular in Germany, Austria, and most of Europe. It's too bad the only places that make Top Ten lists are in the US and UK.
Nena "99 Luftballons"
Both the English and German versions of "99 Luftballons" tell a story of 99 balloons floating into the air, triggering an apocalyptic overreaction by military forces.
If the song is playing while it happens, it won't seem like such a bad time.
Lipps, Inc. "Funkytown"
"Funkytown" is often considered to be one of disco's last stands and a place to go if you need a place to keep you movin' and keep you groovin' with some energy.
The Vapors "Turning Japanese"
The Vapors waited to release "Turning Japanese" as their second single because they feared it might relegate them to one hit wonder status.
Good plan.
Soft Cell "Tainted Love"
"Tainted Love" spent a record breaking forty three weeks on the Hot 100. Things cooled down significantly after that.
Toni Basil "Mickey"
Did you know Toni Basil was 38 years old when "Mickey" was released?
Not too shabby, eh?
A-ha "Take On Me"
Coolest… Music… Video… Ever!
Men Without Hats "Safety Dance"
"Safety Dance" has been used in movies and TV shows like Glee, Family Guy, Hot Tub Time Machine, Bio-Dome, Scrubs, The Simpsons, Futurama, Campus and parodied by Weird Al Yankovic.
Dexy's Midnight Runners "Come On Eileen"
The shop Dexy's Midnight Runners shot the video in front of is now called Brook Drive Mini Market. You can see it using Google Street View. Seriously… check it out HERE and throw in this address: 151 Brook Drive, Kennington London, SE11 4TG.
Aaaaaaand we're spent.
The 80's was loaded with one hit wonders. Now if you'll excuse us, we're a bit tired from all the dancing.
More One Hit Wonders on WCBSFM.com!
Latest from WCBSFM.com >
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- CBS-FM's Top 500 Memorial Day Weekend Countdown


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It's a clean place and the service is pretty good. However, it is located in an area nearby different night clubs and the quality of sleep is really poor. If you have the opportunity to stay at the Howard Johnson, that's a higher standard hotel.
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c-Myc transformed human Burkitt's lymphoma (BL) cells are highly sensitive to TGF-β-induced apoptosis. Previously we demonstrated that TGF-β-mediated cell death in BL cells is regulated via the mitochondrial intrinsic apoptosis pathway, which is dependent on the activation of BAX and/or BAK. TGF-β directly induces transcription of the BH3-only protein BIK and represses expression of the pro-survival factor BCL-XL but has no effect on the direct BAX/BAK "activators" BIM or BID (tBID). Here we show that TGF-β induces the BH3-only activator PUMA to aid induction of the intrinsic cell death pathway. TGF-β also induced PUMA in normal germinal center CD77-positive centroblasts isolated from human tonsil tissue. PUMA was a direct TGF-β target gene in B-cells, and we identify a putative Smad-binding region within the human PUMA promoter that recruits Smad3 and Smad4 in cells in response to TGF-β signaling. Constitutive activity of the isolated Smad-binding region in luciferase reporter assays was dependent on Smad consensus sequences and was partially dependent on endogenous TGF-β signaling and Smad4. Knockdown of PUMA in BL cells using lentiviral shRNA resulted in slower kinetics of the TGF-β-mediated apoptotic response. Analysis of Eμ-Myc cell lines demonstrated that c-myc-driven murine lymphomas are also sensitive to TGF-β-mediated apoptosis. Moreover, Puma−/− Eμ-Myc lines demonstrated significantly delayed kinetics of the apoptotic response when compared with wild type lymphomas. TGF-β therefore induces a polygenic response in Myc-driven lymphomas involving transcription of PUMA, which is necessary for the rapid induction of cell death.
