text:
Click menu bar and click Sidify Apple Music Converter, choose Registration in the drop-down list, you will get the register window. Or directly click the red word "Unregistered" to open the registration window. And then copy and paste your Email address and Code into the registration popup dialog.
Note: The license information is case sensitive. And, please do not add blank at the beginning or the end of the word.
There are two reasons for invalid code.
[1]First, the registration code doesn't match the program. Please make sure you have downloaded the right program.
[2]Second, blank spaces are accidentally added while filling in the registration information. Please mind that license information is case-sensitive. Do not enter any extra blank space to the Email address and license code.
[3]If you have checked that you are running the right program and follow the above tips but still get the error message, please contact our support team via firstname.lastname@example.com for help.
Important Notes: Since Sep. 22nd, 2016, Sidify's products adopt a new registration mechanism in the latest version. New users who purchased Sidify Apple Music Converter for Mac are required to register the program with Email address and registration code. For old users, it's still available to register with registration name and registration code.
It's the limitation of trial version. You can buy full version to unlock the 3-minute conversion limitation.
Sidify Apple Music Converter can convert any audio that can be played in iTunes, including Apple Music tracks, iTunes M4P music, iTunes audiobooks, and Audible audiobooks.
Now, Sidify Apple Music Converter can convert audio files to MP3, AAC, FLAC and WAV format.
[1]Please make sure you've well downloaded all your music from iTunes to your computer and your computer is well authorized with the same Apple ID that you used when downloading the iTunes music.
[2]If you've already downloaded the music but the program fails to read them, please recreate your iTunes Music library.xml for a try:
a. Uninstall Sidify from your computer
b. Go to your home directory –> Music –> iTunes and delete "iTunes Music Library.xml"
c. Download and install Sidify from Download Center (DO NOT launch Sidify)
d. Start iTunes and play a song to generate a new .xml file
Now close iTunes and start Sidify to check whether the issue can be fixed.
Please set the output format as MP3, AAC, FLAC or WAV and slow down the converting speed to 1X for a try. To customize output settings, please open program preferences by clicking Menu bar > Sidify Apple Music Converter > Preferences or directly clicking Setting button on the main interface. Then, set output format, and lower down the conversion speed to 1X in the pop-up setting window. If you are still unsatisfied with the output quality, please contact us at email@example.com for further help.
Please disable the Crossfade Songs in iTunes > Preferences > Playback.
Please disable the Crossfade Songs in iTunes > Preferences > Playback.
Before converting, please click the setting button on the top-right corner, and then check the box of the "Artist/ Album" after Output Organized. Now, Sidify program supports saving the output audio into Artist, Album, Artist/Album or Album/Artist folder for meeting various users needs.
Once choosing MP3 or AAC as output format, the metadata (including title, artist, album, art cover and track number) will be kept by default.
If you want to keep the original chapter in the audiobooks, please select AAC as output format. For more details, please refer to tutorial: How to Convert iTunes Audiobooks to AAC with chapters kept.
[1]At first, please make sure you are running the latest version of Sidify program, otherwise please directly download the latest version of Sidify Apple Music Converter through Sidify Download Center.
[2]Please make sure your original audio can be played on iTunes normally. -- If it is a purchased M4P music from iTunes, please make sure your computer is well authorized with the same Apple ID that you used when downloading the iTunes music.
[3]Please check whether the problem happen to certain music files or not. If it is, please re-convert the music for a try.
[4]Please check whether there is any external audio device connected with your computer including USB, Wi-Fi, Airplay, Bluetooth and HDMI, etc. If it is, please disconnect them and retry.
[5]After clicking "Convert", please open iTunes window and check whether is there any pop-up blocking message (such as download, authorization, update...). If it is, please resolve them and retry.
[6]For Mac OSX 10.13 users, please check whether you have installed other similar music converters. If it is, please try to delete the driver file under "/Library/Audio/Plug-Ins/HAL/MMAudio Device.driver", then open terminal, enter this command "sudo killall coreaudiod" and relaunch Sidify for a try.
Good news comes: the latest version of Sidify Apple Music Converter allows users to convert Apple Music songs at 20x speed under MacOS 10.13. You can click on "How to Convert Apple Music at Faster Speed under MacOS 10.13" for reference.
Some users responded that there was no sound from Mac laptop speakers after downloading and using Sidify. Now you can manage to fix this sound issue by following How to Fix No Sound Issue on Mac after Using Sidify.
Please contact our support team at firstname.lastname@example.com and provide us with detailed info like Mac OS, iTunes version, and program log files for analysis.
Tips: How to find the two log files of Sidify? Just find the menu button on the upper right corner and click on File > Show Log File, A window which contains the log files would pop up. They are iTunes.log and Application.log.


text:
▼ Deceit
Deceit tests your instincts at trust and deception in a multiplayer first-person shooter. You wake up in an asylum to the sound of an unfamiliar voice, surrounded by five others. A third of your group have been infected with a virus, but who will escape? Whilst traversing the building the power will be going out creating darkness, allowing the infected players to transform into their terror form and strike. Find allies and complete objectives around the map to better your chances of survival.
The environment has been specifically setup to cause conflict amongst the group, creating doubt about the true intentions of players. The infected will be trying to cover up their group sabotage attempts, whilst the rest will be keeping an eye out for this suspicious behaviour and attempt to join forces with people they think they can trust.
As you progress through the map you'll come across objectives that will help you survive and progress towards the exit. However, you will need to decide which is most important to you, and whether to collaborate or fight with other players over them. With each decision gives players more information and an indication to what team you're likely to be on. However, the perception of each action won't necessarily be seen as a fair representation to others about your intentions as the truth can be easily skewed.
There are blackout periods across the map which allow for the infected to transform into what's known as their terror form. In this form they are much faster, stronger, and have night vision, with only one weakness: light. In this form the infected are truly terrifying, and with an array of intense killing animations players will be able to create some scary but also funny moments.
Overall, the gameplay involves the frenzy of fast paced combat mixed with strategic map positioning, and of course the psychological aspects of determining who is infected.
Buy it here: http://store.steampowered.com/app/466240/Deceit/
Tell your friends and share Peeps!
It really helps me out!
Horror Trailer 7 T60 - Magnus Ringblom
Music used with permission from Epidemic Sound and Freedom!
▼ Links
My Social Media Links:
Facebook: https://www.facebook.com/Letstrygame
Google+: https://plus.google.com/+Gamefirsttuber
Twitter: https://twitter.com/Letstrygame
YouTube: https://www.youtube.com/user/GameFirstTuber
Watch my live streams on
Mixer: https://mixer.com/Letstrygame
▼ Gaming Computer
GPU: GeForce GTX 1080
CPU: Intel(R) Core(TM) i7-7700K CPU @ 4.20GHz
OS: Microsoft Windows 10 Pro
▼ Editing Computer
GPU: GeForce GT 730
CPU: Intel(R) Core(TM) i5-4690K CPU @ 3.50GHz
OS: Microsoft Windows 10 Pro
▼ Editing Software
MAGIX Movie Edit Pro Premium
▼ Copyright
No copyright infringement intended, all rights go to their respective owners.
All copyrighted/trademarked media is owned by their respective companies

subset: commoncrawl


text:
In a break from tradition, We4Poets take to the stage for an afternoon performance at the extraordinary Lock 91 – possibly the coolest bar in Manchester.
An all star line-up are all set for 3 hours of live music, original spoken word, modern verse and a little soft shoe shuffle.
Free entry and good time guaranteed.
53.474626 -2.250841


text:
Thank you & WELCOME!
Thank you so much for joining the #BookWitch Membership. Come and request access to our private Facebook Group and come and say hi and get stuck in!
#BookWitCH MEMBERSHIP GROUP
If you have any issues, concerns, questions, comments, problems, jokes or good cocktail recipes then please email me at firstname.lastname@example.com
That's pretty much it for now. Go make a celebratory cup of tea. Cheers doll face! xx
Radio JKill
Well done you star! You've made it all the way to the bottom and earned a TUNE!


text:
I love the summer. I love photographing seniors and I love being outdoors. That's why I enjoy my senior sessions so much. I get to hang outside in beautiful places and have fun with some really amazing people. Yes, this is my job. (There's MUCH more to my job than this, but that's a post for another day!)
This featured senior session is special for a few reasons. First, it was with a great girl who I have known for since she was in elementary school. She's fun, has a great smile and an amazingly positive attitude. And she has some pretty great friends. I've learned from her that these things can be a rather powerful combination and can help get you through some rough times. I look at these pictures and they make me smile. : )
This session was also a lot of fun because I had the help of three very creative ladies from Rambling House who styled the session for us. They put together an adorable retro picnic complete with vintage props and books, hand-made banners, fresh fruit, Parisian bread and old fashioned soda pop! This was all set on a gorgeous landscape complete with fruit trees, rambling fields, rustic barns and a few unexpected guests (see photos)! Of course, the beautiful make-up for this senior was done by Flawless Face & Body.
I will be offering a limited number of individualized, styled senior sessions with Rambling House. Contact me if you are interested!


