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Shows: mintCast 307 and LINUX Unplugged 298Submitted by Roy Schestowitz on Wednesday 24th of April 2019 08:11:22 AM Filed under
mintCast 307 – Encryption Part 1
This is Leo and with me I have Joe, Moss, and the return of Rob for this episode! We're recording on Sunday April 21st 2019.
First up, in our Wanderings, I talk Kernel 5.0 and transfer speed, Joe reformats and loses Windows but gains NVidia peace of mind, and finally Moss digests more distros and has some success with migrating Kodi
Then, our news is filled with updates from top to bottom.
In our Innards section, we dive into file and disk encryption.
Blame Joe | LINUX Unplugged 298
This week we discover the good word of Xfce and admit Joe was right all along. And share our tips for making Xfce more modern.
Plus a new Debian leader, the end of Scientific Linux, and behind the scenes of Librem 5 apps.
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Android LeftoversSubmitted by Rianne Schestowitz on Wednesday 24th of April 2019 08:08:30 AM Filed under
Once you use dark mode in these 7 Android apps, you'll never go back
Google I/O 2019: Android Q, Pixel 3a, Stadia cloud gaming and other goodies Google is expected to unveil
LG V35 ThinQ receives Android Pie update in South Korea
Samsung Galaxy Tab S4 Receives Android 9 Pie Update, But Only The Wi-Fi Model
How to clear cache on your Android smartphone for Chrome, Opera, and UC browsers
Android TV box seller fined in landmark copyright infringement case
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Today in TechrightsSubmitted by Roy Schestowitz on Wednesday 24th of April 2019 07:17:54 AM Filed under
- Supreme Court of the UK, Which Habitually Throws Out European Patents, May Overturn Troublesome Unwired Planet v Huawei Decision
- German Federal Patent Court Curbs the Patent Maximalism of the EPO, Which Promotes Patents on Nature and/or Maths Every Single Day
- Any 'Linux' Foundation Needs to Be Managed by Geeks, Not Politicians and PR People
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How to enable SSH access using a GPG key for authenticationSubmitted by Rianne Schestowitz on Wednesday 24th of April 2019 07:14:23 AM Filed under
Many of us are familiar with Secure Shell (SSH), which allows us to connect to other systems using a key instead of a password. This guide will explain how to eliminate SSH keys and use a GNU Privacy Guard (GPG) subkey instead.
Using GPG does not make your SSH connections more secure. SSH is a secure protocol, and SSH keys are secure. Instead, it makes certain forms of key distribution and backup management easier. It also will not change your workflow for using SSH. All commands will continue to work as you expect, except that you will no longer have SSH private keys and you will unlock your GPG key instead.
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today's howtosSubmitted by Roy Schestowitz on Wednesday 24th of April 2019 03:37:56 AM Filed under
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GNU: FS Directory, GNOME Builder and GtkSourceView Moving to MesonSubmitted by Roy Schestowitz on Wednesday 24th of April 2019 03:33:07 AM Filed under
The Free Software Directory needs you! IRC meetups every Friday
The Free Software Directory is an essential catalog of free software online. The Directory is maintained by countless volunteers dedicated to the promotion of software that respects your personal liberty. As with any group composed of volunteers, the informal Directory team has people who come and go, and right now, it could really use some fresh new members to kick our efforts into high gear.
Tens of thousands of people visit the Directory every month to discover free software and explore information about version control, documentation, and licensing. All of this information is also exported in machine-readable formats, making it a valuable source of data for the study of trends in free software. The Directory is powered by MediaWiki, the same software used by Wikipedia.
Builder 3.33.1
Our first 3.33 release has landed as we move towards 3.34. There is a lot to do this cycle in case you're interested in contributing. The best way to get started is to dive into the code. We can help you with that on IRC.
Lots of this release is code behind the scenes, so screenshots won't do them justice. But there are some visible goodies too.
We got a DBus Inspector inspired by D-feet. The long term goal is to merge that new code into D-feet itself.
GtkSourceView moved to Meson
The master branch of GtkSourceView (what will become 4.4) has moved to meson for development. I branched gtksourceview-4-2 for patch releases of 4.2.x which will remain autotools. Today's release of gtksourceview-4.3.1 contains both autotools and meson. However 4.3.2 will remove autotools entirely.
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Ubuntu: GPD Boasting Ubuntu MATE (But Won't Preload), Adwaita Theme on Ubuntu 19.04 and Mark Shuttleworth at Open Infrastructure SummitSubmitted by Roy Schestowitz on Wednesday 24th of April 2019 03:30:04 AM Filed under
GPD Pocket 2 Max is an 8.9 inch laptop with Core m3-8100Y
The computer features a 2560 x 1600 pixel display and it should ship with Windows 10 Home 64-bit software… although it's interesting to see that the company's promotional pictures show the Ubuntu MATE desktop. The folks behind that GNU/Linux-based operating system have made a habit of offering releases customized to support GPD Pocket computers.
How to Try the New Adwaita Theme on Ubuntu 19.04
Learn how to enable the new Adwaita theme in Ubuntu 19.04 Disco Dingo. The updated Adwaita theme is a solid replacement for Ubuntu's Yaru theme.
Canonical at Open Infrastructure Summit -Denver
Open Infrastructure Summit is coming to Denver from April 29th to May 1st, 2019. Will you be there? We sure will! Come and visit us in Booth B1!
Canonical experts will be swarming at the event ready to answer your questions and walk you through our booth demos.
Canonical CEO, Mark Shuttleworth, will be keynoting on Monday, April 29 at 10:25am. Come join us in the main hall to hear Mark's view on open infrastructure and Canonical's approach to driving down the cost of multi-cloud.
WWW and DevelopmentSubmitted by Roy Schestowitz on Wednesday 24th of April 2019 03:26:58 AM Filed under
Acquisition roundabout sees Zend Framework spun off to Linux Foundation
The Zend Framework is to get a new name and a new home, under the auspices of the Linux Foundation, just a few months after its parent co was itself swallowed whole.
Zend – as was – is an open source, object-oriented web application framework implemented in PHP 7. It was synonymous with Zend Technologies, which was taken over by Rogue Wave Software in 2015. Rogue Wave Software was itself acquired by private equity outfit Clear Lake Capital earlier this year.
According to the website for the new organisation, "To take it to the next step of adoption and innovation, we are happy to announce that we are transitioning Zend Framework and all its subprojects to an open source project hosted at the Linux Foundation."
Five RESTful web service client examples for developers
How do you access a RESTful web service? That depends on what you're trying to accomplish.
If you just want to test connectivity, a terminal-based utility like curl is a great RESTful web service client. If you want to inspect the JSON a service returns to you, a browser-based plugin will probably be a better fit. And if you are in the midst of application development, you'll likely need to use JAX-RS, Spring or a similar framework.
5 Best Reasons to Opt for PHP Web Development
Many companies now are choosing PHP web development to realize their IT needs. According to research, almost 83 percent of web services are using PHP, and it is the preferred choice of industry stalwarts such as BlaBlaCar, Slack, and Spotify. PHP is open source and comes with a great community, and it is continuously upgrading. There is no doubt about the same.
It's Complicated: Mozilla's 2019 Internet Health Report
The Report paints a mixed picture of what life online looks like today. We're more connected than ever, with humanity passing the '50% of us are now online' mark earlier this year. And, while almost all of us enjoy the upsides of being connected, we also worry about how the internet and social media are impacting our children, our jobs and our democracies.
When we published last year's Report, the world was watching the Facebook-Cambridge Analytica scandal unfold — and these worries were starting to grow. Millions of people were realizing that widespread, laissez-faire sharing of our personal data, the massive growth and centralization of the tech industry, and the misuse of online ads and social media was adding up to a big mess.
Over the past year, more and more people started asking: what are we going to do about this mess? How do we push the digital world in a better direction?
As people asked these questions, our ability to see the underlying problems with the system — and to imagine solutions — has evolved tremendously. Recently, we've seen governments across Europe step up efforts to monitor and thwart disinformation ahead of the upcoming EU elections. We've seen the big tech companies try everything from making ads more transparent to improving content recommendation algorithms to setting up ethics boards (albeit with limited effect and with critics saying 'you need to do much more!'). And, we've seen CEOs and policymakers and activists wrestling with each other over where to go next. We have not 'fixed' the problems, but it does feel like we've entered a new, sustained era of debate about what a healthy digital society should look like.
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Mesa Graphics: AMD and RADVSubmitted by Roy Schestowitz on Wednesday 24th of April 2019 02:54:12 AM Filed under
RADV Vulkan Driver Lands FreeSync/Adaptive-Sync For Mesa 19.1
While on the kernel-side there has been FreeSync support with the AMDGPU DRM driver since Linux 5.0 and for the OpenGL driver with RadeonSI there has been this functionality in Mesa 19.0 when paired with a supported kernel, the Mesa Radeon Vulkan driver has missed out on this action until now. But landing just in time for the Mesa 19.1 feature freeze is now the FreeSync/Adaptive-Sync enablement for RADV.
A Decade Later, Mesa Wiring In Support For Qualcomm/AMD's ATC Texture Compression
Adding to the list of Mesa 19.1 changes is now AMD_compressed_ATC_texture being plumbed into Mesa/Gallium3D primarily with a focus on the Freedreno driver.