TGF-β is a potent immune regulator, controlling the differentiation, function, and survival of hemopoietic cells including B- and T-cells (1). Control of B-cell homeostasis is mediated through the TGF-β-dependent regulation of cell cycle arrest and apoptosis. We have previously shown that TGF-β signaling contributes to "death by neglect" of spontaneously apoptosing human germinal center centroblasts, as well as inducing apoptosis of malignant lymphomas derived from the germinal center B-cells (c-Myc transformed Burkitt's lymphoma (BL)5 cells) (2, 3). This apoptosis program is induced by the intracellular signaling cascades activated by the binding of TGF-β to the high affinity TGF-β type II receptor. Binding of ligand to the TGF-β type II receptor enables the formation of a heterotetrameric complex between the constitutively active serine/threonine kinase type II receptor and the type I receptor, ALK5. The TGF-β type II receptor phosphorylates and activates ALK5, thereby inducing the canonical Smad pathway and/or several non-Smad pathways (4, 5). ALK5 activation results in C-terminal phosphorylation of the receptor-regulated Smads, Smad2 (Ser-465/476) and Smad3 (Ser-433/435), which then bind to the co-Smad, Smad4. The resulting heteroligomeric Smad complexes accumulate within the nucleus to control expression of genes involved in the regulation of cell survival and apoptosis (6).
In BL cells, TGF-β-induced apoptosis is associated with changes in expression levels of apoptosis regulators upstream of BAX and BAK. Activated BAX and BAK homo-oligomerize in the mitochondrial membrane to cause mitochondrial outer membrane permeabilization (7) and the release of pro-apoptotic factors. The function of BAX and BAK is tightly regulated by the BCL-2 family of proteins, including the pro-survival factors BCL-2 and its homologues, BCL-XL, MCL-1, BFL-1, BOO, and BCL-w. Other members of the family share one region of homology with BCL-2 (BH3-only proteins), are pro-apoptotic, and include BIK, PUMA, BID, NOXA, BIM, BAD, HRK, and BMF. The "activators" of BAX and BAK (8) include BIM, tBID, and PUMA (8–10), which are proposed to directly bind to BAX and BAK in response to apoptotic stimuli. The pro-survival factors such as BCL-2 prevent apoptosis by sequestering the activator proteins, but they themselves may be inhibited by selective interaction with BH3-only proteins (11). These proteins are therefore referred to as apoptosis "sensitizers," which may either liberate activators from the pro-survival factors to enable BAX/BAK activation (12) or, alternatively, block the direct interaction of pro-survival factors with BAX and BAK (13).
Of these BCL-2 family members, TGF-β induces Smad-dependent transcription of the sensitizer BIK and down-regulation of the pro-survival factor BCL-XL in BL cells (3), however, no regulation of the activators BIM or tBID was evident during initiation of the apoptosis program. PUMA (p53-up-regulated modulator of apoptosis) is therefore the only other direct BAX/BAK activator that may have a role in promoting TGF-β-induced apoptosis in this system. PUMA physically associates with BAX via its BH3 domain (residues 127–150) to induce an apoptotic change in conformation of BAX (10), as well as promoting BAX-dependent apoptosis by dissociating BCL-XL from BAX (10, 14). PUMA is transcriptionally regulated by the p53 family (15–17), NF-κB (18), and E2F-1 (19), by activated FOXO3a following cytokine withdrawal (20) and in response to calcium pool depletion of the endoplasmic reticulum (21).
The tumor suppressor functions of p53 are rate-limiting for lymphomagenesis in Eμ-Myc murine models of BL. PUMA plays a major role in both p53-mediated and p53-independent cell death (22, 23) and is required for oncogene-induced cell death caused by c-Myc overexpression (22, 24). Consequently, genetic deletion of Puma accelerates the development of Eμ-Myc lymphomas (24), and the p53/PUMA pathway is often disrupted in Eμ-Myc and BL lymphomas, either by mutation or silencing of p53, by disruption of key p53 regulators (p19ARF and MDM2) (25), or by epigenetic silencing of PUMA itself (24). PUMA is also elevated in activated human and murine primary B-cells and is involved in the regulation of mature B-cell survival following T-cell-dependent B-cell response to antigen (26).
Because activation of PUMA effectively limits lymphomagenesis, in this study we investigate whether PUMA is a downstream effector of TGF-β-induced apoptosis of human and murine c-Myc driven lymphomas. We identify PUMA as a novel direct target of TGF-β signaling in B-cells that mediates rapid induction of apoptosis in response to TGF-β.