text:
We have probably drunk red wine at least once at some point in our adult life. Whether it's just drinking for occasion or drinking for fun, there's something about that glossy red liquid that gets us going. For any wine enthusiast, drinking the rich liquid is more than just its taste. The texture and flavor …Continue reading "Drinking Red Wine For Your Health – Is it Good or Bad?" »
Tag: wellness
How to Achieve Better Sleep For the Sake of Your Health and Well-being!
Getting a good night's sleep is one of the most important habits we must maintain to keep ourselves healthy. It is just as essential as doing regular exercise. This is because our body's damaged cells and tissues during the day regenerate at certain hours of the night when we sleep. Unfortunately, not many of us …Continue reading "How to Achieve Better Sleep For the Sake of Your Health and Well-being!" »
8 Easy Steps on How to Slow Down Aging and Look Young Longer
At some point in our lives, we have probably looked at ourselves in the mirror and noticed that our faces are a little wrinklier than before. We have probably noticed that there are more crow's feet around our eyes and lines on our foreheads now. Or perhaps we have also encountered people treating us …Continue reading "8 Easy Steps on How to Slow Down Aging and Look Young Longer" »


text:
Like many Palestinians of his generation, 76-year-old Yasser Hirbawi felt a new-found pride when a young Yasser Arafat began to campaign for an independent homeland half a century ago. He also sensed a business opportunity.
As Arafat shot into the international spotlight as the leader of the Palestinian Liberation Organisation, Mr Hirbawi realised there was money to be made by manufacturing the keffiyeh, the chequered black and white scarf that became Arafat's trademark headgear.
Today, though, in what many see as a metaphor for the failing state of Palestine's economy, foreign competition threatens to ruin the factory he set up.
The keffiyeh may remain a symbol of Palestinian solidarity and has become a fashion accessory from New York to Tokyo, but its sole surviving Palestinian manufacturer has cut production to a fraction of capacity and is in danger of closing.
"It's the national symbol. It must keep going and it must be produced locally," said Mr Hirbawi, who wears such a scarf over his traditional jalabiyeh robe. "It must be protected."
Mr Hirbawi opened the Hirbawi Textile Factory in 1961, just as Arafat, who died in 2004, was building his Fatah movement in exile in Kuwait. The keffiyeh, without which he was seldom seen, is a traditional desert garment, protecting wearers from hot sun and dust in summer and cold in winter.
Once used largely to denote different tribes, today its various colours and designs carry political connotations - black and white is associated with Mr Arafat's Fatah, while a red and white version is linked to the Islamist organisation Hamas.
It is also now highly fashionable in the West, adorning the shoulders of Palestinian-sympathising students as well as celebrities, including actors Colin Farrell and Kirsten Dunst, the singer Sting and the footballer David Beckham. The fashion chain Topshop sells it in high street stores, and the Spanish fashion house Balenciaga has created a £3,000 designer version for the catwalk.
Over the years, Mr Hirbawi has taken mass orders destined for masked gunmen and political parties as well as souvenir shops, supplying them all from his inventory of chequered scarves. He believes, although he cannot be sure, that Arafat himself wore his keffiyehs.
Ironically, his factory has become a victim of the very cause its product is identified with. With the signing of the Oslo Accords in 1993 came the beginnings of an independent Palestinian economy and trade with the outside world, and before long, souvenir shops were selling scarves made not in the occupied territories, but from factories elsewhere - mainly China, where suppliers offer cut-rate prices.
What remains of the Hirbawi Textile Factory is a cavernous concrete warehouse, with box upon box of unused spools of cotton yarn and a 1970s Volkswagen van rusting in a corner. The automatic looms, brought from Japan with much fanfare in the 1960s and 1970s, sit heavy with dust and largely empty, two of them stopped mid-production with the threads of unfinished scarves still dangling.
Just four of the original 15 machines operate, switched on for just four hours a day to produce 10,000 scarves a year - a far cry from the 150,000 produced annually in the early 1990s. Even so, sales are even lower, and hundreds of keffiyehs are piling up in a storeroom.
Not one is exported, beaten out by overseas suppliers. And the shrinking Palestinian economy, with its high labour and production costs plus heavy barriers on exports, has amplified the struggle.
Not far from this factory is the designated site of a Turkish-backed industrial zone championed by Tony Blair, the Middle East envoy, who took on the task of kick-starting the moribund Palestinian economy when he began his new job last year.
But small family businesses such as that run by Mr Hirbawi - he has five sons and two daughters, all married, and 30 grandchildren - has so far not seen any benefit.
"When I look at Abu Mazen [Palestinian Authority president Mahmoud Abbas] wearing the keffiyeh, I say, maybe he's wearing a keffiyeh from me," said Mr Hirbawi. "But I want to say to him 'Please, Abu Mazen, protect us'. My machines are in good shape. They can start working tomorrow. I just need a market."
Publishers wishing to reproduce photographs on this page should phone 44 (0) 207 931 2921 or email email@example.com


text:
Three fundamentals of a lifestyle business
Ah, the lure of the lifestyle business - working when you want, on stuff you like, working from where you want… Those are the images from the marketing department. But in real life, things look a little different - particularly when you start out. Most lifestyle businesses start out small - usually just the founder of the business. To get your business off the ground you're going to have to work hard (you already know this). You have to get a ton of stuff in place, build a client list, a sales funnel, marketing material - the list goes on.
But the lure is not just a lure - many entrepreneurs realise this dream and live happily ever after. But most struggle because they don't have a solid foundation for their business - the fundamentals are not what they should be.
So what are these fundamentals, and what do you need to build that solid foundation?
Just before we do that, let's get clear on what a lifestyle business is.
What is a lifestyle business?
When we think of a startup we think of business designed to grow huge. There's rounds of investment from venture capital and private equity, the focus is on rapid growth and there's an exit strategy - either an IPO or an acquisition.
But not all of us want that kind of business. We're happy with a business that provides just enough for us, and perhaps a small team, to live a good life.
That's a lifestyle business.
There are some misconceptions out there that a lifestyle business somehow has to limit the amount of revenue it generates. That's not true:
A lifestyle business is simply a business where your decisions about growth take the impact on your lifestyle into account.
You can still grow it big. Elaine Pofeldt's Million Dollar One Person Business has numerous examples of single-person businesses exceeding a million dollars. I've seen many examples of lifestyle businesses with teams - usually small ones, but still designed to give the owner the lifestyle they want.
The successful ones have the fundamentals right.
The three fundamentals of a successful lifestyle business
Here's my formula for a successful lifestyle business:
Lifestyle Business = Life + Positioning + Revenue
Over the last two years or so I developed the Tornado Method - a comprehensive framework for designing, building and running a business. The Tornado Method consists of 11 elements in three layers - and those layers correspond to the fundamentals of a successful lifestyle business.
The top level of the Tornado Method looks like this:
The fundamentals of a successful lifestyle business work as follows:
At the bottom of the Tornado Method is the Getting Stuff Done layer. This is where you get to have a life (or not);
the Building Blocks layer determines your positioning; and
the Revenue Engine is how your business makes money.
Let's look at each in turn.
Fundamental 1: Have a life (a.k.a. getting stuff done)
At the bottom of the tornado is the Getting Stuff Done layer. This is where you have things like team, culture and personal effectiveness - the key to determining whether you're going to have a life as well as a business.
We already know that building a business is hard, and you're going to take the brunt of the work on your shoulders. And this is where you need to make a decision:
Are you going to give up on having a life while you're building your business?
I've been there and done that - a failed marriage is testament to my stupidity. I decided that I'll never go there again, so my answer to this is a firm no - I won't give up on having a life while I'm building my business.
I know that it's going to take longer to build the business because I don't work weekends or evenings. I make up for that to some extent by starting really early and continuously learning how to be more effective.
But there's another reason you don't want to give up on having a life while you're building your business.
Your ability to focus, be effective and productive is dependent on how well you're doing physically, mentally and emotionally. If you're run down, your productivity is going to be low, your ability to focus suffers and the quality of the work you produce is going to be lower than you're capable of.
So to produce more, focus better and build a quality business it actually benefits you to take time off - to have a life. The "honour" badge of 80-hour weeks is a fallacy - you're not producing more or faster, and the quality of your work is lower.
So having a life while building a business actually results in a better business faster. And you enjoy the journey a lot more.
Fundamental 2: Positioning
I work with a lot of lifestyle businesses, especially in the early stages while they're still struggling to gain traction and reliable revenue. Almost without exception, my advice to them is to specialise.
Specialisation is the fastest and best way to become the go-to person for a specific problem. It makes it easier for you to find clients, for them to find you and for you to charge a premium.
In the Tornado Method, I call this positioning.
Positioning is the combination of your specialisation, a kick-ass brand and the products you offer to the market. There's an art and a skill in finding your ideal positioning, and a huge fear that you're going to miss out on other business because you've specialised.
But in practice, I've seen this play out time and time again: the right positioning makes all the difference between competing with the hordes and becoming the recognised expert in a specific field.
Specialisation is the start of positioning. Combine that with a great brand (your business personality) and offer clients a small but well-designed set of products to choose from, and you have great positioning.
That's the second fundamental of a successful lifestyle business.
Fundamental 3: Revenue
Of course, no business is a business without revenue. In the Tornado Method we define a Revenue Engine - the sequence of marketing, lead nurturing, sales, delivery and follow up that makes money for your business.
When I work with lifestyle entrepreneurs I always start with making sure their positioning is well designed. Without somewhat decent positioning your chances of building a reliable Revenue Engine are pretty slim.
But even decent positioning won't deliver the revenue you want if your Revenue Engine is not well-designed - and you don't operate it well. From my experience I've seen two main problems with Revenue Engines:
one or more components of the Revenue Engine don't work very well; or
the different stages of the Revenue Engine are not well connected to each other.
This simply means that (for example) your marketing is not generating enough leads for your business, or there is no clear, simple method for capturing lead details so you can start nurturing them.
A well-designed Revenue Engine has each component running smoothly, and each component helps leads and prospects move to the next stage as easily as possible. For a fun example of how to design a Revenue Engine check out this article.
A checklist: how is my lifestyle business doing?
In the Tornado Method I've defined one question for each of the 11 elements that will tell you how well that part of the engine is doing. Here's a quick checklist you can use:
A successful lifestyle business is based on three fundamentals:
You have to have a well-designed and well-running Revenue Engine to have reliable, consistent revenue streams.
Your positioning is critical to ensure you become the go-to person for a very specific problem, make it easy to find your ideal clients and charge a premium for your products or services.
And finally, you have to have a life so you can enjoy the journey and be in top form when you're working on (or in) your business.
Get these three fundamentals right and you will have a great business - and a life as well.
What you can do now
Download the Business Readiness Workbook to rate yourself!
And have fun building your business!