AMD_compressed_ATC_texture is the extension worked on a decade ago by AMD/Qualcomm for ATC compressed texture formats. ATC was AMD's proprietary compression algorithm with a focus on mobile devices for power and memory bandwidth savings. That was right around the time ATI/AMD Imageon IP was sold off to Qualcom to form the Adreno graphics processors for the company's SoCs.
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Security LeftoversSubmitted by Roy Schestowitz on Wednesday 24th of April 2019 01:14:44 AM Filed under
How secure are your containerized apps? [Ed: Why does SJVN promote the Microsoft-connected anti-FOSS firm Snyk?]
IPFire 2.23 - Core Update 131 is available for testing
Finally, the next major version of IPFire is ready to testing. We consider our new Intrusion Prevention System such an important change, that we are calling it "IPFire 2.23" from now on. This update also contains a number of other bug fixes and enhancements.
How hacking threats spurred secret U.S. blacklist
U.S. energy regulators are pursuing a risky plan to share with electric utilities a secret "don't buy" list of foreign technology suppliers, according to multiple sources.
The move reflects the federal government's growing concern that hackers and foreign spies are targeting America's vital energy infrastructure. And it's also raised new questions about the value of top-secret U.S. intelligence if it can't get into the hands of power industry executives who can act on it to avoid high-risk vendors.
Joseph McClelland, director of the Federal Energy Regulatory Commission's Office of Energy Infrastructure Security, told a Department of Energy advisory committee last month that officials are working on "an open-source procurement list" for utilities to use when deciding where to source their software and equipment.
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Programming: Python, Rust and MozillaSubmitted by Roy Schestowitz on Wednesday 24th of April 2019 01:12:59 AM Filed under
What do companies expect from Python devs in 2019?
Python is everywhere.
According to the 2019's StackOverflow's Developer Survey, it is the 2nd most loved programming language in the world.
Sending Emails With Python
PyCoder's Weekly: Issue #365 (April 23, 2019)
Rust's 2019 roadmap
Each year the Rust community comes together to set out a roadmap. This year, in addition to the survey, we put out a call for blog posts in December, which resulted in 73 blog posts written over the span of a few weeks. The end result is the recently-merged 2019 roadmap RFC. To get all of the details, please give it a read, but this post lays out some of the highlights.
Why AI + consumer tech?
After talking to nearly 100 AI experts and activists, this consumer tech focus feels right for Mozilla. But it also raises a number of questions: what do we mean by consumer tech? What is in scope for this work? And what is not? Are we missing something important with this focus?
At its simplest, the consumer tech platforms that we are talking about are general purpose internet products and services aimed at a wide audience for personal use. These include things like social networks, search engines, retail e-commerce, home assistants, computers, smartphones, fitness trackers, self-driving cars, etc. — almost all of which are connected to the internet and are fueled by our personal data. The leading players in these areas are companies like Google, Amazon, Facebook, Microsoft and Apple in the US as well as companies like Baidu, TenCent, and AliBaba in China. These companies are also amongst the biggest developers and users of AI, setting trends and shipping technology that shapes the whole of the tech industry. And, as long as we remain in the era of machine learning, these companies have a disproportionate advantage in AI development as they control huge amounts for data and computing power that can be used to train automated systems.
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Devices: Airtop3 With Linux Mint and Debian's Jonathan McDowell Studies a PCBSubmitted by Roy Schestowitz on Wednesday 24th of April 2019 01:09:59 AM Filed under
Airtop3 Manages A Passively-Cooled Core i9 9900K + Quadro RTX 4000
CompuLab today announced the Airtop3, the latest in their series of industrial-grade, excellently built fanless PCs. The CompuLab Airtop we benchmarked back in 2016 while showing its age now with the Core i7 5775C Broadwell processor is still running strong with its original design and even after what's been hundreds if not thousands of hours of benchmarking workloads still is running strong. Then again, that isn't too surprising as we continue to be improved by their build quality now after benchmarking their systems with Linux for the past decade.
Fanless mini-tower runs Linux Mint on up to 5GHz octa-core i9-9900K
Compulab's passively cooled, Linux-friendly "Airtop3" mini-tower builds on a 9th Gen, octa-core Intel Core i9-9900K with Quadro RTX 4000 graphics plus up to 128GB DDR4, NVMe and SATA storage, triple displays, 2x GbE, 6x USB 3.1, and -40 to 70°C support.
Compulab has launched a redesigned Airtop IoT edge server that accomplishes the challenging task of passively cooling Intel's high-end, 9th Gen Core i9-9900K processor. The Airtop3 is "nearly" twice as powerful as the 7th Gen Kaby Lake based Airtop2 mini-tower while maintaining the fanless, embedded-oriented design, says Compulab. Linux Mint and Windows 10 Pro are available.
Jonathan McDowell: Making my first PCB
I then started to notice I was getting JLCPCB ads while web browsing, offering 10 PCBs for $2. That seemed like a good deal, and I thought if I did things right I could find the right case and then make sure the PCB fitted in it. I found a small vented white case available from CPC. This allows me to put a temperature sensor inside for some devices. KiCad seemed like a good option for trying to design my basic schematic and lay it out, so I installed it and set to work trying to figure out what I wanted to put on the board, and how to actually drive KiCad.
As the core I chose an ESP-07 ESP8266 module. I've used a few of them before and they're cheap and convenient. I added an LDO voltage regulator (LD1117) so I could use a 5V PSU (and I'm hoping with a heatsink I can get away with a 12V supply as well, even if it's more inefficient). That gave enough to get a basic schematic once I'd added the necessary pull-up/down resistors for the ESP8266 and decoupling capacitors. I included a DC barrel jack for the PSU, and pin headers for the serial port, SPI pins and GPIO0 for boot selection. One of my use cases is to make an LED strip controller, so I threw in a screw terminal block for power + control - the board is laid out to allow a MOSFET for a simple white 12V LED strip, or the same GPIO taken straight to the terminal block for controlling a WS2812 strip. By including a couple of extra pull-up resistors I added the option of I2C for further expansion.
After I had the basic schematic I moved to layout. Luckily Hammond provide 2D CAD files for the case, so I figured I would import them into KiCad's PCB layout tool to make sure things would fit. That took a little bit of effort to go from DWG to DXF and trim it down (I found a web tool to do the initial conversion and then had to strip out the bits of the case that weren't related to the PCB size + mounting points). I wasn't confident enough that the edge cuts layer would include the mounting holes, so I manually placed some from KiCad over the right spots.
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today's howtosSubmitted by Roy Schestowitz on Wednesday 24th of April 2019 01:07:22 AM Filed under
How to back up the Dropbox sync folder on Linux
How to automatically start programs on Mate
Learn Linux by Doing with New Linux Hands-On Training!
How to install feren OS 2019.04
What is Load Balancing and How Does it Work?
From the earliest days of the Internet era, it was painfully obvious that having a single application on a single server can't handle high-traffic situations. This limited set up simply could not provide high-availability.
No matter how powerful the underlying infrastructure of a single server instance of a given application or website, the reality is that concurrent traffic can overwhelm a single server. A solitary instance for application availability is quite literally a single point of failure.
To handle this load, the technique commonly referred to as load balancing was developed. It's an approach that enables organizations to execute workload balancing across different server and compute resources. The benefit of load balancing is that traffic and workload delivery are "balanced" or spread across available resources in an effort to provide higher resilience and availability.
In recent years, load balancing has become a feature of a broader class of technology, known as Application Delivery Controllers (ADC) which aims to provide multiple advanced load balancing features to ensure workload balancing, along with an overall high quality of application delivery.
Ansible: Generate Crypted Passwords for the User Module
Brute force against SSH and FTP services: attacking and defending SSH and FTP
Bash For Loop With Examples
Ansible Interview Questions & Answers for Devops
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Games LeftoversSubmitted by Roy Schestowitz on Wednesday 24th of April 2019 12:33:26 AM Filed under
Killer Chambers looks like an insane challenge to make you throw your gamepad, now available on Linux
Think you're made of tougher stuff? Killer Chambers will probably test you, given how you need some seriously slick reaction times and a good memory to survive it.
BATTLETECH is going to the city with the Urban Warfare expansion due in June
Paradox Interactive and Harebrained Schemes have announced that you will soon be blowing up cities in BATTLETECH, with the Urban Warfare expansion.
I have to admit, this one does sound rather exciting especially considering they're saying every single building in a city will be "fully destructible". Considering your line of sight can be blocked, taking down a few buildings is going to end up essential to your survival in this. Not just that, there will also be things of interest littered around like explosive fuel trucks, so battles here will be quite intense.