BL cell lines were maintained in RPMI 1640 (Invitrogen) supplemented with 5–10% (v/v) heat-inactivated FCS, 2 mm glutamine, and antibiotics. Murine Eμ-Myc lymphoma lines were generated as detailed previously (27) and maintained in DMEM (Invitrogen) supplemented with 10% heat-inactivated FCS, 2 mm glutamine (Invitrogen), 1 mm pyruvate (Invitrogen), 50 μm β-mercaptoethanol (Sigma), 200 μm asparagine (Sigma), and antibiotics. Analyses were performed on four cell lines derived from four independent Eμ-Myc lymphomas isolated from Puma+/+ mice and on four cell lines derived from four independent Eμ-Myc lymphomas isolated from Puma−/− mice. The cells were treated as required with 1–5 ng/ml TGF-β1 (Peprotech), 50 μm Z-VAD.FMK (Calbiochem), 5 μg/ml etoposide (Sigma), or 10 μm SB-431542 (Tocris). Protein synthesis inhibition was carried out by preincubation for 2 h with 50 μg/ml cycloheximide and 100 μm anisomycin (Sigma). Inhibition of transcription was carried out by pretreatment of cells for 1 h with 2.5 μg/ml actinomycin D (Sigma). Inhibition of c-Myc activity was carried out by treatment of cells with 25 μm of the c-Myc selective inhibitor 10058-F4 (5-[(4-ethylphenyl) methylene]-2-thioxo-4-thiazolidinone) (Sigma).
5 × 106 CA46 BL cells were transfected in triplicate with 3 μg of firefly luciferase reporter plasmid and 1 μg of Renilla luciferase or β-galactosidase expression plasmids as internal controls for transfection efficiency using an Amaxa nucleofector (solution T, program A-23) as recommended by the manufacturer. Treatment with SB-431542 (10 μm) or solvent vehicle was carried out after transfection if required. Transfected cells were incubated overnight before being treated with TGF-β (5 ng/ml) for 6 h. The ratio of firefly/Renilla luciferase activity was determined using the dual luciferase assay system (Promega) and expressed as the means ± S.D. ratio relative to untreated control samples. The Smad4 shRNA plasmid (TRC 40028) was obtained from Open Biosystems.
CD77+ve cells were purified as described previously (3). Following isolation, the cells were incubated with or without TGF-β in the presence of z-VAD-fmk.
Radioimmune precipitation assay lysates were analyzed by SDS-PAGE. Antibodies used in Western blotting were mouse monoclonals against PARP (BD Biosciences); Smad2/3 (BD Biosciences); actin (Sigma); rabbit polyclonal antibodies recognizing phospho-Smad 2 (Ser-465/476), phospho-Smad3 (Ser-433/435), and PUMA (Cell Signaling/Enzo Life Sciences); and a rabbit monoclonal against Tubulin (Cell Signaling). Secondary HRP-conjugated antibodies were obtained from Dako/GE Biosciences. Bound immunocomplexes were detected by enhanced chemiluminescence (ECL; Amersham Biosciences) or Immobilon western chemiluminescent substrate (Millipore).
The cells were fixed in 80% ethanol, labeled with propidium iodide and analyzed by flow cytometry for less than 2 n DNA content. Murine lymphoma lines were stained with FITC-conjugated annexin V (1 μg/ml, kindly provided by Dr. P. Duriez) and propidium iodide (5 μg/ml) in binding buffer (10 mm HEPES, pH 7.4, 140 mm NaCl, 2.5 mm CaCl2) and analyzed by flow cytometry.
HEK293FT cells were co-transfected with lentiviral packaging plasmids and pLKO.1-based shRNA constructs (TRC 33610 or TRC 33613; Open Biosystems, termed sh1 and sh2). Lentivirus-containing supernatants were collected, filtered, pooled, and used to infect BL cells. After 48 h, the cells were selected in 0.4 μg/ml puromycin. The resulting stably transduced BL30 cells were routinely passaged in growth medium (RPMI 1640 plus 20% FCS) containing 0.6 μg/ml puromycin. Experiments to analyze TGF-β-induced apoptosis were carried out in the absence of selection medium.