text:
Are you looking for a PTC site to earn money online? There several of them available on the Internet. So many users get confused which one to choose that is legit & gives the highest paying opportunity.
Do you want to know about Minerva-Clicks? It is also a PTC site that is growing at a rapid rate, and here you'll find a comprehensive review and a guide to Minerva-Clicks. All your queries and doubts will be cleared, and you'll be able to take the correct decision whether to join this platform or not.
What is Minerva-Clicks?
Minerva-Clicks is a PTC site (Paid To Click website) which was founded in 2018. It is relatively a new PTC site that has been getting a great response after its introduction. The main business model is to provide commission to their members for clicking, viewing ads & from referrals.
Minerva-Clicks is similar to all the other PTC sites, they act as a middlemen between the advertisers and the internet users. The advertisers pay Minerva-Clicks to display their advertisment on their website. Those Internet users who are interested in earning some extra money join those PTC sites. The users have to click and view the advertisements and a small amount gets credited into their account.
Is Minerva-Clicks legit or Spam?
Although Minerva-Clicks came up recently and is a new player but it is NOT a scam website. It is legit and has become a top competitor to the other PTC sites within a short span of time.
Minerva-Clicks has crossed over 18,000 members and has paid them more than $26,000. The number is not staggering, but given the time period they took to touch the same is remarkable. The members have given a good feedback and provided several payment proofs stating that Minerva-Clicks is a genuine website.
How does Minerva-Clicks work?
Minerva-Clicks is a PTC site as already mentioned. So there will be two categories of people who will look forward to join Minerva-Clicks.
The first category will be the advertisers who want to advertise their website or product on the Internet. These advertisers don't have a high budget and also the quality of their site is low. Therefore they choose PTC sites for advertisment where traffic is bound to arrive on their site irrespective of its status. The advertisers will pay Minerva-Clicks a fees for displaying the ads. The major part of revenue of all the PTC sites comes from the advertisers.
The second category of people are those who are looking to earn extra income. They join PTC sites as members and click and view the advertisments of the advertisers to earn some money.
Minerva-Clicks will act as middlemen between them and pay their members a small commission from the fees paid by the advertisers. They currently offer only PTC tasks and not GPT tasks. The payment methods with its terms & conditions for advertisers and members are explained further.
Are there any charges for registering as Member with Minerva-Clicks?
No, there isn't any charges to register yourself with any PTC site including Minerva-Clicks. It is initially absolutely free to join the network.
The members after registration get a free account, but mostly all PTC sites offer their members the option to upgrade their accounts to premium by paying some charges. The premium account members get additional benefits and higher payment as compared to the free account users. It is completely dependent on the individuals choice whether to upgrade their account or not.
How to Sign Up for Minerva-Clicks?
- The interested users can directly visit the homepage from this link provided here.
- They can also visit their homepage via any of the search engines.
- There you have to click on the registration option provided on the top of the page.
- Fill in the form and provide your correct email address.
- After clicking on the REGISTER option, Minerva-Clicks will send an email to the email address that you have entered.
- Visit your inbox and click on the activation link to activate your account.
- You'll then become a member of Minerva-Clicks who can earn money via viewing and clicking their advertisements.
How to Make Money with Minerva-Clicks?
The main method to earn money on Minerva-Clicks is to click on the advertisements that are displayed. After clicking the member will be redirected towards the advertisers site where you have to view it for atleast 10 seconds.
The other method is by using referrals, when you refer others to join Minerva-Clicks using their banners or your referral link then you will earn a commission.
How much Can I Earn From Minerva-Clicks?
You are NOT going to get rich by using this or any other PTC sites. These sites including Minerva-Clicks requires lots of efforts and time to earn a meaningful income without investment. Those members who upgrade their accounts to paid plans earn more and easily than compared to the free members.
The per click rate of Minerva-Clicks is not fixed and is in anywhere near $0.001 for free account users. You can do the math and guess the rough estimate that you'll earn from this PTC site.
Payment methods of Minerva-Clicks
The minimum payout balance requirement in Minerva-Clicks is $4 for the first payout, it will increase by $1 on every payout till it reaches the maximum limit amount of $10.
The payment processors supported by Minerva-Clicks are as follows :-
- PerfectMoney
- Payeer
- Skrill
- Cryptocurrencies – Bitcoin, Ethereum & Dash
There are several payment proofs provided by the members which makes them a trusted PTC site that is genuine.
Pros and Cons of Minerva-Clicks
Everything has both good and bad, even Minerva-Clicks is not perfect. After reading this you will get an insight into the picture.
Pros of Minerva-Clicks
- It is a completely free to join platform.
- There are lots of postive reviews from their members.
- It is among the fastest growing PTC sites and has become quite reliable.
- They offer email support and assistance to resolve the issues faced by their members.
- Low minimum payout of $4 with multiple payment methods.
Cons of Minerva-Clicks
- The owner of the website is still fixing the bugs and yet it isn't a completely established PTC site.
- It permits only one account per household, those who violate this condition get all their accounts terminated.
- The website is not well designed and isn't mobile friendly.
- The advertisments that are displayed by the advertisers is not of the highest quality. It looks like spam and is not very useful.
- Low rate of commission per click.
Minerva-Clicks is new in this PTC business, but is looking like a solid player from its prospect. If you are searching for the absolute best PTC sites to make money. Here are the Top & the best PTC sites that are recommended to join.
The decision to enroll and create an account on any of the PTC sites is relatively dependent on an Individual's choice. The free account members will generally get their pay equal to the amount of peanuts. So, if you are not willing to invest your time and money. Then the reward is certainly very less.
If you have any other doubts or queries on Minerva-Clicks, feel free to write below in the comment section to get it solved.


text:
How home business brings freedom for entrepreneurs with disabilities
- Written by Karen Meager
Entrepreneurs with disabilities are finding freedom working from home despite the lack of a safety net which full-time employment provides
"There aren't many people who are truly happy on their own for very long periods of time. This is not specific to people with disabilities. "
Read the full article:


text:
ON AIR - The Love Zone
with Rob Allen
The 'Pitch Perfect' star - whose naked pictures were posted online by hackers earlier this year alongside other stars like Jennifer Lawrence and Kate Upton - is glad the episode encouraged people not to blame those who were victims.
She said: ''I'm happy that it seems like more people are coming around. Because it happened en masse in that way, it forced people to come around and go, 'Everybody does this and victim-blaming is not helping.'
''Because before that, there was a lot of, ''If you didn't want naked photos of yourself on the Internet, well, you shouldn't have taken them.''
The 29-year-old actress has admitted that even her friends had changed their views, after realising how many celebrities had their personal photographs hacked.
She explained to The Daily Beast: ''So, the one and only good thing that came out of it is that it really shifted the conversation.
''Friends of mine that had taken the other attitude were like, 'People should be able to do whatever they want, and it's f***ed up that that happened to them.' That's the lone positive that came out of that whole thing.''
Source Entertainment.ie | 06:10 | Friday 28th November 2014


text:
Here's the deal...when you're friends get engaged and you're all together on an island in the middle of the Pacific Ocean...you take photos to prove it all happened. Mandy + Forrest have been living on the big island of Hawaii working with the same missions organization that I am with - YWAM (Youth With A Mission). They are so in love it's adorable. But, you don't need me to tell you that...the photos will speak for themselves.
Prepare yourself for some Hawaiian beauty from the big island.
And without further adieu, here are Mandy + Forrest's engagement photos.


text:
Many of us are stuck in our current location - whether we are bounded by work, school, or money. This doesn't mean we can't fantasize about escaping our routines and it certainly hasn't stopped female vocalist Akua. The young artist recently released 'Far Away', a slow and well put together track produced by Golden Child.
Lyrics such as, "Take me away, I've been so sick of this town. Hold my hand and let's go down", describe Akua's lust of journeying far away. Tonally, the Milwaukee native's voice is comparable to TDE artist SZA - seductive and delicate. The airy and ambient track is held together by a slow and heavy bassline, accompanied by interesting percussion that wavers in and out throughout the song. What makes this song especially blissful is the many layers of melodies Akua sings throughout.
Her atmospheric R&B endeavors continue with her latest track 'The Other Side' featuring Genesis Renji. Allow yourself to step back and give Akua a good ear. You will not be disappointed.


text:
Lee Health Announces Executive Promotions
(Fort Myers, Fla. – May 3, 2017) --- Lee Health is proud to announce the promotions of Lisa Sgarlata, DNP, FACHE to Chief Patient Care Officer; K. Alex Daneshmand, D.O., MBA, FAAP to Chief Patient Safety Officer and Alex Greenwood, MBA to Vice President of Lee Health – Coconut Point.
Beginning June 1 with the retirement of Donna Giannuzzi, RN, MBA, NEA-BC, Lisa Sgarlata will assume the role of Chief Patient Care Officer. With more than 25 years of experience in various areas of health care, Sgarlata's most recent roles include Associate Chief Patient Care Officer of Lee Health and Chief Administrative Officer for Lee Memorial Hospital. She also has an extensive background in forensic and emergency nursing, and has served previously as director and executive director of Emergency Services and Vice President of Patient Care Services. Sgarlata earned her Doctorate in Nursing Practice and recently completed a Fellowship with the Robert Woods Johnson Executive Nurse Program.
Also beginning June 1, Dr. Daneshmand will begin serving as Lee Health's Chief Patient Safety Officer. Dr. Daneshmand currently serves as System Medical Director of Pediatric Clinical and Quality Services at Golisano Children's Hospital of Southwest Florida. Since joining the hospital in 2007, Dr. Daneshmand has served as a physician in the pediatric intensive care unit, medical director of the pediatric procedural sedation center, committee member of the oversight system medical staff quality services committee and as chairman of the pediatric medical staff quality committee. Since June 2016, Dr. Daneshmand has been medical director of quality for the children's hospital. During this time, he developed quality initiatives, organized quality subcommittees and promoted the common goals of patient safety and excellent clinical outcomes. His efforts and collaboration led to the hospital being named one of Leapfrog's Top Children's Hospitals—one of only nine in the country to earn this distinction.
Alex Greenwood currently serves as Orthopedic Service Line Director for Lee Health. He will continue in that role along with his recent promotion to Vice President of Lee Health – Coconut Point. As Vice President, Greenwood will support and work to ensure the vision of Coconut Point as a health and wellness destination for residents of Southwest Florida comes to fruition when the facility opens in 2018. Greenwood has been employed by Lee Health since 2006. In addition to his leadership role with Orthopedics, Greenwood also served as System Director of Operations at Gulf Coast Medical Center and Cape Coral Hospital.
"Lee Health is proud to promote from within," explains Larry Antonucci, M.D., Chief Operating Officer. "We are very fortunate to have many loyal employees who know our system well, commit to our mission, vision and values, and work hard every day to propel their careers, and our health system, forward."
About Lee Health
Since the opening of the first hospital in 1916, Lee Health has been a health care leader in Southwest Florida, constantly evolving to meet the needs of the community. A non-profit, integrated health care services organization, Lee Health is committed to the well-being of every individual served, focused on healthy living and maintaining good health. Staffed by caring people, inspiring health, services are conveniently located throughout the community in four acute care hospitals, two specialty hospitals, outpatient centers, walk-in medical centers, primary care and specialty physician practices and other services across the continuum of care. Learn more at www.LeeHealth.org
- Press contact information
- Pat Dolce
- Public Affairs Specialist
- Phone: 239-343-4801
- Cell: 239-989-2758
- After Hours: 239-789-5786
- Fax: 239-343-4802
Visit us on the web at: http://www.leehealth.org/publicaffairs/press-releases.asp
Visit us on Twitter at: http://twitter.com/Lee_Health
Visit us on Facebook at: http://www.facebook.com/LeeHealth