Minecraft just had a seriously huge update named Village & Pillage
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Android LeftoversSubmitted by Rianne Schestowitz on Tuesday 23rd of April 2019 10:31:06 PM Filed under
How to Install Google Chrome Extensions/Plug-Ins On Your Android Phone
Pre-installed Apps on Android Show Why Privacy is No Longer Guaranteed
Sony confirms pricing on latest Android TV models, tops out w/ $70,000 98-inch 8K HDR TV
Xiaomi Mi 6 receives its first Android Pie beta
US Galaxy Tab S4 Wi-Fi model now receiving Android 9 Pie update
Exclusive: Chrome screenshots confirm 'touchless' Android based on Oreo [Gallery]
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Ubuntu: Pop!_OS 19.04 is here!Submitted by Rianne Schestowitz on Tuesday 23rd of April 2019 10:25:35 PM Filed under
It's spring again! Leaves are budding and updates are blooming for Pop!_OS. Here's what's new in Pop!_OS 19.04:
-The Slim Mode option maximizes your screen real estate by reducing the height of the header on application windows
-Dark Mode gives your applications a relaxing ambience for nighttime viewing. Both Dark Mode and Slim Mode can be activated in the Appearance settings menu.
Also: System76 Releases Pop!_OS 19.04 for Its Linux PCs, Based on Ubuntu 19.04
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Red Hat Breathes New Life Into JavaSubmitted by Rianne Schestowitz on Tuesday 23rd of April 2019 10:18:11 PM Filed under
Red Hat is the new keeper of the keys to two popular versions of the open source Java implementation, OpenJDK 8 and OpenJDK 11. The company has taken over stewardship from Oracle, it announced last week.
Oracle ended commercial support for Java 8 and the Oracle JDK 8 implementation of Java SE last year. Oracle left the enterprise Java business when it transitioned support and maintenance of Java Platform to the Eclipse Foundation, where it is now known as "Jakarta EE."
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Netrunner Rolling 2019.04 releasedSubmitted by Rianne Schestowitz on Tuesday 23rd of April 2019 06:50:29 PM Filed under
Like its cousin, the Debian based version, Netrunner Rolling also ships a dark Look and Feel theme including the Kvantum theme engine.
Using the Kvantum Theme engine plus the Alpha-Black Plasma Theme allowed us to create a more 3D-looking design.
Moving the mouse into the lower right corner now visibly activates the "Minimize all Windows to show Desktop" function by a light glow.
For those who prefer the classic look, going back to the well-known LNF is a three-button click and explained under "Tips" in our current Readme Section.
Also: Debian-Based Netrunner Linux Gets April 2019 Release with New Look and Feel
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Android LeftoversSubmitted by Rianne Schestowitz on Tuesday 23rd of April 2019 04:56:31 PM Filed under
Best Android TV Boxes
Qualcomm's new 5G chip helped create a face-mounted Android tablet
Android tablet market shrinks even smaller as ASUS reportedly exits
Best Samsung Tablets
Galaxy S8, Note 8 Caller ID Not Working After Android 9? Try This
Does the Withings Move work with Android?
How to Get Flash on Android
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Server: Cloudwashing by SUSE and Openwashing by Red HatSubmitted by Roy Schestowitz on Tuesday 23rd of April 2019 04:29:24 PM Filed under
Why Hybrid Cloud is About to Get a Whole Lot Easier
It seems like analysts, vendors and IT decision makers have been talking about "hybrid cloud" for the longest time. The concept has been around for at least a decade – and that's a really long time in the IT industry. Is it still important? Absolutely.
Almost every piece of cloud market research I read shows the majority of enterprises are focusing on a hybrid cloud strategy. Why? Because they all need increased agility, innovation and productivity, better cost optimization and improved customer experience.
The Open Organization guide to Red Hat Summit 2019 [Ed: The 'Open Organization' slant in Red Hat Summit 2019 with Microsoft CEO as keynote because it's all about money, not "open" or "free" (just proprietary and expensive]
When Red Hat CEO Jim Whitehurst published The Open Organization in 2015, he didn't just release a book. He catalyzed a global conversation about the ways open principles are reshaping organizational culture and design.
Developing distributed applications and services for tomorrow: a proof of concept
Innovation is accelerating across the automobile industry, bringing advances in the in-vehicle experience. Connected vehicle technologies are opening up new business models and providing a whole range of new software and data-driven services.
When it comes to new software and data-driven services, the possibilities are immense. But there is one trend many use cases have in common: they are becoming more distributed. To provide a great user experience, connected in-vehicle services often need to integrate increasingly diverse data.
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OpenBSD 6.5 Released With RETGUARD, OpenRSYNC
OpenBSD 6.5 was released today, about one week ahead of schedule for this security-minded BSD operating system. OpenBSD 6.5 is bringing several prominent new features including RETGUARD as its new stack protector and OpenRSYNC as its ISC-licensed in-progress replacement to rsync. OpenBSD 6.5's new RETGUARD functionality aims to be a better stack protector on x86_64 and AArch64 with instrumenting every function return with better security properties than their traditional stack protector. Also: OpenBSD 6.5
Development kit showcases Cortex-A76 based Snapdragon 855
Intrinsyc has launched a 96Boards CE form-factor "Snapdragon 855 Mobile HDK" that runs Android 9 on a 7nm, octa-core Snapdragon 855 with GNSS, WiFi/BT, and optional touchscreens and cameras. Intrinsyc's Qualcomm Snapdragon 855 Mobile Hardware Development Kit is now available for $1,149, offering a development window into Qualcomm's powerful Snapdragon 855 SoC. The new HDK runs the latest Android 9.0 Pie release.
Sad News! Scientific Linux is Being Discontinued
Scientific Linux, a distributions focused on scientists in high energy physics field, will not be developed anymore. It's creator, Fermilab, is replacing it by CentOS in its labs.
today's leftovers
Older Stories (Next Page)
- Shows: mintCast 307 and LINUX Unplugged 298
- Android Leftovers
- Today in Techrights
- How to enable SSH access using a GPG key for authentication
- today's howtos
- GNU: FS Directory, GNOME Builder and GtkSourceView Moving to Meson
- Ubuntu: GPD Boasting Ubuntu MATE (But Won't Preload), Adwaita Theme on Ubuntu 19.04 and Mark Shuttleworth at Open Infrastructure Summit
- WWW and Development
- Mesa Graphics: AMD and RADV
- Security Leftovers
- Programming: Python, Rust and Mozilla
- Devices: Airtop3 With Linux Mint and Debian's Jonathan McDowell Studies a PCB
- today's howtos
- Games Leftovers
- Android Leftovers
- Ubuntu: Pop!_OS 19.04 is here!
- Red Hat Breathes New Life Into Java
- Netrunner Rolling 2019.04 released
- Android Leftovers
- Server: Cloudwashing by SUSE and Openwashing by Red Hat


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Caruso's Brahmi 9000 One A Day 50T
Help clear your mind and bring some focus to your day with Caruso's Brahmi 9000 One A Day 50T. Brahmi has been used in Traditional Ayurvedic Medicine for centuries to assist with the maintenance of memory and to support the nervous system. Caruso's Brahmi 9000 One A Day 50T may help to support memory and maintain healthy brain function, whilst helping to support mental clarity and improve concentration and learning.
Learn more
Caruso's Brahmi 9000 One A Day 50T may help to maintain healthy brain function including memory, learning and cognitive function. It may also be useful in promoting mental concentration and focus. Brahmi has been used traditionally in Ayurvedic medicine as a nerve tonic to support memory and mental clarity.
Customers who would use Caruso's Brahmi 9000:
- People who wish to improve their learning ability
- Those looking to improve concentration and focus
- Elderly people who wish to support brain health and memory
- People who wish to improve their cognitive performance, mental function and memory recall
- Students who wish to improve their learning and mental capacity
|Caruso's Natural Health
- May support healthy brain function
- May enhance mental clarity, concentration and focus
- May improve learning and cognitive performance
- May improve short-term memory and memory recall
- May maintain a healthy nervous system
Adults and children over 12 take 1 tablet a day with food or as advised by your healthcare professional
Each tablet contains herbal extracts equivalent to:
Bacopa monnieri dry extract…………………...450mg
derived from Bacopa Monnieri dried whold plant 9g
Standardised to Bacoside A….................….202.5mg
Warnings and Contra-Indications:
- Always read the label and follow directions for use. If symptoms persist, worsen or change unexpectedly, talk to your health professional
- Not recommended for use while pregnant and/or breastfeeding
- Not suitable for children under 12 years of age
- Contains Soy
- Caution with anti-hypertensive and blood pressure medication, and hyperthyroidism
- Take at least two hours away from pharmaceutical medication
- Caruso's Testorod 60TCARUSOS NATURAL HEAL$94.95$69.9526%OFF RRP
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- Caruso's Saffron Two A Day 60TCARUSOS NATURAL HEAL$29.95$21.9527%OFF RRP


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Dow Kokam Breaks Ground on $322M Battery Plant in Midland, Michigan
"We're making history today for Michigan and for the adoption of alternative energy by the automotive industry," said Dow Kokam CEO Ravi Shanker. "In just nine short months, Dow Kokam has moved from formation to construction of a manufacturing facility to meet the needs of the next generation of the auto industry."
Construction will be completed in two phases, which are supported by federal grants from the American Recovery and Reinvestment Act (ARRA). The first phase of $322 million building will receive $161 million in federal support. Vice President Joe Biden and Michigan Governor Jennifer Granholm were both on hand to celebrate the groundbreaking.
The project will create 320 jobs in its first phase, and ultimately 800 positions by 2015. Operations will begin in 2012.
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Atlassian Sourcetree is a free Git and Mercurial client for Windows.