Ecotropic Phoenix cells were treated with 5 μm chloroquine 30 min prior to calcium: phosphate precipitation-mediated transfection with the pMIH vector (modified pMIG, GFP cassette exchanged for hygromycin resistance) encoding a human BCL-2 transgene, or vector alone, alongside the helper vector pCL.Eco. After 48 h, virus-containing supernatants were removed and filtered, and Polybrene was added to 4 μg/ml. Eμ-Myc lymphomas were plated at 6 × 105 cells/well in a 12-well plate, and 1.5 ml of virus-containing medium was applied. The plates were centrifuged at 480 × g at 37 °C for 45 min. Selection was then carried out using 250 μg/ml hygromycin for 7 days. Subsequently, BCL-2 expression levels were analyzed by flow cytometry and Western blot.
Total cell RNA was isolated using TRIzol as recommended by the manufacturer. cDNA and qRT-PCRs were prepared using SYBR green two-step qRT-PCR kits (Finnzymes) and specific primers for each gene (Qiagen). Amplified products were analyzed by Chromo4 continuous fluorescence detector and Opticon Monitor3 software. The relative amount of RNA for each gene is expressed after normalization to the amount of 18 S rRNA in each sample. RNA isolated from murine lymphomas was converted to cDNA using a first strand synthesis RT-PCR kit (Invitrogen). Quantitative PCRs were performed using qPCR Supermix-UND (Invitrogen) and TaqMan gene-specific primers (Applied Biosystems). Amplification and analysis was achieved via use of a C1000 thermal cycler fitted with a CFX96 fluorescence detector (Bio-Rad) and CFX96 manager software (Bio-Rad).
2 × 107 CA46 cells were treated with 1 ng/ml TGF-β for 1 h and fixed in 1% formaldehyde for 15 min. The formaldehyde was quenched with glycine, the cells were washed in PBS, and nuclear extracts were prepared. The extracts were sonicated to fragment the DNA and incubated overnight with 20 ng of goat anti-Smad3 or 4 (Santa Cruz) or isotype-matched control antibodies. Immune complexes were captured using anti-goat IgG-coated agarose beads, the samples were washed, and the DNA was eluted off the beads. DNA cross-links were reversed by incubation at 65 °C in 0.3 m NaCl, and the samples were then digested with proteinase K. DNA was extracted using phenol/chloroform and ethanol-precipitated. PCRs using specific primers for human PUMA (5′-AGACTTGCTTGAACCCAGGA-3′ and 5′-AATCGCCTCACCAGAGTGAC-3′) were performed for 35–50 cycles, and the PCR products were analyzed on 2% agarose gels stained with SYBR Green.
We previously showed that TGF-β treatment of BL cells results in the transcriptional regulation of members of the BCL-2 family of apoptosis regulators including BIK and BCL-XL (3). Analysis of other BH3-only proteins that might contribute to the apoptotic program induced by TGF-β was carried out on subsequent TGF-β treatment time courses (Fig. 1). Apoptosis was evident, as detected by Western blotting for cleavage of the caspase-3 substrate PARP, as early as 6–8 h after TGF-β addition (Fig. 1A). During this period, the induction of BIK mRNA was confirmed; however, we also detected a transient increase in transcripts of the apoptosis activator PUMA (Fig. 1B). There was no concurrent increase in expression levels of the apoptosis sensitizers, NOXA, BAD, HRK, or BMF. The induction of PUMA mRNA in response to exogenous TGF-β addition was confirmed across a panel of BL cell lines (Fig. 2A) and at the level of protein expression (Fig. 2B). The Western blot analysis shown in Fig. 2B indicates that the α isoform but not the β isoform of PUMA was induced in response to TGF-β signaling.