text:
Content Marketing World, September 6-9, is the one event where you can learn and network with the best and the brightest in the content marketing industry.
You will leave with all the materials you need to take a content marketing strategy back to your team – and – to implement a content marketing plan that will grow your business and inspire your audience.
Meet the ion interactive team — Asa, Megan, Donald and Jay — at Booth #28. And, make an appointment with ion's VP of Accounts, Mary Ward.
For Content Marketing World event details and to SAVE $100 on registration, use the code ION100 here.


text:
My So-Called Two-Wheeled Adventure in Afghanistan →
It is hard to explain why I took a job in Afghanistan. One reason is because I wanted a bit of adventure after living in Singapore for two years and hoped to take photos of Kabul in my free time. People always ask me if I felt safe in Kabul. Well, I was uneasy on most days and on some days, darn right lucky. On my second day at my office, rocket propelled grenades (RPGs) were launched at the government building next door for 18 hours. A few days later, seven people including six kids and one Nepalese Gurkha were blown up at a nearby compound. There were many other close calls that may be labeled as happenstance but were downright eerie.
Taking photos brought a sense of normalcy to living in a war zone. During my long commute across dusty and bumpy Kabul, I saw fathers take care of their daughters, kids throw paper airplanes off of mountains, and lots of bikes. There were tons of dirty, old Chinese bikes that Afghans rode everywhere. There were cops on bikes and food deliverymen on bikes. I even spotted the 'Bookseller of Kabul' peddling with a mountain of books strapped to his back wheel. Sadly, they were even suicide bombers on bikes. I only saw one woman on a bike because in Afghanistan women riding bikes is taboo and illegal. The Afghans like to keep things closed and shut, so to speak!
Out of the thousands of miles that I spent in the car, there was only one female biker in Kabul. I am very proud to have caught the young lady cruising on her bike in her school uniform. Kabul is a high desert surrounded by the gorgeous 5,000-meter high mountains of the Hindu Kush. From my speeding armored vehicle, she looked like she was riding on the moon.
I turned down a chance to ride an oversized, junky bicycle in my hotel compound. I had not been in the saddle for months. Out of respect for the Afghan guards who would be watching me ride, I declined. You can get away with some things in Afghanistan and riding a bicycle inside the walls is low risk.
Instead of going on a short spin around the yard, I showed the Afghan security guards my photos of Kabul. They liked my photos and noticed my bicycle shots. One guard, Qais snipped that he sees these street scenes all the time.
Exactly. Repetitive. Boring. Normal. Perfect.


text:
Product Description
Christmas Tree with lip balm - Orange & Clove - Dainty hanging decorations, the year's smallest present, a stocking filler or simply a just-because-I-can-gift. There are a multitude of reasons why we love these little cherubs. Inside you'll find your own little Lip balm with a taste of Orange, Clove & Cinnamon. Sun-ripened oranges and dried cloves merge with cinnamon and a pinch of vanilla to give a warm, spicy and utterly seductive aroma. The Christmas taste!
Product Reviews
Write Review


text:
Cross country shifters might seem expensive. On the other hand, they will do a great deal of things for you. Below are a number of factors to take into consideration spending the cash.
Use The Most Suitable Out Of State Mover Near Beaumont TX 77710
It is never the ideal thing to start packing without figuring some things out. Firstly, you need to make the in-depth list of everything that will be loaded and moved. Many of the time, moving is done by professional packers and movers, or by hiring a car. In that case, it is important that we understand if anything is lost.
Now, pack just packages with items you consider to be friends. You don't have as much of these as you thought, most likely! Now, buy your moving truck size based on the area you will have to load only these items. You're saving money currently!
Door To Door To Nearly Anywhere
For interstate mover, some special containers can also be acquired, in which clothing with hangers can be stored; so that there is no have to pack and unpack your whole wardrobe.
Me and my partner are workers of a huge pharmaceutical company. This year we were invited to move for work in Montreal on really beneficial terms. We have actually discussed thoroughly this question and lastly chose to accept this attractive proposition and to relocate to Montreal. We have had several arguments to do so: first off, as I have actually pointed out above job conditions were extremely advantageous. Besides our extremely buddies, as well as my partner sibling and his family live in Montreal.
It would not fast to change in the new regional community – to locate college for your young children, support them make good friend, and lastly gel in the regional neighborhood. It would not be uncomplicated to get started your life all more than all over again in the brand-new metropolis. For that you will want earnings, and god understands how a lot. So why not begin saving something. Perfect from the time you direct interstate movers in Beaumont TX 77710?
The Beaumont Texas 77710 Long Distance Movers Everyone Could Certainly Turn To
The San Francisco Moving Business is the solution to you moving problem. Fist of all they will have for you the ideal truck so that all your things will go into. The San Francisco Movers supply expert services that include long distance moving. This is extremely important, because if the moving remains in the Bay Area is OKAY to make more that a person trip, however otherwise one journey only would be the very best thing.
X.Now the selection is made and we move on to packing. Companies like Movers NY leave cages and packing cases a couple of days before transportation. This permits you to attempt your hand on packaging. On the designated day they take few hours to achieve the job with such qualified movers.
Bottom Line
The info in this article actually only represents a small portion of all there is to learn about long distance movers. When they dig deeper into this subject, individuals tend to have more reliable outcomes and feel more satisfied. There is just too much to cover in this short informational essay, and we shift into more deeper treatment in a minute. You will find it to be really beneficial in numerous ways, and a few of it is really particular to your particular requirements.


text:
Two weeks ago I turned 30. To some people this is nothing, to some others it's a...Read More
Counting Down 20 Years
Life for the past 20 years are filled with mostly school and many things I'm not...Read More
My Own Wedding, part 2/2
Fast forward my last entry in Musing, today is one year anniversary of my own...Read More
My Own Wedding, part 1/2
So, when people see me recently, they'd be like "Hey! How is wedding life?!" This applies to...Read More
5 Years and Growing
We are now officially 5 years in business and still going. I survived the crucial beginning...Read More
a Girl a Wife a Mother
She used to be a very cute & happy little girl too. No worries no responsibilities, everyday is...Read More
First Half of My 20s'
So it's gone. First half of my 20s' ended 8th September and I am now officially 26 years...Read More
No Sweat Wedding
It was our very first wedding of the year in 2013 where I traveled farthest away from home till...Read More
Colorblind Malaysians
During our childhood, we had 'colorblind' vision towards people in front of us. We don't even...Read More


text:
Morehead Planetarium and Science Center is a 2nd Friday ArtWalk venue, featuring an evening star show and science activities on the sundial out front! This month's star show is 7pm: Wildest Weather and 8pm: Carolina Skies. Parlez-Vous Crepe food truck will be there too! www.moreheadplanetarium.org
Back to All Events
Earlier Event: March 14Paintings by Dan Smith at Studio Design Gallery
Later Event: March 14Woodwork by Bill Neville at the Ackland Museum Store


text:
Fall is the perfect time to get your spring bulbs in the ground. We want to help you with your fall bulb planting with our tips and suggestions for how to do it correctly.
Of the many garden maintenance tasks you need to attend to this fall, bulb planting can be an easy task if you know the correct way to do it. With our bulb planting strategy, we'll teach you how to plant your spring bulbs the right way.
Listen to our quick segment on this "DIY Garden Minute" to learn about these planting tips and tricks to save you time.
Fall Garden Task Series
For more information on various fall garden tasks, check out our 7-episode Fall Garden Tasks podcast series!!
Our 7-episode, podcast series will outline 8 different garden tasks you should be doing this fall or should consider adding to your list. All of these garden maintenance tasks are crucial to the health of your garden.
The FALL GARDEN TASKS episodes are:
- Ep. 22: Introduction to the 8 Fall Garden Tasks and our own Fall Garden Bootcamp Challenge!
- Ep. 23: Fall Weeding Tips and Effortless Leaf Clean Up
- Ep. 24: Fall Garden Mulching and Lawn Maintenance
- Ep. 25: Fall Garden Pruning and Beneficial Leaf Clean Up
- Ep. 26: Fall Planting, Transplanting, and Fertilizing
- Ep. 27: Fall Equipment Maintenance
- Ep. 28: Fall Garden Task Series Wrap-Up and Next Steps
Need Pruning Supplies For Your Fall Pruning Tasks??
- Check out our Best Pruning Shears post here to learn what makes a good pruning shear and what we recommend!
- Check out our Best Loppers post here to read our recommendations!!
Want to Learn to Prune Like a Pro?
Subscribe to our Spoken Garden community and receive your FREE cheat sheet! Included in the cheat sheet for you: Tools you will need for proper pruning, different types of pruning cuts, steps to prune successfully, and pro-tips!
And hey—before you head out—if you want to connect with us and subscribe to our newsletter go to Spoken Garden's free resource page! We have FREE garden content we are so excited to give to you, like cheat sheets, plant profile sheets, etc! See you there!
Thanks for Listening!!!
Share Your Thoughts:
- Leave a note in the comment section below.
- Leave comments/ suggestions at email@example.com
- Share our show on Twitter, Facebook, or Pinterest.
To Help Out The Show:
- Leave a review on iTunes. Your ratings and reviews really help us out!
- Subscribe on iTunes or Spotify.
*Spoken Garden is a participant in the Amazon Services LLC Associates Program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to Amazon.com.
Subscribe on iTunes
Today's Topic:
Fall Bulb Planting
Learn how to plant your fall bulbs correctly now so you can enjoy them in the spring!
Pruning Cheat Sheet at our Free Resources Page
What You'll Learn:
- Correct strategies for planting your fall bulbs.
- Examples of bulbs you can plant in the fall.
Some of the resources and products below may be affiliate links, meaning we might get paid a commission (at no extra cost to you) if you use that link to make a purchase
- DIY Garden Minute Ep. 6: Fall Perennial Planting Tips, Demystified!
- Ep. 21: Proactive Weed Control Strategies For Any Season!
- How to Mulch: 6 Basic Steps for Mulching Anytime Of Year
- Top 7 Reasons Mulching Your Garden is Beneficial
Products Related To This Podcast:
- Best pruning shears
- Loppers
- Hand saw: folding or fixed
- Blower: hand-held or backpack, gas or electric
- Leaf Rake: metal or plastic
- Hard Rake: metal
- Pitch Fork
- Tarp
- 521Shares