Atlassian Sourcetree is a free Git and Mercurial client for Mac.
The Configuration Foundation (Confound) library provides a lightweight yet powerful model for accessing various types of application configuration facilities via Csar.
More complex configuration may be using
Confound.setDefaultConfigurationConcern(ConfigurationConcern) with the concern of choice, as in the following example:
Confound.setDefaultConfigurationConcern(new MyConfigurationConcern());
Quick Start
This initial release of Confound only supports system variables and environment variables.
An application can easily integrate Confound configuration support with just a few steps:
- Include the dependency
- Pass configuration information to the application using an environment variable such as
FOO_BARor a system property such as
- Call
Confound.getConfiguration().getXXX("foo.bar")to retrieve the type of parameter you desire. This will retrieve the system property
foo.baror, if not present, the environment variable
FOO_BAR, using the appropriate case automatically.
Note: Confounded also provides a mixin interface
Confounded that allows an instance of
MyClass to call
Other Confound implementations in addition to system and environment variables will be available in the future.
Confound is available in the Maven Central Repository in group io.confoud.
Issues tracked by JIRA.
- 0.5.0: First release.


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As the back-to-the-future American food movement implores U.S. food policymakers to look to Old World models of local and artisanal production, the Old World itself is changing beyond recognition. This week Europe ended the decade much as it began it--buffeted by food scares. In the wake of the storm comes a stark sanitize-or-perish ultimatum for food producers that could spell the end for many small farmers, butchers and cheese makers.
The latest panic was sparked when a new diagnostic test revealed cases of bovine spongiform encephalopathy (BSE), the lethal mad cow disease, in slaughterhouses in Spain, France and Germany. Continental beef consumption plummeted by 40%, cattle markets emptied, sweetbreads were scrubbed from menus and the world's press bore down on even the tiniest Bavarian bratwurst stalls.
Ten years before that, charges over listeria in French cheese and salmonella in British eggs sparked a cross-Channel trade dispute likened in the right-wing French press to the Hundred Years' War.
If the paroxysms have become familiar over the years, government reactions to them could scarcely have changed more radically. When the era of food scares first dawned in the 1980s, the stock official response was to deny or downplay the danger. This French disclaimer about listeria in 1989 was typical: "It would take a lot to convince a Frenchman that his beloved Camembert, which he had been eating since childhood, was poisonous, even if it was--which it isn't."
Today, the requisite response is calls for accelerated testing programs, ever-tighter hygiene protocols and the formation of tough new watchdog agencies.
It took an unprecedented disaster involving a deadly new peril to precipitate the shift. In 1996, the deaths of 10 young people from the human form of mad cow disease forced the terrifying realization: Cattle products were in such universal use in food, pharmaceuticals and cosmetics that 59 million Britons had potentially been exposed to an incurable, mind-rotting disease.
The following year, when Tony Blair's Labor Party swept to power in the U.K. on the back of the crisis, the first day in office it unveiled plans for a new Food Standards Agency. Elsewhere in Europe, Ireland and France also created new agencies. To top it off, last month in France, leaders of the 15 member states of the European Union just approved plans for an overarching European Food Authority.
Safety Rules
For the reformers, uncompromising regulation is the only way to safeguard food and buoy consumer confidence in a globalized industry. This galvanized new safety movement has also fired a series of warning shots toward Europe's American trading partners, expressing fear and loathing of genetic modification of crops and hormone-treated beef.
"We have to regain public confidence in the capacity of the food industry and in public authorities to ensure that food is safe," insists EU Health and Consumer Commissioner David Byrne.
But to the minds of an array of Europe's most traditional food handlers--butchers, cheese makers, chefs, cookery writers and even safety experts--U.S. genetically modified crops and drug-enhanced beef are far from the only threat to consumer confidence. They charge that the new European safety movement itself is also wreaking irreparable damage. Holding local artisans to the same standards as global companies, they say, is a death sentence for small producers and timeless regional traditions.
Their objections come in the face of a formidable PR blitz. Byrne, a lawyer by training, has spent much of the last year flying from European capital to capital promoting the benefits of the new "farm to fork" regulation, "rapid alert system" and "critical hazard control checks." The party line: "Safety is the most important ingredient in our food."
Sources close to Byrne are, if anything, even more enthusiastic. "It is only positive and not negative," insists one spokesperson. "I don't see how anyone could possibly be against safe food."
But among Byrne's most passionate critics are his own countrymen in Ireland. "David Byrne needs to come back over here and meet real people who are trying to produce safe, quality food," says Darina Allen, proprietor of the Ballymaloe Cooking School in Cork County and a best-selling cookbook author.
"He needs to follow them around and find out what their lives are really like. Quality food producers are being hassled out of existence."
A Butcher's Tale
She points to Ed Hick, a fourth-generation Irish butcher.
"Quality is a word I wouldn't use anymore," says Hick. "It doesn't necessarily mean good; it means clean or spotless."
Hick, who used to supply his Dublin butcher shop with pork that he and his brother processed at a tiny nearby slaughterhouse, no longer slaughters his own pigs. The silky caul he used to produce for terrines, the chitterlings, the kidneys that used to come with the loins are all unavailable.
According to Hick, it's not any one rule that forced his family to close the slaughterhouse where they killed at most 25 pigs a week, but a succession of them. First, the local pig-keepers began to disappear.
"We've seen pretty much all the small guys we used to deal with go out of business," he says. "There was a coal man and a bus driver and just local people for whom it was a tradition to keep pigs on the side. These guys would have six or eight pigs at a time. They fed them on vegetable waste that they collected from the hotels. But then there was legislation requiring these guys to tool up with thousands and thousands of pounds' worth of equipment just to cook swill for the pigs."
Then came the 1992 structural requirements of the European Union Fresh Meat Directive that would condemn thousands of slaughterhouses across Europe. "Nobody came out and told us to close," he says. "It just became more and more apparent. We had people asking us to open up doorways through walls and others asking us to close them. There was quite considerable cost, as well. I think we figured out it was costing us [$7,000 to $10,000] a year."
If he found it hard to comply with regulations, he admits that he was also hard to regulate. Toward the end, he says, it was not uncommon for three inspectors--a veterinarian, a meat inspector and a hygiene inspector--to be dispatched to supervise him and his brother at work. "If we had taken them on in football, we would have lost," he says. "We were seen as a huge waste of veterinary time and manpower."
The meat Hick now sells comes from massive EU-approved slaughterhouses that process more than a thousand pigs a day. While he does not question the hygiene in large plants, Hick is not impressed by the animal husbandry or quality of the meat.
"I don't get as long a shelf life out of my product," he says. "Bad handling of the animal while it's alive, bad handling when it's killed and after it's killed all reduces the quality of the meat. A sympathetic hand probably tacks another two day's shelf life onto your meat. It's to do with the animals being rested and not too stressed."
His craft, he says, is dying. "Because of the way things have gone and this policy of centralization and intensification, basically the trade aspect of slaughtering has gone by the board," he says. "There isn't a huge amount of training. One person works one section of a line only."
When it comes to traceability of foods--a hot topic in the wake of massive recalls sparked by both BSE and a 1999 Belgian dioxin in poultry and pork scare--Hick is unimpressed by the new systems. "To be honest, I think we offered more traceability in the old days than all these new tattoo export animals. My brother and I slaughtered animals that we bought from local people then sold the meat back to local people," he says.
Death of the Slaughterhouse
British food safety advisor Richard North chronicled the death of the local slaughterhouse in the United Kingdom in the 1993 pamphlet "Death by Regulation" for the London-based Institute of Economic Affairs. Since 1980, by his count, the number of slaughterhouses has fallen from 1,500 to 350.
"The net effect of scares is always to concentrate the industry into fewer and fewer hands," he says.
EU officials counter that concentration of food industries is an international trend, not a result of their policies.
Whatever the cause, nobody disputes that food production is intensifying at a fierce pace. When Britain joined the European Union in 1972, it had more than 100,000 dairy farms. Today it has an estimated 30,000. While farm numbers plummet, herd sizes rise and animals once given names like Daisy and Dewdrop are now more likely to be called Cow 133 and 134.
Among its shopkeepers, a network of 35,000 butcher shops is now down to fewer than 14,000, with closures since the 1996 BSE crisis estimated at seven a week. Meanwhile, in the country Napoleon derided as a "nation of shopkeepers," six major supermarkets now control sale of more than 75% of the food.
Worst are the farm failures. December figures released by the British Ministry of Agriculture show that in the last year, more than 450 farmers and farm workers were driven out of business every week in what is now being described in the British press as "the worst collapse in farm incomes for 60 years."
North, a confessed Europhobe and now a research director in Brussels for the policy group Europe of Democracies and Diversity, argues that the new regulations can only speed the collapse. He points to the egg farmers whom he consulted during the 1989 salmonella scare. Having only just paid off the sizing machines to grade "legal" eggs, they must now buy identification machines to comply with new tracing regulations. "These can cost about [$100,000] apiece," he says. "Farm incomes are so marginal, this could effectively wipe out small producers."
Modernizing Craftsmen
In Spain, these are the village traders. Alastair Brown is an Englishman who bottles organic chestnuts near Seville. His business is new, and he had no choice but to tool up to meet the latest requirements. He says while at first he resented the stringent protocols, now he respects them. "However," he adds, "where the regulations can become a nonsense is for the smaller artisanal producers."