BLs are derived from centroblasts within the germinal centers of secondary lymphoid tissue. By isolating CD77-positive centroblasts from human tonsil tissue (as described in Ref. 3), we were able to demonstrate a small but consistent increase in PUMA mRNA expression in TGF-β treated cells relative to untreated controls. No comparable increase in either NOXA or HRK mRNA expression was evident (Fig. 2C). Increased abundance of PUMA RNA transcripts in the presence of TGF-β was also evident throughout a 24-h time course of treatment of CD77-positive primary cells (Fig. 2D). PUMA can therefore be induced in response to TGF-β in both BL cell lines and in the cell of origin isolated from primary tissue. Induction of PUMA mRNA in response to exogenous TGF-β addition could also be detected in the pro-B-cell line RS4;11, but not in non-B-cell lineages including mouse embryo fibroblasts and HaCaT epithelial cells (data not shown).
We next investigated the mechanism of TGF-β-mediated regulation of PUMA expression first by testing whether PUMA is an immediate early target of TGF-β signaling. BL cells were pretreated with cycloheximide and anisomycin and were subsequently treated with exogenous TGF-β for the times indicated. Induction of PUMA occurred even in the absence of de novo protein synthesis (Fig. 3A), but blocking transcription with actinomycin D resulted in the complete ablation of PUMA mRNA induction (Fig. 3B). c-Myc overexpression is characteristic of BL cells, and c-Myc itself is known to repress PUMA transcription following growth factor stimulation (28). We tested the effect of c-Myc overexpression on TGF-β-induced activation of PUMA by pretreating BL cells with the c-Myc inhibitor 10058-F4 at concentrations previously shown to induce BL cell death (29). Inhibition of c-Myc modestly increased basal PUMA transcription (especially in L3055 cells) but did not significantly affect PUMA induction in response to TGF-β (Fig. 3C). Our data indicate that PUMA is a direct target of TGF-β signaling and, as such, implies that PUMA transcription is likely to be regulated by Smad complexes binding to a cognate binding element within the gene promoter. Smad-binding regions (SBRs) frequently contain two copies of the Smad-binding element (SBE) sequence 5′-GTCT-3′ or its reverse complement 5′-AGAC-3′ (30). We identified a potential SBR (5′-agactctgtct-3′) (Fig. 3D) ~1.9 kb upstream of the PUMA transcription start site, contained within a region of the promoter bearing striking homology to the SBR we previously identified within the human BIK promoter (Fig. 3D and Ref. 3). ChIP analysis using primers spanning this region demonstrated TGF-β-dependent binding of Smads 3 and 4 to the endogenous PUMA promoter in vivo (Fig. 3E).
We next analyzed the activity of a luciferase promoter reporter construct containing ~2 kb of the 5′ region upstream of the PUMA transcription start site (31). The PUMA promoter construct was constitutively active in CA46 BL cells and could not be further induced by the addition of exogenous TGF-β (Fig. 4A), suggesting that the endogenous level of TGF-β signaling in these cells may be sufficient for maximal activity of the isolated promoter region. We therefore tested whether inhibition of basal TGF-β signaling using 10 μm of the ALK5 inhibitor SB-431542 would affect PUMA promoter activity (Fig. 4B). We observed an approximate 50% decrease in luciferase activity following SB-431542 treatment, indicating that TGF-β signaling is required for maximal promoter activity.
To examine the activity of the potential Smad-binding region more directly, we generated firefly luciferase reporter constructs containing concatemerized (2×) versions of wild type and mutant sequences shown in Fig. 4C. Consistent with the 2-kb promoter fragment, the 2× wild type SBE sequence was active in CA46 cells. This activity was dependent on the consensus SBEs because mutation of the sequence agactctgtct to atattctatat reduced reporter activity to near basal levels (Fig. 4D). Inhibition of endogenous TGF-β signaling also reduced activity of the 2× wild type SBE reporter (Fig. 4E).
We next transiently transfected a shRNA construct targeting Smad4 to determine whether Smad4 is required for PUMA promoter activity. Because BL cells are renowned for their low transfection rate, we monitored efficacy of Smad4 knockdown by inhibition of the Smad4 dependent CAGA12-luciferase reporter response to TGF-β addition (Fig. 4F). We were able to achieve a partial inhibition of CAGA12 activity and likewise observed a small but significant decrease in PUMA activity (Fig. 4G). Taken together, these findings indicate that regulation of the PUMA promoter by TGF-β signaling is most likely dependent on Smad binding at the SBR at positions −1923 to −1885 and that proper regulation of the endogenous gene depends on the correct repressive chromatin context.