text:
My delivery day is Thursday, so often times we get subs for certain items. This week, instead of green beans, we got peas in our Local Box. I wasn't complaining, as I love adding peas to stir-frys.
My husband's favorite dish when we go to PF Changs is ginger chicken, so I wanted to make it for him at home, too. This version is a little different (I feel like PF Changs uses a ton of high-fructose corn syrup in theirs), but he still likes it.
Adapted from Simply Recipes
- 1 bunch green onions
- 2 Tbsp. white wine vinegar
- 2 Tbsp. + 1/2 Tbsp. olive oil
- 1/2 Tbsp. cardamom
- 1-2 Tbsp. grated fresh ginger, depending on how gingery you like it (or 1 tsp. ground ginger)
- 1.5 lb. chicken breast or tenders
- 1/2 c. low-sodium chicken stock
- 1/3 c. mango jam. (The original version calls for mango chutney. I used the rest of the peach-mango jam that I made last week.)
- 1 Tbsp. minced garlic
- Chop the light and dark green tops of the onions and set aside. Trim the peas and set those aside as well.
- Combine the white wine vinegar, olive oil, cardamom and ginger.
- Trim the chicken of fat and cut it into small cubes. Add to the ginger mixture. Marinate for 15-30 minutes.
- Add 1/2 Tbsp. olive oil to a pan over high heat. When the olive oil is hot, 1/2 c. of the green onions and the peas to the pan. Saute for ~4 minutes, until the peas are al dente. (If the peas are very large, you might want to blanch them instead.) Remove the onions and peas from the pan and set aside.
- Add the remainder of the green onions to the pan. After about 30 seconds, add the chicken and the ginger mixture to the pan. Brown over high heat.
- While the chicken is browning, combine the chicken stock, mango jam and garlic in a small bowl. After the chicken is browned, add the chicken stock mixture to the saucepan. Reduce the heat, add the peas to the pan again, and simmer for 3 minutes.
- Serve with white rice and broccoli.
The peppery bite of the ginger combines nicely with the sweet mango jam in this dish. It's a quick, healthy dinner that's always a winner in our house. The great thing about stir-frys is that you can add whatever veggies you want in here. If you don't have peas, asparagus, string beans or squash would be good additions as well.