These, he says, have been disappearing before his eyes at the local market. "The local market used to be a thriving, boisterous place with half a dozen fish stalls, a handful of butchers and various fruit and veg people. It has been completely neutered by having all sorts of remodeling done to comply with new regulations. The result is that rents have gone up and less than a third of the stall holders are back. So it has pushed what was a pretty marginal activity for people beyond the point of nonexistence."
Andrea Petrini, a French food writer in Lyons, reports that a now constant feature of the city's famous La Croix-Rousse organic food market are fluttering petitions complaining about the same sort of new refrigeration, plumbing and structural requirements. "These cost lots of money," he says.
But again, the EU objects. "The standards that are set are very basic," retorts an official from Brussels. "They consist of temperature control for milk or meat and things, like in a market you have to have hot water and soap. It's really very basic stuff." France and Italy, they add, have been actively assisting traditional food businesses cope with the regulatory burdens.
The most unpopular aspect of the new regulations is the paperwork. Every food handling establishment, from farm to restaurant, must now abide by a process called HACCP, an acronym for Hazard Analysis Critical Control Point. This requires that crucial actions are routinely logged. Local inspectors will then check the records, and their scrutiny will be double-checked periodically by the new European authority.
Irish butcher Hick describes the burden of having to log one's every move as the equivalent of "having to do a written test on the rules of the road while you are driving."
Scottish chef Jeremy Lee agrees. He runs the Blueprint Cafe in London, a 120-seat restaurant above the Design Museum that is part of a larger chain owned by Sir Terence Conran. Lee estimates that logging fridge temperatures, cleaning schedules, training and risk assessment takes 15 hours a week. "We're lucky to be part of a large organization," he says. "You'd be running ragged to keep up with it if you were a wee independent."
But the new policies also have determined supporters. David Hicks--no relation to the butcher--runs a 15-year-old restaurant meat supply business in Dublin. "We have benefited tremendously from the EC [European Commission]," he says. At his newly revamped factory, meat arrives by the ton already boned, shrink-wrapped and boxed. Hicks likes it that way. "The meat factories have got to get it right in the farms, the abattoirs, the factories--the whole lot--so that the consumer can be assured that the product leaving Ireland is coming from the best conditions," he says.
Representatives of Europe's most powerful cooperatives also welcome the plan. David Biltchik, U.S. representative for the Consortium of Prosciutto di Parma argues that the new rules provide "equivalency" of safety measures in the world market. He says that since the 1970s, the 200 ham producers belonging to his consortium have "spent millions" on upgrades and safety research that has protected, not destroyed, a traditional product. "I do not accept for a minute that the quality or the tastiness of Italian food is being destroyed by all this regulation. Nonsense."
French officials, once so skilled at denying risk in eating their soft, raw milk cheeses, also now concur. In 1999, seven years after an EU directive instructed it to adopt a zero-tolerance standard for listeria in soft cheese, France has finally complied, causing a storm among producers of Epoisses, Maroilles, Saint-Felicien and Camembert.
At the same time, the Wall Street Journal reported the rise of pasteurized "vrai faux" cheeses: industrial cheese in rustic packaging.
French officials are unrepentant. "It would be very difficult to say to the consumers that the rules should not be the same because of the size of the company," says a delegate to the EU. "Food safety is food safety."
Effectiveness Unknown
Whether or not harmonization of food safety standards will check the impact of bacterial pathogens such as salmonella, listeria and campylobacter is not only unknown, it may be unknowable. The EU's Scientific Committee on Veterinary Measures relating to Public Health reported in May that existing food poisoning statistics from member states are too inconsistent to be reliable.
But safety expert North, who charted the British salmonella-in-eggs scare for his doctoral thesis, argues that we had long lived with these bugs quite peaceably, that the dangers have been grossly overblown and public fear cynically manipulated, not least by the media. " . . . in the night imagining some fear, how easy is a bush supposed a bear," he says, quoting Shakespeare.
Yet North also acknowledges that in the shadow of the BSE crisis, the scale of the impact of these old, largely gastrointestinal scourges now hardly matters. Since the first 10 of now almost 90 human deaths linked to mad cow disease were announced in 1996, the argument has not been just over consumer welfare, but liability when something goes wrong. Who is to blame in case of another food catastrophe?
Dodging Blame
BSE forever changed old notions of risk and responsibility. Multinational rendering and livestock feed companies may have recycled the lethal pathogen in contaminated feed, but last October a public inquiry into BSE by the British government could not find fault with their behavior. Technically, they had behaved legally, even when they had knowingly exported suspect rations for almost a decade after feed was determined to be the cause of BSE in Britain.
The inquiry did, however, censure U.K. officials for dozens of regulatory failures and "sedating" consumers with unfounded reassurances. Last October, in the wake of the inquiry, the British government only managed to preempt a class action lawsuit brought by families of the now almost 90 human victims by promising a no-fault compensation package. The cost, now estimated in millions of dollars, could rise to billions, depending on the final human toll.
Under the new European Food Authority, the diligence of complying member states may well be unassailable. Bang up front in the hygiene section of the new legislation is the warning: "The leitmotif throughout the recast of the hygiene rules is that food operators bear full responsibility for the safety of the food they produce."
But to the mind of North, while the mandatory implementation of HACCP might prove a death sentence for small producers, it could serve as the ultimate get-out-of-jail-free card for businesses big enough to devote the time to it.
"Failures in food safety will be legally defendable provided the paperwork has been maintained," he says. "Strict adherence to HACCP makes it legally impossible to prosecute anybody for causing food poisoning."
John Jackson of the Times library contributed to this report.
1981, SPAIN: Adulterated olive oil is recorded as the cause of more than 600 deaths and more than 25,000 illnesses, though arguments persist that the actual cause might be pesticide poisoning.
1988-93, UNITED KINGDOM: A junior health minister remarks on a TV news show, "We do warn people now that most of the egg production in this country, sadly, is now infected with salmonella." Panic ensues. The minister is sacked. Egg consumption plummets; 3.5 million hens are killed, 9,000 producers go out of business and the incidence of salmonellosis continues to rise. The sacked minister writes a romance novel.
1983-89, EUROPE: Hundreds die across Europe from listeriosis. Soft cheese made on the French-Swiss border, known to have killed 31, is blamed. British health authorities warn pregnant women against eating French soft cheeses. French editorials retort that Britain's petits pois are not petits, its milk products are adulterated with vegetable fats and its sausages short on meat, and that it is flooding France with bad eggs. Germany and Sweden also ban some French cheeses. The main culprit behind many of the deaths proves to be Belgian pa^te.
1990, EUROPE: Discovery of benzene in Perrier causes withdrawal of 160 million bottles throughout Europe.
1990, NORTHERN IRELAND: Cattle carcasses held back by butchers because of "unusual characteristics" lead to exposure of widespread abuse of Clembuterol, a hormone banned by the EU since 1988. By the time a detection test is developed in 1993, two farmers are dead from inhaling it, customs officials have made dozens of seizures of the drug and Irish beef livers are recalled from more than 200 supermarkets in the Republic of Ireland and Great Britain.
1991-94, ITALY: The country's Istituto Superiore di Sanita (its National Institute of Health) reports that the most frequently implicated dish in food-borne salmonellosis cases is tiramisu.
1992, FRANCE: At least 29 people die in France from a Listeria outbreak traced to cured meat products.
1993, GERMANY: More than 1,000 people, mainly children, contract salmonellosis from contaminated paprika and paprika-powdered potato chips.
1994, SPAIN: 48 cases of salmonellosis in 14 regions traced to powdered infant formula.
1995, UNITED KINGDOM: The first of now more than 80 deaths among young people from the human form of bovine spongiform encephalopathy, or mad cow disease. By 1996, the scare is global. The European Union bans British cattle products from the world market, Britain begins destroying more than 4 million cattle. In 1997, the Conservative government, in power for the duration of the BSE epidemic, falls.
1996, SCOTLAND: More than 300 are sickened and more than 10 senior citizens die from E. coli O157 infections contracted from food served at a church social.
1997, SWEDEN: Swedes charge that their salmonella-free status is threatened by contaminated foods, including turkey kebabs, which had been approved by the EU and imported from France, Denmark and Belgium.
1999, BELGIUM: Oil containing PCBs and dioxins is criminally dumped into cooking fats awaiting pickup for recycling into chicken and pig rations. Deformities noticed in chickens are determined to be due to dioxin poisoning. Belgian egg and meat products are swept from shops and banned from the world market. "Chickengate" costs the ruling prime minister his office. No human illness or death is noted, but a venerable and theoretically safe practice of recycling cooking fat in animal feed is banned and fat disposal quickly becomes an environmental issue.
1999, FRANCE: Classic soft cheeses Epoisses, Maroilles, Saint-Felicien and Camembert are recalled when France finally adopts a 1992 EU zero-tolerance directive for listeria detection in any 1-ounce sample of cheese.
2000, EUROPE--Beef consumption plummets by 40% across the continent when it is revealed that meat from a herd with a case of BSE has been sold in French supermarkets, and new cases are discovered in German and Spanish herds. A ban on the suspected carrier of BSE, meat and bone meal, is extended to rations for pigs and poultry. Europeans consider replacing the MBM protein constituent with dreaded imports of genetically modified soy beans from the U.S.