To determine the functional consequence of PUMA induction by TGF-β, we used lentiviral transduction of shRNA constructs to stably knockdown PUMA in BL30 cells. Transducing BL30 cells with a combination of two commercially available shRNA constructs (shRNA1 + 2) reduced the basal and TGF-β-inducible PUMA levels to below the background levels observed in the control line (Fig. 5A). Two PUMA knockdown stable cell pools (shRNA1 + 2#1 and shRNA1 + 2#2) were analyzed for apoptosis induction by TGF-β using propidium iodide staining and flow cytometry. Knockdown of PUMA resulted in a significant reduction in the amount of apoptosis induction by TGF-β after 24 h of treatment (Fig. 5B). We noted, however, that at later time points (48 h) there was little discernable difference between apoptosis induction in control versus PUMA knockdown cell lines (data not shown). We therefore carried out a more detailed kinetic analysis of early apoptosis induction using PARP cleavage as a measure (Fig. 5C, top panel). The Western blots revealed that although all lines retained equivalent TGF-β signaling, shown by equal levels of phosphorylation of the receptor regulated Smad2, the apoptotic response in PUMA knockdown lines was retarded when compared with the control line, 22 and 18% PARP cleavage in the knockdown lines after 8 h of treatment compared with 50% (summarized in the graph shown in Fig. 5C, bottom panel).
Given that PUMA expression levels in the stable knockdown cell lines were reduced to below the basal levels detected in untreated controls (Fig. 5A), the lack of any significant effect on the percentage of apoptotic cells at later time points is unlikely to be a consequence of the incomplete inhibition of PUMA induction in response to TGF-β. However, to exclude this possibility, we extended our study to compare wild type and Puma null murine B-lymphoma cells. Eμ-Myc murine lymphoma lines are a model of BL. We first determined whether isolated wild type Eμ-Myc lymphoma cells undergo apoptosis in response to TGF-β addition in vitro. Annexin V staining of Eμ-Myc cells revealed TGF-β-induced cell death after 24 h of treatment (Fig. 6A). Apoptosis was confirmed by cleavage of PARP in TGF-β-treated wild type lymphoma cells (Fig. 6B). Cell death in response to TGF-β was dependent on the TGF-β type I receptor ALK5 because pretreatment of lymphoma cells with SB-431542 inhibited death induced by TGF-β but had no effect on the cellular response to the DNA damage-inducing agent etoposide (Fig. 6, C and D). Apoptosis was inhibited by overexpression of BCL-2 in murine lymphoma lines, indicating the involvement of the intrinsic apoptosis pathway (Fig. 6, E and F). In addition, TGF-β treatment resulted in induction of Puma shown by quantitative RT-PCR (Fig. 6G) and by Western blot (Fig. 6H). These data indicate that wild type murine Eμ-Myc lymphoma cells mimic the response of human BL cell lines to TGF-β and undergo ALK5-dependent intrinsic apoptosis involving PUMA induction.
shRNA knockdown of PUMA in human BL cell lines delayed the onset of apoptosis in response to TGF-β (Fig. 5C). We observed a similar correlation in the murine Eμ-Myc line 16, which had slower Puma RNA induction after TGF-β addition (Fig. 7A) and lower levels of apoptosis by 12 h of TGF-β treatment (Fig. 7B). However, to definitively investigate the role of PUMA in this response, we compared the response of wild type Eμ-Myc cell lines with Puma null lines (Puma−/−) to TGF-β. All lines responded to TGF-β because they showed similar levels of Smad2 phosphorylation following exogenous TGF-β addition (Fig. 7C). When the effects of TGF-β treatment were directly compared in wild type versus Puma null cells, there were significantly fewer apoptotic Puma null cells than apoptotic wild type cells at early time points following exposure to TGF-β (Fig. 7D) but not at later time points (Fig. 7E), thus confirming the data obtained in human BL lines. PUMA is therefore selectively required for efficient, rapid TGF-β-induced apoptosis.