text:
Virus-specific cytotoxic T lymphocytes (CTL) with high levels of functional avidity have been associated with viral clearance in hepatitis C virus infection and with enhanced antiviral protective immunity in animal models. However, the role of functional avidity as a determinant of HIV-specific CTL efficacy remains to be assessed. Here we measured the functional avidities of HIV-specific CTL responses targeting 20 different, optimally defined CTL epitopes restricted by 13 different HLA class I alleles in a cohort comprising 44 HIV controllers and 68 HIV noncontrollers. Responses restricted by HLA-B alleles and responses targeting epitopes located in HIV Gag exhibited significantly higher functional avidities than responses restricted by HLA-A or HLA-C molecules (P = 0.0003) or responses targeting epitopes outside Gag (P < 0.0001). The functional avidities of Gag-specific and HLA-B-restricted responses were higher in HIV controllers than in noncontrollers (P = 0.014 and P = 0.018) and were not restored in HIV noncontrollers initiating antiretroviral therapy. T-cell receptor (TCR) analyses revealed narrower TCR repertoires in higher-avidity CTL populations, which were dominated by public TCR sequences in HIV controllers. Together, these data link the presence of high-avidity Gag-specific and HLA-B-restricted CTL responses with viral suppression in vivo and provide new insights into the immune parameters that mediate spontaneous control of HIV infection.
A small number of human immunodeficiency virus (HIV)-infected individuals, comprising approximately 0.5% of all cases, are able to control the infection in the absence of antiretroviral treatment. Such individuals exhibit low to undetectable virus loads in the peripheral blood and show either no or minimal signs of HIV disease progression; they are therefore referred to as "controllers" or "long-term nonprogressors" (LTNP) (26). Elucidating the mechanisms that are involved in the relative control of viral replication in these individuals is essential for our understanding of HIV pathogenesis and could crucially inform vaccine development (18).
CD8+ cytotoxic T lymphocytes (CTL) are considered an important component of immune defense against HIV and viral pathogens in general. The most compelling evidence for their protective role in HIV infection comes from the following: (i) studies that show a temporal association between the appearance of CTL responses and the initial early decline of plasma viremia (45), (ii) CD8 T-cell depletion experiments in the nonhuman primate simian immunodeficiency virus (SIV) model (70), and (iii) the finding that certain HLA class I alleles are enriched in subjects with delayed disease progression (reviewed in reference 59). Interestingly, a recent genome-wide association analysis comparing HIV controllers and noncontrollers identified specific amino acids in the peptide binding grooves of HLA-B molecules that could enable these alleles to restrict particularly potent CTL responses (64). However, despite the large number of studies characterizing HIV-specific T-cell responses, the hallmarks of a truly protective immune response remain unclear.
Neither the total breadth nor the magnitude of the overall virus-specific CTL response associates with the outcome of HIV disease (1, 32). However, CTL responses restricted by specific HLA-B alleles and responses that target the Gag protein appear to be particularly effective in vivo (43, 87). Nonetheless, some individuals with progressive disease also carry protective HLA alleles and mount broad Gag-specific CTL responses, indicating the presence of other factors that determine in vivo immune efficacy. One parameter that has repeatedly been associated with the control of viral replication in animal models is the functional avidity of virus-specific CTL (2, 9, 27). High-functional-avidity T cells feature a superior ability to recognize virally infected cells even at low surface antigen densities and deploy effector functions at lower cognate antigen concentration thresholds than do low-avidity T-cell populations (10, 14). In humans, such high-avidity CTL have been associated with clearance of hepatitis C virus (HCV) infection and broad recognition of HCV epitope variants (86). Although not necessarily directly linked, these data suggest that high-avidity CTL responses may be especially effective at controlling infections with highly variable pathogens, since variant recognition may be crucial for the limitation of immune escape (25). Furthermore, in HIV infection, the Nef protein downregulates HLA class I molecules on the surfaces of infected cells; in this setting, high-avidity CTL responses could provide a crucial advantage by recognizing HIV-infected cells despite the reduction in surface antigen density (8, 9, 24). The presence of high-avidity CD4+ T cells in HIV controllers and the emergence of escape mutants as a direct consequence of immune selection pressure mediated by high-avidity CTL provide support for the important role of functionally avid responses in the control of HIV/SIV replication (49, 61, 82).
In the current study, we directly tested the role of functional avidity as a determinant of CTL efficacy in the control of HIV replication. Furthermore, we examined the relationship between this parameter and antigen specificity, in terms of both the targeted HIV-derived epitopes and their associated HLA class I restriction elements.
Study subjects.All study participants were recruited from previously described cohorts in the Boston, MA, area (33, 64). HIV loads were determined using Roche Amplicor tests with a lower detection limit of 50 copies/ml. HLA typing was performed as described previously using sequence-specific primer PCR (SSP-PCR) (30). The Partners Human Research Committee at Massachusetts General Hospital approved all human subject research protocols, and all subjects provided written informed consent prior to enrollment. Responses were compared between 44 subjects with spontaneous control of HIV replication to less than 2,000 RNA copies/ml ("controllers"; median viral load < 50 copies/ml; range, <50 to 2,000 copies/ml) and 68 untreated subjects with viral loads greater than 3,000 RNA copies/ml ("noncontrollers"; median viral load, 38,450 copies/ml, range, 3,900 to 604,000 copies/ml). For experiments investigating the effects of highly active antiretroviral therapy (HAART)-induced viral suppression on CTL function, the functional avidities and magnitudes of CTL responses were determined for 14 treated individuals before and after the initiation of HAART; the median time on treatment at the second time point was 56.4 weeks (range, 1 to 150 weeks).
Peptide epitopes.A total of 20 well-defined and commonly recognized HIV CTL epitopes listed at the Los Alamos HIV Immunology Database were included in the present study (15, 33). These epitopes are restricted by 13 different HLA-A (n = 4), HLA-B (n = 7) and HLA-C (n = 2) molecules and are summarized in Table 1. The antigenic peptides were selected to allow testing of epitopes with the following characteristics: (i) they are restricted by alleles commonly found in both HIV-controllers and noncontrollers, (ii) they are targeted by a substantial proportion of individuals expressing the restricting allele(s), and (iii) they can be presented in the context of different alleles (15, 33). For additional experiments on the role of HLA alleles as determinants of functional avidity, epitopes for which promiscuous presentation on multiple class I alleles has been demonstrated (33) and which are also presented by HLA alleles associated with controlled and noncontrolled HIV infection (60) were used as indicated in the respective section.
IFN-γ ELISpot and functional avidity assays.Peripheral blood mononuclear cells (PBMC) were isolated from whole blood using density gradient centrifugation and tested ex vivo in gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assays using single epitopes at a concentration of 14 μg/ml (11). Functional avidity was determined as described previously (86). In brief, unexpanded PBMC were thawed and rested for 4 h at 37°C and then stimulated in IFN-γ ELISpot assays with peptides added in serial 10-fold dilutions ranging from 100 μg/ml to 10 pg/ml (33). A positive (phytohemagglutinin) control well and at least four negative (no peptide) control wells were included on each plate. Stimulations were set up in duplicates or triplicates if cell availability allowed. Results are expressed as spot-forming cells (SFC) per million input cells. The threshold for a positive response was defined as at least five spots per well at a peptide concentration of 10 μg/ml and ≥3× mean background. Functional avidity was calculated by sigmoidal dose (SD)-response curves fitted to the ELISpot data using the GraphPad Prism software program (version 4.0).
T-cell receptor (TCR) repertoire analysis.Antigenic stimulation was performed on thawed PBMC. Individual HIV peptides were used at 10 μg/ml to stimulate HIV-specific CD8+ T cells for 14 h at 37°C in the presence of costimulation with anti-CD28 and anti-CD49d monoclonal antibodies (1-μg/ml [each] final concentration; BD Biosciences). In every experiment, a negative control (costimulation alone with anti-CD28/anti-CD49d) was included to account for spontaneous stimulation, and a positive control (Staphylococcus enterotoxin B [SEB], 1 μg/ml, final; Sigma-Aldrich) was included to ensure that the cells were responsive. After stimulation, the cells were surface stained with the following monoclonal antibodies: anti-CD3-fluorescein isothiocyanate (FITC), anti-CD8-Pacific Blue, anti-CD14-Cy5PE, anti-CD19-Cy5PE, anti-CD25-phycoerythrin (PE), and anti-CD69-allophycocyanin (APC) (all from BD Biosciences). Viable antigen-specific CD8+ T cells were identified on the basis of CD3, CD8, CD25, and CD69 coexpression as described previously for reactive T-cell populations (66). Staining for CD14 and CD19 was used to exclude macrophages and B cells from the analysis. Responding cell populations were sorted at 70 lb/in2 directly into 1.5-ml microtubes (Sarstedt) containing 100 μl RNAlater (Applied Biosystems) using a modified FACSAria flow cytometer (BD Biosciences); sort purity was >98% in all cases, and at least 1,000 cells were collected for each experimental condition. Unbiased clonotypic analysis of all expressed TRB gene products was then conducted using a nonnested template-switch-anchored reverse transcriptase PCR (RT-PCR) as described previously (28, 67). The entire procedure was performed blinded to the clinical status of the subjects in all cases.
Statistical analysis.The GraphPad Prism (version 4.0) software for Macintosh was used for the 50% SD (SD50) determinations. The Mann-Whitney test was used to compare the functional avidities of CTL responses between the two study groups; the Kruskal-Wallis test and Dunn's posttest were used to compare the functional avidities of CTL responses across different restricting HLA alleles and different viral proteins. A Spearman rank test was used to assess the relationship between the functional avidities or magnitude of the CTL responses and the time after initiation of HAART.
HLA-B-restricted and Gag-derived epitopes are targeted by CTL responses with high levels of functional avidity.A number of studies in humans and animal models have aimed to understand the impact that different major histocompatibility complex (MHC) class I molecules exert on the quality and quantity of the evolving CTL response (15, 42, 54, 64). This issue is of particular importance in HIV infection, for which specific HLA class I alleles have been associated with improved in vivo control of viral replication (reviewed in references 59 and 60). However, direct links between the attributes of CTL-mediated immunity and the clinical control of HIV infection have been addressed only in the context of a few selected HLA alleles and individual epitopes or on the basis of responses elicited by peptide pools spanning whole viral proteins (4, 5, 13, 14, 40, 44, 49, 51, 55). To overcome these limitations, we assessed the functional avidities of CTL responses to 20 optimally defined HIV-derived epitopes in a cohort of 112 HIV-infected individuals with variable disease progression rates and heterogeneous HLA backgrounds. The epitopes included in this study spanned CTL targets in the HIV Gag, Pol, Nef, and Env proteins, thereby also enabling comparison of functional avidities across specificities from distinct viral gene products (Table 1). Of the 112 HIV-infected individuals tested, functional avidities were defined for a total of 199 distinct CTL responses, including 121 responses detected in 68 noncontrollers and 78 responses detected in 44 controllers.
To test whether functional avidity was affected by the restricting HLA molecule or the viral protein from which it was derived, SD50 values were compared between responses restricted by HLA-A, -B, or -C alleles and between epitopes located in different viral proteins. CTL responses restricted by HLA-B exhibited significantly higher functional avidities (median, 93.9 ng/ml; range, 8.37 × 10−8 to 16,312 ng/ml) than those restricted by HLA-A (median, 761.6 ng/ml; range, 1.45 to 625,806 ng/ml; P < 0.001). Although the median SD50 was also higher than HLA-C allele-restricted responses, this comparison did not reach statistical significance (median, 651.5 ng/ml; range, 0.01 to 17,255 ng/ml) (Fig. 1A). When responses to different HIV proteins were compared, Gag-specific responses displayed higher functional avidities than responses directed against other individual proteins or all non-Gag-derived epitopes combined (P < 0.0001) (Fig. 1B and C). Together, these data support an important role for HLA-B-restricted responses in HIV control (42) and suggest that the significantly higher functional avidities of Gag-specific responses might contribute to their previously described efficacy (43, 87).
HIV controllers mount CTL responses with higher functional avidities than noncontrollers.To further assess the relevance of functional avidity in HIV control, we determined whether epitope-specific CTL responses in controllers exhibited intrinsically higher avidities than those in noncontrollers. Where possible, comparable numbers of CTL responses were tested for each epitope in either group (Fig. 2A). When the SD50 values of all responses for HIV controllers (n = 78 responses) and noncontrollers (n = 121 responses) were compared, significantly higher levels of functional avidity were detected for controllers (median, 140.3 ng/ml; range, 8.37 × 10−8 to 625,806 ng/ml) than for noncontrollers (median, 327.5 ng/ml; range, 0.066 to 23,168 ng/ml; P = 0.047) (Fig. 2B). Stratification by HLA allele demonstrated that the functional avidities of responses restricted by HLA-B (P = 0.0175) but not HLA-A (P = 0.38) or HLA-C (P = 0.43) were significantly higher for HIV controllers than for noncontrollers (Fig. 2C). In both groups, we found no significant correlation between the time since initial HIV infection and functional avidity (data not shown). Furthermore, when responses were grouped according to epitope source, only Gag-specific responses exhibited significantly different functional avidities between HIV controllers and noncontrollers (P = 0.0143) (Fig. 2D).
Functional avidities and CTL response magnitudes decline with suppression of viral replication by antiretroviral treatment.Previous studies have suggested that viral load may drive some of the observed differences in HIV-specific T-cell reactivities (32, 73). To test whether low functional avidities could be driven by high viral loads and consequently would be restored to higher levels of functional avidity by antigen removal, the functional avidities of CTL responses to 8 different epitopes were assessed for 12 untreated individuals with chronic HIV infection over a period of up to 3 years (median, 56.4 weeks; range, 1 to 151 weeks; total number of epitope-specific responses tested, 14) both before and after the initiation of highly active antiretroviral treatment (HAART). As expected from previous reports (23, 62, 63), the magnitude of 13/14 responses declined over time during the treatment period (P = 0.003) (Fig. 3A). Indeed, the duration of HAART was inversely associated with the reduction in CTL response magnitudes, supporting previously described gradual decreases over the period of suppressed antigenemia (P = 0.012) (Fig. 3B). Rather unexpectedly, however, the functional avidities of these responses were higher at the pretreatment time points (median SD50, 146.3 ng/ml) than for samples obtained during HAART (median SD50, 324.9 ng/ml; P = 0.051) (Fig. 3C). Functional avidity seemed to decline in relation to the duration of HAART treatment; this correlation, however, was not significant (Fig. 3D) (P = 0.16). Although treatment-induced viral suppression may not have the same effects on viral avidity as viral suppression by the immune system, these data suggest that antigen suppression during the chronic stages of uncontrolled HIV infection does not restore higher levels of functional avidity and that CTL response magnitude and functional avidity could be differentially regulated.
CTL responses to protective epitopes restricted by HLA-B27 and HLA-B57 exhibit the highest functional avidities.Among the many peptides presented by HLA class I alleles associated with superior HIV control, only a few optimally defined epitopes have been specifically implicated in the suppression of viral replication in vivo (21, 31, 35, 36). The most consistent of these are the HLA-B27-restricted KRWIILGLNK epitope (B27-KK10) and the HLA-B57-restricted KAFSPEVIPMF epitope (B57-KF11), both of which are located in HIV Gag p24. Since responses to these epitopes may contribute disproportionately to the observed association between Gag-specific responses and relative HIV control, we assessed the functional avidities of these responses in detail. Consistent with their apparent in vivo efficacy, both the B27-KK10 and B57-KF11 epitopes elicited in vitro responses with significantly higher functional avidities than those for the other epitopes, independent of clinical status (P = 0.0003 and P = 0.0006 for B27-KK10 and B57-KF11, respectively) (Fig. 4A and B). Of note, the median functional avidities of responses to these two specific epitopes were not significantly different for controllers from those for noncontrollers (13.46 ng/ml versus 201.4 ng/ml for B27-KK10 [P = 0.268]; 152.0 ng/ml versus 443.9 ng/ml for B57-KF11 [P = 0.200]; data not shown). This could be attributed partly to smaller samples sizes for this subanalysis but may also indicate that other mechanisms contribute to the lack of viral control in the rare individuals who express protective HLA alleles but fail to control the infection (20, 57).
To assess whether CTL responses to these two epitopes were also driving the overall associations between high levels of functional avidity, HLA-B restriction, and the targeting of epitopes in Gag, we repeated the analyses after excluding B27-KK10 and B57-KF11 from the data set. Despite the reduced number of CTL responses analyzed, HLA-B-restricted responses still exhibited higher functional avidities than responses restricted by HLA-A alleles (P = 0.02). Similarly, the statistically significant difference in the functional avidities of responses to Gag-derived versus non-Gag-derived epitopes persisted (P = 0.0069). Thus, while the B27-KK10 and B57-KF11 responses showed the most pronounced effects, in line with their particular protective efficacy in vivo, the remaining HLA-B-restricted and Gag-derived epitope-specific responses still showed significantly higher functional avidities than responses to epitopes restricted by HLA-A alleles and responses to non-Gag-derived epitopes, respectively.