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Here, we provide an optimized protocol for the isolation and cloning of the scarce T-cell entity of peripheral Vδ1+CD4+ T cells that is, as we showed recently, an extrathymic αβ T-cell progenitor. This technique allows to quantitatively isolate, clone and efficiently expand these cells in ex vivo culture.
Cite this ArticleCopy Citation | Download Citations
Welker, C., Handgretinger, R., Schilbach, K. Isolation and Ex Vivo Culture of Vδ1+CD4+γδ T Cells, an Extrathymic αβT-cell Progenitor. J. Vis. Exp. (106), e53482, doi:10.3791/53482 (2015).
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The thymus, the primary organ for the generation of αβ T cells and backbone of the adaptive immune system in vertebrates, has long been considered as the only source of αβT cells. Yet, thymic involution begins early in life leading to a drastically reduced output of naïve αβT cells into the periphery. Nevertheless, even centenarians can build immunity against newly acquired pathogens. Recent research suggests extrathymic αβT cell development, however our understanding of pathways that may compensate for thymic loss of function are still rudimental. γδ T cells are innate lymphocytes that constitute the main T-cell subset in the tissues. We recently ascribed a so far unappreciated outstanding function to a γδ T cell subset by showing that the scarce entity of CD4+ Vδ1+γδ T cells can transdifferentiate into αβT cells in inflammatory conditions. Here, we provide the protocol for the isolation of this progenitor from peripheral blood and its subsequent cultivation. Vδ1 cells are positively enriched from PBMCs of healthy human donors using magnetic beads, followed by a second step wherein we target the scarce fraction of CD4+ cells with a further magnetic labeling technique. The magnetic force of the second labeling exceeds the one of the first magnetic label, and thus allows the efficient, quantitative and specific positive isolation of the population of interest. We then introduce the technique and culture condition required for cloning and efficiently expanding the cells and for identification of the generated clones by FACS analysis. Thus, we provide a detailed protocol for the purification, culture and ex vivo expansion of CD4+ Vδ1+γδ T cells. This knowledge is prerequisite for studies that relate to this αβT cell progenitor`s biology and for those who aim to identify the molecular triggers that are involved in its transdifferentiation.
In vertebrates, adaptive immunity that is structured in the cellular and a humoral part of immunity plays a major role in the defense against pathogens. The recognition of a wide range of antigens is mediated by hyperpolymorphic T- and B cell receptors (TCR/BCR), which with regard to T cells are assumed to be produced mainly in the thymus1. Thereto, hematopoietic stem cells (HSCs), derived from bone marrow, seed the thymus and differentiate along well-defined stages finally giving rise to all T cell lineages. Thymus seeding progenitors are CD4- and CD8- and thus constitute the immature, double negative (DN) thymocyte fraction. Thymus-derived signals then induce their lineage commitment and the differentiation into either αβ or γδ T cells. The expression of functionally rearranged TCR-γ and TCR-δ chain genes in DN2/3 thymocytes leads to γ δTCR complexes, which drive cellular proliferation and promote differentiation into γ δT cells2,3. In contrast, the rearrangement of a functional TCR-β chain, that can pair with preTα to build a preTCR pT, induces the transcriptional silencing of the TCR-γ chain in DN3 thymocytes and their transition into CD4+CD8+ double-positive thymocytes4. At this stage, recombination of the TCR-α chain occurs, deleting the TCR-δ locus that nestles within the TCR-α locus, thus abrogating the production a γδTCR in these cells irrevocably5-9. Rearranged αβTCRs are subsequently selected for their ability to bind self-MHC weakly (positive selection), which may not exceed a certain threshold to avoid autoimmunity (negative selection). According to their capacity of binding MHC class I or II, the selected αβT cells develop into single-positive CD4+ or CD8+ T cells, which exit the thymus as naïve T cells.
However, involution of the thymus starts early in life leading to exponentially reduced output of naïve T cells that is almost extinguished post-adolescence10. Nevertheless, the size of the T cell pool remains constant throughout life, which can be explained only in part by post-thymic homeostatic proliferation of T cells and the proliferation of long-lived immunologic memory11. Consequently, extrathymic T cell development must occur. Recent research has gained substantial attraction that characterized αβT cell progenitors, which-at extrathymic sites-gave rise to functional αβ T cells12-17. Yet, detailed knowledge about extrathymic αβT cell precursors that independent from a thymus differentiate into αβT cells is as fragmentary as the background that we have on the route they take thereby.
We recently identified the small T-cell entity of Vδ1+ CD4+γδT cells as an extrathymic αβT cell prognitor18, which when isolated from peripheral blood of healthy human donors can transdifferentiate into αβT cells in a mild inflammatory environment. Interestingly and contrary to the homeostatic proliferation of post-thymic T cells, transdifferentiation of Vδ1 CD4+ cells generates new T cell receptors, thus broadening the repertoire diversity, so that potentially new antigens can be recognized and may protection the host against newly acquired pathogens. This adds to the plasticity of T cells and adds a so far unappreciated new pathway for extrathymic T cell development.
The quantitative isolation from lymphocytic sources, the generation of single-cell clones and their efficient expansion are essential for the objective to identify those markers and molecules that trigger this αβT cell precursor`s extrathymic development.
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Ethic Statement: All procedures were carried out according to the Declaration of Helsinki and were approved by the Clinical Ethics Committee at the University of Tübingen (projects 38/2009B02 and 470/2013B02).
1. Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
- Take 50-100 ml from a healthy volunteer via venipuncture using a 50 ml syringe containing 1,000 IU heparin sulfate and dilute the blood 1:2 with PBS (pH = 7.2).
- Carefully layer 35 ml of the blood:PBS solution on 15 ml blood separating solution such as Biocoll (d = 1.077 g/ml) in a 50 ml conical tube. Centrifuge for 15 min at 800 x g at RT.
NOTE: Brake must not be used.
- Collect the cells of the lymphocytic layer with a pipette and wash the cells twice in at least 50 ml PBS (centrifuge for 12 min; at 400 x g). Remove the supernatant after centrifugation with a pipette.
- Lyse remaining erythrocytes by suspending and incubating the lymphocytic fraction with 1-5 ml of a hypotonic buffer solution for 2-4 min.
NOTE: Duration of exposure and volume of lysing buffer depend on the used lysing buffer and on the amount of red blood cells left in the lymphocyte fraction.
- Dilute the cells with PBS (1:10) and centrifuge at 250 x g for 12 min and subsequently resuspend and wash the cell pellet once in 10 ml PBS at 300 x g for 12 min.
- Resuspend the lymphocytes in PBS and count the cells in a Neubauer counting chamber using Trypan blue solution.
NOTE: Usually, >1 x 108 lymphocytes are received from 100 ml freshly drawn peripheral blood.
2. Isolation of Vδ1 T Cells
- Take <1 x 106 isolated lymphocytes for an initial FACS determining the frequency of Vδ1 CD4 T cells.
NOTE: Population size of this T-cell entity may differ greatly between individuals and according to their immunologic state. Typically, about 1% of T cells are Vδ1+ and about 1-5% of Vδ1+ cells are CD4+.
- Dilute the cells with 1 ml FACS-buffer, which consists of PBS containing 2% FCS and 5 mM EDTA. Centrifuge at 660 x g for 2 min and decant the supernatant. The reflux volume of about 100 µl FACS buffer is sufficient for the subsequent staining. Briefly vortex and incubate the cells with 5 µl Fc-Blocking Reagent for 5 min at RT for increased specificity of the FACS staining.
- Add human monoclonal antibodies directly to the cells without removing the Fc-Blocking Reagent. Make sure to stain the cells with antibodies against Vδ1 (1:50), CD3 (1:50), CD4 (1:200), CD8 (1:200) and TCRαβ (1:25). Prepare the isotype control with the corresponding immunoglobulin isotypes. Incubate cells with antibodies for 20 min at 4 °C in the dark.
- Wash the cells in 1 ml FACS buffer (centrifuge for 2 min; at 660 x g), decant the supernatant and proceed to FACS acquisition in the remaining buffer volume.
- Pelletize the cells (that are not used in the FACS analysis) by centrifugation for 12 min; at 300 g; 8 °C. Remove the supernatant and resuspend the cell pelletin 60 µl MACS-buffer per 1 x 107 cells. Add 20 µl Fc-Blocking Reagent per 1 x 107 cells and incubate for 5 min at 4°C.
- Add 10 µl FITC-conjugated anti-human Vδ1 antibody per 1 x 107 cells directly to the cells. Incubate for 12 min at 4 °C in the dark.
- Wash the cells using 14 ml MACS-buffer (centrifuge for 12 min; at 300 g; 8 °C). Discard supernatant completely and resuspend the cells in 80 µl MACS buffer per 1x107 cells.
- Take 1-2 x 105 cells and dilute them in 100 µl FACS buffer for verification of the labeling by FACS analysis. No further additives are needed for this step. Make sure that there is a sufficient difference in the brightness for Vδ1- and Vδ1+ cells. If this is not the case, repeat steps 2.3) and 2.4).