In this study we demonstrate a direct link between pro-apoptotic TGF-β signaling and transcriptional up-regulation of the apoptosis activator PUMA in c-Myc-driven B-cell lymphomas. PUMA was required for the effective, early induction of apoptosis in both human and murine lymphomas, but its induction does not require c-Myc activity. The involvement of PUMA in this process was demonstrated by stable knockdown of PUMA in human BL, but to be confident that we could assess the effect of complete lack of PUMA on TGF-β-induced apoptosis, we extended our analysis to compare Eμ-Myc lymphomas derived from Puma null and wild type mice. We believe this is the first demonstration that murine Eμ-Myc lymphomas are susceptible to TGF-β-induced apoptosis. Stroma-derived TGF-β from infiltrating immune cells has previously been reported to mediate a tumor suppressor function in vivo by inducing senescence of Eμ-Myc lymphoma cells (32). Our data suggest that in addition to senescence, TGF-β expressed in the tumor microenvironment would also be likely to induce ALK5-dependent apoptosis of malignant cells.
We report that a complete loss of PUMA in knock-out Eμ-Myc cells and knockdown of PUMA in human BL cells delay cell death but do not ultimately prevent cells from undergoing apoptosis. This observation is consistent with the idea that PUMA has a role in priming cells for apoptosis as part of a polygenic apoptotic response that may have a role in "sensing" the apoptotic signal (33). As well as PUMA activation, the apoptotic program involves the early activation of BIK in human cells followed by down-regulation of BCL-XL (3). Such changes in expression levels of the other BCL-2 family members presumably are sufficient to induce apoptosis, albeit with slower kinetics. As predicted previously, we have seen no TGF-β-dependent activation of BIK in mouse cells because the Smad-binding element identified within the human BIK promoter is not conserved (Ref. 3 and Fig. 3). BCL-XL, however, is down-regulated in response to TGF-β in murine Eμ-Myc lymphoma cells.6 This suggests that loss of BCL-XL function may ultimately be sufficient in murine lymphoma to induce apoptosis. but we cannot exclude the possibility that other BH3-only sensitizers might also be induced in response to TGF-β in the mouse.
The mechanism of PUMA induction by TGF-β in this system requires some further analysis. We have determined that PUMA is induced even in the presence of protein synthesis inhibitors, demonstrating that de novo protein synthesis is not required for PUMA transcriptional up-regulation in BL cells and also that in vivo, activated Smad3/Smad4 is recruited directly to a SBR within the endogenous promoter. Constitutive activity of the isolated putative Smad-binding region was dependent on consensus SBE sequences in transient transfection assays, and it was partially inhibited by blocking endogenous TGF-β signaling and lowering Smad4 levels; however, exogenous addition of ligand did not induce its activity. These data suggest that chromatin structure and/or transcriptional repressors may be important for correct regulation of the endogenous gene.