High functional avidities to specific epitopes relate to the presenting HLA allele.Several previous studies have demonstrated that promiscuous presentation of specific CTL epitopes by different HLA class I alleles is not uncommon (22, 30, 33, 51, 53, 77). Indeed, the vast majority of known optimal CTL epitopes derived from HIV and Epstein-Barr virus (EBV) are effectively presented by several different and unrelated HLA class I alleles (33). Employing such promiscuous epitopes allows the impact of the presenting HLA class I molecule on the quality of the epitope-specific CTL response to be studied more directly, avoiding potential biases related to protein abundance and epitope processing. Based on an earlier report (33), epitopes presented by HLA alleles that are differentially associated with HIV disease control were selected for this purpose (52, 60). The functional avidities of responses to five such epitopes were compared in the context of their originally described and alternative restricting alleles (Fig. 5A to H). As controls, an additional three epitopes were included that are promiscuously presented by HLA class I alleles for which no strong association with HIV control, either good or bad, has been reported. In all cases tested, the alleles associated with better control of HIV replication restricted responses with higher functional avidities than the alleles associated with a less beneficial HIV disease outcome, showing a strong trend toward statistical significance (P = 0.08) (Fig. 5I). The functional avidities of CTL responses restricted by the original or alternative alleles were not significantly different (P = 0.2) (not shown), demonstrating that responses restricted by "alternative" HLA class I alleles can represent functionally relevant responses. Collectively, these data indicate that the nature of the presenting HLA allele can profoundly influence the functional avidities of the CTL responses that it restricts. This observation is in line with recent data describing an important role for the presenting HLA molecule in HIV control (64) and a previous analysis of HIV clade C-derived epitopes presented in the context of different alleles in the HLA-B7 supertype, in which the more beneficial allele restricted responses with higher functional avidities (49).
High-avidity responses in controllers exhibit narrow and highly skewed TCR repertoires.Next, we examined the functional avidities of CTL responses in relation to their composite T-cell receptor (TCR) repertoires, which act as counterparts to the targeted peptide/MHC complex and determine the consequences of antigen engagement. The definition of functional avidity directly ex vivo is based on the antigen concentration-dependent reactivity of the entire epitope-specific CTL population, which potentially comprises multiple cognate TCR clonotypes. Some of these clonotypes may be shared between individuals, even in CTL populations that target the same epitope presented by different HLA molecules (51, 77, 79). Such "public" clonotypes display variable degrees of interindividual sharing and have been associated with protective effects in response to a conserved immunodominant Gag-derived epitope during acute SIV infection (65, 78, 81). To determine whether the high-avidity responses in controllers were linked to particular features of the corresponding antigen-specific TCR repertoires, quantitative molecular analyses of clonotype composition were conducted for three paired epitope-specific responses that were each detectable in one HIV controller and an HIV noncontroller: (i) the HLA-B53/B58-restricted QW9 epitope (Fig. 6, top panels), (ii) the HLA-B42-restricted TL9 epitope (Fig. 6, middle panels), and (iii) the HLA-B27-restricted KK10 epitope (Fig. 6, bottom panels). For all three epitope-specific responses, the HIV controllers exhibited narrower TCR repertoires than the noncontrollers. Furthermore, these narrower repertoires were markedly skewed in two of the controllers (Fig. 6, F7174 and L8190). The public TCR sequences that were shared between the three pairs of individuals in all cases dominated the TCR in the HIV controllers.
The functional avidities of the KK10-specific responses restricted by HLA-B27 and the QW9-specific responses restricted by HLA-B53/58 were higher in the controllers than in the noncontrollers (data not shown), despite similar peptide affinities for the different restriction elements in the latter case (46 nM versus 9.7 nM) (15). In contrast, the functional avidity of the TL9-specific response restricted by HLA-B42 for the noncontroller was slightly higher than that of the corresponding response for the controller (27.7 ng/ml versus 79.5 ng/ml; data not shown). However, in this case, the noncontroller displayed a narrow and highly skewed TCR repertoire (Fig. 6, F723). Thus, although limited to three pairs of epitope-specific responses, these data indicate that high-avidity CTL responses tend to be mediated by narrower TCR repertoires, which are not invariably associated with, but may contribute to, superior in vivo HIV control.
A number of recent studies have suggested an important role for the functional avidity of CTL responses in the control of viral infections (7, 86). However, many reports have been based on responses to single epitope/HLA allele combinations or have used T-cell cultures after in vitro expansion, which could bias the resulting avidity measurements (4, 38, 86). Furthermore, a comprehensive assessment of HIV-specific CTL responses with respect to the role of functional avidity in the control of viral replication is lacking. To address these issues, we studied unexpanded CTL directly ex vivo in a cohort of more than 100 HIV-infected individuals with varying disease status and broad HLA class I allele distribution. Our data confirm previous reports based on selected epitopes that link the presence of high-avidity CTL populations to viral control and further support the important role of HLA-B-restricted responses targeting Gag-derived epitopes in antiviral immunity (4, 6, 16, 38, 42, 44, 46).
Although directly supporting human experimental data are scarce, it is likely that several different mechanisms are responsible for such a superior immune function of HLA-B-restricted CTL responses and the induction and maintenance of responses of elevated functional avidity. These possibly include efficient epitope processing, the characteristics of the presented peptide on B alleles, and a particularly effective TCR recognition of presented epitopes in the context of HLA-B alleles. The HLA-B-restricted HIV epitope repertoire is thought to be enriched for epitopes with increased sequence conservation, which might limit viral escape from CTL-mediated immune pressure and select for a narrower TCR repertoire of high avidity (29). Such selective presentation of more conserved epitopes on HLA-B alleles could result from the need for specific anchor residues for B alleles that require amino acids that pose stringent functional and structural constraints on the viral protein and thus complicate viral escape. Whether this would be specifically true for the protective HLA-B alleles remains to be tested. Other than the need for specific anchor residues, it is also plausible that structural differences of the MHC molecules may influence the interaction between the TCR and the peptide-MHC complex (pMHC) (76), which could explain CTL responses of higher avidity to epitopes presented by HLA B alleles (78, 83).
Aside from HLA restriction, the fine specificity of HIV-specific CTL has also been associated with relative control of viral replication. In accordance with several previous studies (43, 53, 87), the data presented here also link Gag-specific responses with relative viral control and, likely of relevance in mechanistic terms, with high levels of functional avidity. Since Gag degradation products from the infecting viral particle are presented very early after infection, high-avidity responses to such cross-presented antigens could mediate the effective elimination of infected cells at a relatively low surface density of the represented epitopes and before the production of progenitor virus (69). Furthermore, Gag is one of the most conserved proteins in HIV and may contain a disproportionally high number of epitopes that bind to alleles associated with superior HIV control, particularly HLA-B27 and HLA-B57. Since these epitopes are among the most immunodominant ones in individuals expressing these alleles (15, 33), they may be focusing the most beneficial response onto this protein (17). Taken together, our data thus might suggest that high-avidity CTL to Gag-derived, HLA-B-restricted epitopes contribute to relative HIV control by targeting epitopes that (i) are expressed early in infection, (ii) could elicit cytolytic effector functions at low epitope abundance on the surface (74), (iii) are located in conserved regions with restricted viral escape pathways, and (iv) might result in a more efficacious TCR activation in the context of HLA-B alleles.
The differences in the median functional avidities between controllers and noncontrollers, although statistically significant, were surprisingly modest. This may be due in part to the evident variability in SD50 values between different epitope-specific responses. In addition, IFN-γ production is relatively preserved in exhausted T cells and may be subject to comparatively low activation thresholds (13, 73). Thus, determining SD50 values for additional effector functions may reveal more marked differences between study groups, epitopes, and restricting HLA alleles. Furthermore, functional avidity as tested in our present study reflects the summation of CTL response and epitope presentation, without taking into consideration several additional factors that are likely important in determining the in vivo efficiency of these responses. For example, epitope abundance on the surface of an infected cell will also contribute to the in vivo effectiveness of the CTL response independently of the experimentally measured functional avidity using synthetic peptides. This amount of presented epitopes on the cell surface is in turn impacted by several factors, such as antigenic protein expression levels, epitope processing preferences, intracellular epitope stability, HLA binding, HLA expression kinetics, and likely others (75). Data on these factors are incomplete to date and are difficult to take into account experimentally all at the same time, thus limiting the conclusions that can be drawn from analyzing individual factors in isolation. In vitro testing of functional avidity in the context of HIV-infected cells might be one approach to bypass some of the limitations of synthetic peptide-based assays and to reflect the in vivo situation more directly. Practical limitations, however, would limit the feasibility of such studies for larger, genetically heterogeneous host populations. Furthermore, epitopes with the highest functional avidity in the present study are not necessarily the epitopes with the highest intracellular stability (47), which suggests that epitope stability is a possibly independent factor, aside from avidity and others, that will define the in vivo effectiveness of a CTL response.
Further complicating our understanding of the relative importances of specific CTL populations and HLA restriction for HIV control are factors such as epitope competition in individuals with different HLA backgrounds, variable epitope dominance patterns, the possible effects of other coinfections, and often extensive sequence variation in the targeted HIV epitope itself (19). In the present study, we attempted to eliminate some of these potential biases by assessing functional avidities for CTL responses targeting the same epitopes presented by several different HLA class I alleles, particularly those associated with different HIV disease progression rates. For all tested epitopes with promiscuous HLA restriction, the alleles associated with superior HIV disease control restricted responses with higher functional avidities than the less beneficial alleles, even if the responses in the beneficial alleles were tested in samples from a noncontroller individual. These findings suggest that the restricting allele may be a more potent determinant of functional avidity within the CTL population than the bound peptide, which would be in line with recent data that link specific residues within beneficial HLA allele sequences with relative viral control (64).
Functional avidity is an indirect measure of the strength of the interaction between the T cell and its target cell (e.g., an HIV-infected cell) and depends among other factors on the monomeric affinity of the clonotypic TCR for cognate pMHC antigen (72). In paired analyses of epitope-specific CTL responses, high levels of functional avidity were associated with narrower TCR repertoires in our study. This observation is consistent with interclonal competition for antigen, which has been shown to drive oligoclonal and highly skewed epitope-specific TCR repertoires in persistent DNA virus infections (67). Previous work has associated public TCR usage in response to the protective Mamu-A*01-restricted Gag CM9 epitope during acute infection with relative control of SIV replication (65). The observation may be due to an intrinsically higher level of cross-reactivity among public clonotypes, which are also present in the naive T-cell pool at greater precursor frequencies and can therefore be mobilized more readily (68). It remains to be determined, however, whether the greater representation of public clonotypes in controllers is associated with functional avidity per se and how the allele's intrinsic ability to restrict cross-reactive responses could impact these results. In addition, differential TCRα chain pairing (56) could potentially complicate the overall interpretation. Indeed, such pairing promiscuity could explain why the same public TCRβ sequences were subdominant in the three noncontrollers studied here. Alternatively, these clonotypes may be inefficiently selected in noncontrollers due to greater immune dysregulation or could perhaps be waning due to higher rates of senescence and clonal turnover under conditions of rampant viral replication (4). Such ongoing viral replication also increases viral diversity, and this might lead to the emergence of escape variants that can induce new T-cell populations at later time points in infection (3, 37). These variant-specific CTL populations may possess functional avidity different from that of the wild-type-specific CTL population and may broaden the TCR repertoire of the epitope-specific response. This possibility cannot be ruled out in the present study, where responses were not studied longitudinally and under inclusion of all autologous viral epitope variants. As a consequence, the elevated functional avidity in HIV controllers may reflect a certain bias caused by measuring epitope-specific responses in HIV controllers that target nonmutated epitopes and score with a higher avidity than responses in HIV noncontrollers that may harbor more-avid responses reactive with autologous epitope variants but which may not be detected in our assay system using consensus-based peptides.
Most studies aimed at understanding the characteristics of protective immune responses to HIV are confronted with difficulties in disentangling cause versus consequence of the tested markers. In the SIV model, a progressive decline of functional avidity has been observed with increasing time of infection (61), which has been suggested to reflect early escape of epitopes targeted by high-avidity responses. This would be in line with recent data on a selected HLA-B*27 epitope showing the gradual escape from high-avidity CTL responses (39). To indirectly address this point in our analyses and to test whether or not removal of antigen would result in the (re)emergence of high-avidity responses, chronically HIV infected individuals were studied before and during HAART therapy. Somewhat surprisingly, a decline rather than a recuperation of functional avidity was observed, suggesting that reduction of high antigen doses is not sufficient to select for high-avidity responses. Obviously, HAART-induced suppression and spontaneous viral control are very different situations, and the data may simply be a reflection of the previously active viral replication and its effects on the CTL function during the pre-HAART period. In particular, ongoing viral replication could have led to immune alterations in chronic progressive infection, which might no longer be reversible upon HAART initiation, including post-TCR signaling pathway dysfunction, increased cell turnover, and likely other mechanisms (41, 71). Alternatively, high-avidity effector CTL may be more affected by antigen removal than their low-avidity counterparts and/or the persisting memory pool may be skewed toward certain clonotypes upon the initiation of HAART. However, the clonotypic composition of epitope-specific T-cell populations is thought to remain relatively stable during HAART (84), which would tend to invoke interclonal qualitative differences. In this regard, one potential explanation for the observed reduction could be the preferential activation of memory T cells that are cross-reactive with other persistent pathogens and thus might not exhibit the highest functional avidities for the cognate HIV antigen (19). Clearly, longitudinal studies of untreated individuals starting in acute infection simultaneously controlling for changes in TCR repertoire, viral sequence evolution, phenotypic T-cell characteristics, immune dysfunction, and functional avidity will be required to resolve these questions.
Despite some limitations, the present study provides strong evidence, on a cross-sectional basis and in the context of a broad HLA background, for a direct association between the presence of high-functional-avidity CTL responses and relative control of HIV replication in vivo. These findings have substantial implications for HIV vaccine design, which may need to focus on the induction of CTL responses with high levels of functional avidity through the mobilization of optimal epitope-specific TCR repertoires and targeting highly conserved regions to reduce immune escape.
This work was funded by a National Institutes of Health grant to C.B. and D.E.K. (R01 067 077) and in part by the Spanish FIPSE grant 36-0737-09. C.T.B. is supported by a research grant from the University of Basel, Basel, Switzerland, and the Janggen-Pöhn Foundation, Switzerland. D.A.P. is a Medical Research Council (United Kingdom) senior clinical fellow. B.M. holds a research fellowship grant from the FIS (Rio Hortega; CM08/00020), Madrid, Spain. C.B. is a senior ICREA research professor.
C.T.B. thanks C. Hess for ongoing support.
- Received 7 March 2011.
- Accepted 30 June 2011.
- Accepted manuscript posted online 13 July 2011.
- Copyright © 2011, American Society for Microbiology. All Rights Reserved.
- 1.↵
- 2.↵
- 3.↵
- 4.↵
- 5.↵
- 6.↵
- 7.↵
- 8.↵
- 9.↵
- 10.↵
- 11.↵
- 12.↵
- 13.↵
- 14.↵
- 15.↵
- 16.↵
- 17.↵
- 18.↵
- 19.↵
- 20.↵
- 21.↵
- 22.↵
- 23.↵
- 24.↵
- 25.↵
- 26.↵
- 27.↵
- 28.↵
- 29.↵
- 30.↵
- 31.↵
- 32.↵
- 33.↵
- 34.↵
- 35.↵
- 36.↵
- 37.↵
- 38.↵
- 39.↵
- 40.↵
- 41.↵
- 42.↵
- 43.↵
- 44.↵
- 45.↵
- 46.↵
- 47.↵
- 48.↵
- 49.↵
- 50.↵
- 51.↵
- 52.↵
- 53.↵
- 54.↵
- 55.↵
- 56.↵
- 57.↵
- 58.↵
- 59.↵
- 60.↵
- 61.↵
- 62.↵
- 63.↵
- 64.↵
- 65.↵
- 66.↵
- 67.↵
- 68.↵
- 69.↵
- 70.↵
- 71.↵
- 72.↵
- 73.↵
- 74.↵
- 75.↵
- 76.↵
- 77.↵
- 78.↵
- 79.↵
- 80.↵
- 81.↵
- 82.↵
- 83.↵
- 84.↵
- 85.↵
- 86.↵
- 87.↵