- Add 20 µl anti-FITC microbeads per 1 x 107 cells to the remaining cells, mix well and incubate for 15 min in the dark at 4 °C. Thereafter, wash the cells using at least 10 ml MACS buffer (centrifuge for 12 min, at 300 x g; 8 °C).
- During the centrifugation, equilibrate the pre-cooled magnetic column placed in a magnet with 500 µl MACS buffer.
- Resuspend the cells in 500 µl MACS-buffer (for cell numbers higher than 1 x 108, scale up this volume accordingly) and apply the cells carefully onto the column. Collect the flow-through containing the Vδ1- cells.
- Wash the column three times with 500 µl MACS-buffer and collect the flow-through again in the same tube. Note that the reservoir must be empty before applying buffer onto the column.
- Remove the column from the magnetic device and place it into a 15 ml conical tube. Quickly flush the column with 1 ml MACS buffer by firmly pushing the plunger into the column. Collect the eluate in a tube.
NOTE: In order to obtain a purity of >98%, it is usually necessary to repeat steps 2.7-2.9) with a second column.
- Verify the purity of the cells by FACS analysis18 with the same antibody panel as before (see 2.1.2) and count the cells.
3. Isolation of Vδ1CD4+ T Cells
- Resuspend 25 µl of CD4 beads with a vortex for >30 sec. Wash the beads (the minimum amount as depicted by the manufacturer) in 1 ml MACS buffer in a 1.5 ml reaction tube by placing the tube into a magnet for 1 min. Discard the supernatant carefully with a small-scale pipette (200 µl) and resuspend the beads in the original volume of MACS-buffer.
- Pelletize Vδ1+ cells (centrifuge for 12 min; at 300 x g) and aspirate the supernatant and resuspend all the Vδ1+ cells in 500 µl MACS-buffer and add them to the tube containing the beads. Mix vigorously and incubate for 20 min at 4 °C with constant tilting (e.g., in a rotator).
- Place the tube containing the cells in a magnet for 2 min. Make sure that there are no remnants in the lid.
NOTE: CD4+ cells will have bound the magnetic beads and attach to the side of the tube facing the magnet.
- Carefully open the lid while keeping the tube inside the magnetic device and collect the supernatant which contains the CD4- Vδ1+ cells using a small-scale pipette. Place CD4- Vδ1+ cells into a separate tube and place this tube into the magnet again to avoid possibly remaining CD4 cells or beads from the population.
- Wash the CD4- cells in 5 ml MACS-buffer (centrifuge for 12 min; at 300 x g) and resuspend them in a concentration of 1 x 105 cells per 100 µl media. CD4- cells can be readily cultivated under the conditions given below (4.2).
- Place the tube with the CD4+ cell targets outside of the magnetic field, resuspend the cells in 500 µl MACS-buffer and put them back into the magnetic device. Repeat steps 3.3-3.5 twice to obtain a higher purity. Remove supernatant by pipetting
- Resuspend the CD4+ cells in 100 µl culture media (RPMI 1640, 10 % FCS, 1% L-Glutamine, 1% Penicillin/Streptomycin) and add 10 µl of a bead-detaching solution. Incubate at RT for 45 min with constant tilting (e.g., in a rotator).
- Place the cells in a magnet for 1 min. Carefully collect the supernatant containing bead-free Vδ1+CD4+ cells using a small-scale pipette.
- Outside of the magnet, resuspend the beads with 100 µl culture media and repeat steps 3.6 and 3.7 twice to obtain higher cell numbers of CD4+ cells.
- Pelletize the cells (centrifuge at 300 x g, for 12 min) and discard the supernatant completely by pipetting. Resuspend cells in fresh, pre-warmed media and count them. Examine the purity of the Vδ1+CD4+ cells by FACS analysis depicted in steps 2.1.1-2.1.3.
4. Single-cell Cloning by Limited Dilution
- Isolate peripheral blood mononuclear cells (PBMCs) from an allogeneic donor as depicted in 1.1-1.6.
- Irradiate 2.5 x 107 allogeneic PBMCs with 80 Gy in 25 ml culture media using γ-radiation.
- Add IL-2 (200 U/ml), IL-7 (20 ng/ml) and PHA (2 µg/ml) to the irradiated feeder cells and distribute them in 96-well U-form plates, 5 x 104 feeder cells in 50 µl per well.
- Dilute the Vδ1+ CD4+ cells to a concentration of 0.3 cells per 50 µl. Pipette 50 µl of the cell solution to each well harboring the irradiated cells and cytokines.
NOTE: Thus, the cytokines are diluted to the final concentration of 100 U/ml IL-2, 10 ng/ml IL-7 and 1 µg/ml PHA. Incubate the 96-well plates in an incubator at 37 °C, 5% CO2 humidified atmosphere.
- Optionally, cultivate remaining purified Vδ1+ CD4+ cells as bulk culture under the same culture conditions as the clones.
- Supply the cells every 3-4 days with cytokines and fresh media. For this, exchange half of the medium in the wells with 50 µl fresh medium by pipetting. Add 2-fold concentration of cytokines to every supplementation. Add 5x104 irradiated feeder cells per well every other round of feeding.
- Using a microscope, observe first clones become visible after 3-4 weeks, as they metabolize and thus change the color of the culture medium and form colonies.
NOTE: For further cultivation, resuspend the clones well and transfer them to separate 96-well plates. Analyze the cells via FACS as indicated in 2.1.1-2.1.3. Clones kept under these conditions may eventually change their TCR from Vδ1 to αβTCR. The time point for this transdifferentiation varies from clone to clone.
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Representative Results
Figure 1 depicts the different stages and the outcome of the isolation of Vδ1 T cells from peripheral blood. Figure 1A shows a typical distribution of Vδ1+ cells in CD3+ lymphocytes, as well as the co-receptor expression of the Vδ1+ population. In this donor, the frequency of Vδ1+ cells (red) is 2.3% of total lymphocyte counts and the CD4 expression (green) of Vδ1+ lymphocytes is 2.6%. Altogether, the target population for isolation represents 0.06% of total lymphocytes in this donor. Figure 1B shows an optimal staining intensity for Vδ1+ cells before proceeding to the magnetic labeling. Staining with the primary antibody should result in clearly distinct Vδ1negative and Vδ1positive populations so that the Vδ1+ cells can efficiently be labeled and separated with the magnetic beads. Figure 1C depicts Vδ1+ cells after the isolation procedure by FACS analysis. The purity of Vδ1+ is usually >99% of CD3+ cells. To increase purity, isolated cells are applied onto a second isolation column. This results in considerable cell loss however this steps leads to quantitative elimination of potentially contaminating TCRαβ + CD4+ cells.
The results of the CD4+ isolation are shown in Figure 2. Due to the low numbers of CD4+ Vδ1+ cells, and since the cells are expanded in a limited dilution single-cell cloning approach purity of CD4+ cells less than 100% can be tolerated. In the experiment shown here, the purity of the Vδ1+CD4+ cells was about 90% (Figure 2A, right blot) and subsequent single-cell cloning resulted in more than 60 Vδ1+CD4+ T cell clones. Contrarily, in the Vδ1+CD4- fraction no CD4+ cells remained (Figure 2B), which highlights the efficiency of this isolation strategy. Of note, the CD4+ cells (blue) express Vδ1 at a distinguishable lower level than CD4- cells (red) (Figure 2A, both blots). Thus, staining of Vδ1+ cells must be excessive-as described in the protocol-for a sufficient labeling and successful isolation of the CD4+ fraction within the Vδ1+ T cell pool.
In Figure 3A, the typical phenotype of an emerging clone is presented. The TCR is composed of a Vδ1 and a Vγ9 chain and the clonal cells express CD3 and CD4. Figure 3B depicts the process of transdifferentiation of one clone isolated with the presented technique. Transdifferentiation includes the downregulation of the V´1-chain to a low expression level, which is paralleled by the de novo expression of an αβTCR. This leads to a transient TCR-double-positive phenotype which eventually gives rise to single-positive αβTCR-expressing T cells. The co-receptor expression may also change during the course of transdifferentiation. Similar to thymocytes, a CD4+CD8+ DP phenotype may occur, which eventually develops into either SP CD4+ or CD8+ αβ T cells. Under the culture conditions presented here, transdifferentiation is a rare event-only one out of 50 clones changed their Vδ1+ TCR into αβTCR.
Figure 1. FACS analyses monitoring all stages of the isolation process of Vδ1 T cells from peripheral blood. (A) Distribution of Vδ1+ cells (red) in CD3+ cells of peripheral blood lymphocytes and their co-receptor expression. CD4+ Vδ1+ cells are indicated in blue (circle). (B) Staining of the Vδ1 fraction before proceeding to magnetic labeling. (C) FACS analysis of Vδ1+ cells of positive fraction after isolation. Numbers indicate percentage of gated cells. Please click here to view a larger version of this figure.
Figure 2. FACS analyses of Vδ1 CD4+ isolates. (A) CD4+ fraction and (B) CD4- fraction after CD4+ cell isolation. Numbers indicate percentage of gated cells. Please click here to view a larger version of this figure.