The induction of PUMA-mediated apoptosis by TGF-β superfamily members has been described previously in oligodendrocytes (induced by activin A but not by TGF-β) and by TGF-β in a gastric cancer cell line. In both cases, however, elevated PUMA levels were dependent on p53 family members. A p53 family inhibitor pifithrin-α blocked activin A-induced apoptosis of oligodendrocytes (34), whereas TGF-β-induced apoptosis of SNU-16 gastric cancer cells was due to the induction of p73, which presumably bound p53 consensus sequences on the PUMA promoter (35). In these studies, there was no evidence that Smads were recruited directly to the PUMA promoter as detected in BL cells. However, it is possible that a p53 family member might still be involved in TGF-β-induced regulation of PUMA in our system by acting as co-activators with the Smad proteins. Smads are regulated by numerous post-translational modifications and cooperate with many other transcription factors (including the p53 family) to regulate gene transcription, thus ensuring that their function is highly context dependent (6). p53 and p73, for example, directly bind Smads 2 and 3 and can stabilize Smad-DNA complexes on specific gene promoters (36). It would seem unlikely that p53 is involved in TGF-β/Smad activation of the PUMA promoter because all but one of the BL cell lines used in this study express mutant p53 (BL2 cells express wild type p53 (37)). Transcripts of p73 are expressed in BL cells but are not increased by TGF-β treatment.6
So far, direct activation of PUMA by TGF-β has been demonstrated in B-cell subsets (lymphomas and human primary centroblasts) but not in murine embryonic fibroblasts or HaCaT epithelial cells. The reason for the selective response in certain cell types is currently unclear. It is possible that the proteome of B-cells uniquely allows PUMA activation in response to TGF-β. The panel of BL cells examined here lack expression of SLUG, a transcription factor involved in the repression of PUMA transcription.6 Expression of SLUG in hematopoietic progenitor cells is responsible for protecting progenitor cells from DNA damage-induced apoptosis by repressing p53-mediated PUMA transcription (38). TGF-β may be able to induce PUMA in B-cells by virtue of its lack of repression. Another possibility is that PUMA is regulated by a B-cell-specific transcription factor in conjunction with TGF-β-activated Smads.
A study published during the preparation of this manuscript implicates the activation status of B-cells as a factor in PUMA production, with PUMA being involved in the regulation of antigen specific memory B-cells both in vitro and in vivo. PUMA protein expression was detected in histological sections of human lymph nodes, potentially as a result of antigenic stimulation, because stimulation of cells in vitro with the mitogens Staphylococcus aureus Cowan or LPS, or alternatively following ligation of the B-cell receptor or CD40, all resulted in up-regulation of PUMA. Provided sufficient pro-survival signals are received to maintain BCL-XL levels, the effect of PUMA induction in this system is negated (26). The mechanism of PUMA induction was not identified by Clybouw et al. (26) but was determined to be independent of p53 because p53−/− B-cells produced similar levels of PUMA to wild type cells.
Given our finding that PUMA is induced in vitro by TGF-β treatment of centroblasts (Fig. 2) and that active, phosphorylated Smad2 is detectable throughout the germinal center (3), we can speculate that PUMA detected in the tonsil sections could result from TGF-β signaling. This may arise as a result of the establishment of an activation-induced TGF-β-autocrine feedback loop (39). There are several lines of evidence to support this hypothesis. TGF-β mRNA is detectable in both resting and activated B-cells (40). Stimulation of human tonsil cells with S. aureus Cowan does not significantly affect TGF-β mRNA levels but does result in a 7-fold increase in TGF-β protein secretion (41). Similarly, LPS treatment of murine splenocytes induces the production of bioactive TGF-β (42). The induction of TGF-β in activated B-cells is thought to limit the expansion of the activated population. Without this autoregulatory feedback loop severe autoimmunity develops (43, 44). Our data presented here indicate that regulation of PUMA plays a role in the apoptotic response of B-cells exposed to TGF-β and suggest that this may play a role in the efficient control of activated B-cell proliferation. This notion may provide an explanation for the elevated levels of IgA observed in Puma null mice (45) because IgA class switching is promoted by TGF-β (46). The intriguing possibility that activation-induced PUMA production occurs because of an autocrine TGF-β/ALK5/Smad pathway and the relative role, mechanisms, and kinetics of regulation of BCL-XL and other BCL-2 family members in B-cell survival warrants further investigation.
We acknowledge Dr. P. Duriez (Cancer Research UK Protein Core Facility, Southampton) for the preparation and provision of annexin V-FITC (following the kind gift of the annexin V plasmid from Prof. S. Martin), as well as Dr. C. Scott and Prof. A. Strasser (Walter and Eliza Hall Institute) for the provision of murine lymphoma material, K. Cox and Dr. E. Williams for help with the generation and characterization of the Eμ-Myc cell lines, and Dr. G. V. Helgason for advice on the preparation of lentivirus.
*This work was supported, in whole or in part, by National Institutes of Health Grants CA129829 and U01-DK085570 (to J. Y.).
6L. C. Spender, unpublished observations.
5The abbreviations used are:
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