Figure 3. Phenotypic analysis of a clone right after detection. (A) TCR composition and co-receptor expression of a freshly isolated clone. Vδ1 and Vγ9 TCR is co-expressed with CD3 and CD4. (B) Process of transdifferentiation in a CD4+ Vδ1+ clone. Change of TCR expression (top), and change of co-receptor expression (bottom)18. Please click here to view a larger version of this figure.
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To study the phenotype, biology and function of a scarce (T-) cell entity, namely Vδ1+CD4+ T cells, we used two markers: Vδ1 and CD4 for its positive magnetic cell isolation. Vδ1 is an orphan receptor, whereas CD4 is expressed on T helper cells, at a lower level on monocytes and dendritic cells, and at a very low level on hematopoietic progenitor cells.
Techniques for the enrichment and selection of cells in high purity include Fluorescence activated cell sorting (FACS), a technique that separates a population of cells into sub-populations based on fluorescent labeling. Cells stained using fluorophore-conjugated antibodies can be separated from one another depending on which fluorophore(s) they have bound. Sorters can achieve purity close to 98%19, which is useful if the only necessity is the sorting itself. Drawbacks of this technique include that sorted cells show a decrease in cell viability, and in our target population also a reduced potential for transdifferention. A reduced cell viability after sorting is hypothesized to be due to the shearing forces and hydrodynamic stress that the FACS imposes upon cells. Moreover, to keep cells viable and without contamination for subsequent culture, the experiment should be undertaken in aseptic sterile conditions which is hard to manage. In addition, FACS sorting recommends high numbers of cells for sorting.
Isolation Kits have been developed from several companies for direct magnetic labeling and the sorting of cells according to multiple surface markers. Their strategy relies on the removal of the magnetic label from the cells after the first sort by using a release reagent, which either enzymatically or competitively removes the magnetic label from the structure to which it was bound. This allows a second magnetic labeling and separation of the cells for another surface marker of interest that is achieved by using either direct or indirect labeling with magnetic beads. Removal of the label recommends working at low temperatures (on ice, at 2-8 °C) for extended periods of time, exposure to a chemical and repeated washing steps. If the target cells of the second parameter separation are present in a low concentration after selection (<10% target cells in the positive fraction after the first separation) even a second round for removal ofany residual magnetically labeled cells is needed. This procedure is-besides having a substantial loss of target cells due to repeated washing steps-at high risk for inducing cellular damage and loss of function due to mechanical and physiological stressors. Compared with FACS sorting however cell preparations remain sterile and smaller cell numbers can be sorted.
The strategy that we pursue combines two consecutively executed positive selections, yet the removal of the first label is not required as the magnetic labels differ in the magnitude of their magnetic forces by several log phases. While the first anti-fluorochrome magnetically-labeled bead type is small, with a diameter of 50 nm, the second anti-fluorochrome magnetically-labeled bead type measures 1 to 4.5 µm, thus exceeding the volume/strength of the first magnetic label 20 to 90-fold. Additionally, the second positive sort omits cell stress, that is associated with magnetic column retention/release as cells remain in an 1.5 ml vial that is put in a magnetic device so that labeled cells are attached to the wall of the vial. Washing procedures can be kept on a small scale. This elaborated protocol reduces cell loss and increases cell viability since cells experience less manipulation and pass the isolation process faster than protocols using multisort isolation kit systems.
Consequently, this protocol allows to separate even a small number of cells and another advantage: sorted cells remain sterile throughout the procedure. Subsequent cloning experiments using limiting dilution allows the efficient expansion of individual members of the isolated cells, here the very scarce Vδ1+CD4+γδ T cell entity18. Sorted cells show excellent viability and clonogenicity and remain fully functional.
For future applications, use this strategy to quantitatively select and successfully purify small T cell entities from diverse human sources including freshly drawn peripheral blood, (mobilized) leukapheresis products, cord blood and bone marrow by combining two consecutively performed positive selections using magnetic labels that differ by magnitude in their magnetic forces and thus retention capacity.
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The authors have nothing to disclose.
Christian Welker is funded by a grant provided by the Jürgen-Manchot-Stiftung.
|Catalog Number
|Biocoll Solution
|lymphocyte separating solution
|Lysing Buffer
|BD BioSciences
|lysis of erythrocytes
|Phosphate-buffered Saline
|Sigma Aldrich
|MACS buffer
|Miltenyi Biotec
|supplement with BSA and pre-cool before use
|Miltenyi Biotec
|not mandatorily from this supplier
|anti-human Vd1 FITC (clone: TS8.2)
|Thermo Scientific
|not mandatorily from this supplier
|anti-human CD3 PerCP (clone: SK7)
|BD BioSciences
|not mandatorily from this supplier or this flurochrome
|anti-human TCRab PE (clone: T10B9.1A-31)
|BD BioSciences
|not mandatorily from this supplier or this flurochrome
|anti-human CD4 VioBlue (clone: M-T466)
|Miltenyi Biotec
|not mandatorily from this supplier or this flurochrome
|anti-human CD8 APC-H7 (clone: SK1)
|BD BioSciences
|not mandatorily from this supplier or this flurochrome
|Anti-FITC MultiSort Kit
|Miltenyi Biotec
|yields better results than anti-FITC MicroBeads
|MS columns
|Miltenyi Biotec
|pre-cool before use
|MiniMACS Separator
|Miltenyi Biotec
|CD4 Positive Isolation Kit
|life technologies
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- Krangel, M. S., Carabana, J., Abbarategui, I., Schlimgen, R., Hawwari, A. Enforcing order within a complex locus: current perspectives on the control of V(D)J recombination at the murine T-cell receptor alpha/delta locus. Immunol. Rev. 200, 224-232 (2004).
- Hawwari, A., Krangel, M. S. Role for rearranged variable gene segments in directing secondary T cell receptor alpha recombination. Proc. Natl. Acad. Sci. U.S.A. 104, (3), 903-907 (2007).
- Hawwari, A., Krangel, M. S. Regulation of TCR delta and alpha repertoires by local and long-distance control of variable gene segment chromatin structure. J. Exp. Med. 202, (4), 467-472 (2005).
- Hawwari, A., Bock, C., Krangel, M. S. Regulation of T cell receptor alpha gene assembly by a complex hierarchy of germline Jalpha promoters. Nat. Immunol. 6, (5), 481-489 (2005).
- Hager, E., Hawwari, A., Matsuda, J. L., Krangel, M. S., Gapin, L. Multiple constraints at the level of TCRalpha rearrangement impact Valpha14i NKT cell development. J. Immunol. 179, (4), 2228-2234 (2007).
- Linton, P. J., Dorshkind, K. Age-related changes in lymphocyte development and function. Nat. Immunol. 5, (2), 133-139 (2004).
- Sprent, J., Tough, D. F. Lymphocyte life-span and memory. Science. 265, (5177), 1395-1400 (1994).
- Guy-Grand, D., et al. Extrathymic T cell lymphopoiesis: ontogeny and contribution to gut intraepithelial lymphocytes in athymic and euthymic mice. J. Exp. Med. 197, (3), 333-341 (2003).
- McClory, S., et al. Evidence for a stepwise program of extrathymic T cell development within the human tonsil. J. Clin. Invest. 122, (4), 1403-1415 (2012).
- Jbakhsh-Jones, S., Jerabek, L., Weissman, I. L., Strober, S. Extrathymic maturation of alpha beta T cells from hemopoietic stem cells. J. Immunol. 155, (7), 3338-3344 (1995).
- Garcia-Ojeda, M. E., et al. Stepwise development of committed progenitors in the bone marrow that generate functional T cells in the absence of the thymus. J. Immunol. 175, (7), 4363-4373 (2005).
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- Maillard, I., et al. Notch-dependent T-lineage commitment occurs at extrathymic sites following bone marrow transplantation. Blood. 107, (9), 3511-3519 (2006).
- Ziegler, H., et al. Human Peripheral CD4(+) Vdelta1(+) gammadeltaT Cells Can Develop into alphabetaT Cells. Front Immunol. 5, 645 (2014).
- Mollet, M., Godoy-Silva, R., Berdugo, C., Chalmers, J. J. Computer simulations of the energy dissipation rate in a fluorescence-activated cell sorter: Implications to cells. Biotechnol Bioeng. 100, (2), 260-272 (2008).
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Smooth, fluid lines and lithe shape suggest the languorous movement of a serpent. A shape so supple and graceful, it looks almost as if it would respond to touch. Shown left to right: 5 7/8 serpentine vase, 7 7/8 serpentine vase. Also available: 9 7/8 serpentine vase.
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Rectangle Tables, 96 in
Daily: $6.99
Weekly: $25.99
Monthly: $59.99
Additional Information
- Seats 8 people or 10 if you use the ends, 30 inches wide
- Line/Tablecloth Sizes Available:
- 60 by 120 - lap length
- 90 by 156 - floor length
* We will beat any local competitors price or sale! Just ask!
* The daily rate for PARTY items is considered a 3-day rate: pick up on day one, event on day two, and return on day three.
* Give us a call at (304) 728-6657 or email us, and we'll be happy to answer any questions you may have about our rectangle tables 96 in in Kearneysville, WV, serving West Virginia, Maryland, and Virginia.
* Prices subject to change without notice. Taxes, delivery, and other fees are not included in price estimates.