text:
FREE Shipping on Education and Farm orders $99 or more Exclusions Apply
Jacquard® Pearl Ex Powdered Pigments - Set of 8
Product Number: 9735599
No longer avaliable
This item has been discontinued
No longer available.
Can be mixed with any medium and can be applied to virtually any surface. Extremely colorfast and stable. Nontoxic. Neutral pH. Includes one 0.50-oz. jar each of citrine, scarlet, magenta, shimmer violet, sapphire blue, apple green, emerald, and dark brown.
of 0 Reviews
Your guide to an exceptional shopping experience.
Learn More


text:
Trial Lawyer Arash Homampour talks about the work that goes into large cases and the resulting relationships that are built with co-counsel.
If you'd like to watch more videos like this, subscribe to the Homampour YouTube Channel. We publish videos of Arash that deliver real insight into different areas of the law. Click here to subscribe.


text:
Celebrate the Holidays and Enter Our $1,000 Gift Card Giveaway!
Celebrate the Holidays with 12 Days of Christmas Giveaways! The holiday season is quickly approaching and to celebrate, we are giving our loyal Rewardz members by giving away $1,000 in gift cards for the 12 Days of Christmas. Starting on Monday, December 10th, we will be drawing a name every day. To enter, all […]


text:
Thursday, December 06, 2007
Bush On Iran No Nukes ; How Long Might He Have Been Lying For?
We know a bit more know on the process of the release of info on the 'Iran stopped it's nuclear programme'. George Bush was told there was new information on Iran's nuclear programme several months ago.
However, it's then said that he didn't know what the information was. Plus it's not been confirmed whether the Director Of National Intelligence, Mike McConnell told the President that all previous intelligence could be considered invalid.
Tell The Truthiness ; So, did the President know? If he did, how long ago did he know? If the intelligence was faulty, was Mr Bush told this, or did he choose to continue ratcheting up the non-story on Iran in absence of any valid information? Mr Bush, you couldn't stop talking on Monday, want to talk out the answers to these questions? We're ready to listen.
Posted by Swindoncentric at 12:29 am
No comments:
Subscribe to: Post Comments (Atom)


text:
A Closer Look At Hard Cast Lead Bullets
The sport of shooting is a time honored tradition in many parts of the country as well as around the world. Despite all of the new technology around weapons, or perhaps because of it, many gun owners have continued to use traditional types of shooting practices, including the use of hard cast lead bullets.
The Definition of Hardness
For those new to the term, there is a difference in hard cast lead bullets and lead bullets. Lead itself is a very soft material and is only rated as a 5 on the Brinell Hardness scale.
To put that in perspective, pine and other softwoods have a hardness of 1.6 on the same scale, while actual hardwood varieties can have a score of 2.6 to 7. Copper has a hardness rating of 35, and stainless steel is at 200.
The Brinell hardness of a hard cast bullet will be between 11 and 30, depending on the specific mix of tin and antimony that is used in the development of the bullet. Most manufacturers have slight variations that remain consistent to provide a uniform shot.
The Benefits
The problem with the softness of the lead is that it leaves deposits in the barrel, resulting in fouling and loss of accuracy with a weapon. By shooting with hard cast lead bullets, there is significantly less fouling in the barrel and no corresponding drop in accuracy, even with a much larger number of rounds fired between cleanings.
Additionally, many hunters prefer to use the hard cast bullet option due to their action on impact. Instead of mushrooming and stopping very closer to the level of impact, they maintain their shape deeper into the target, resulting in a cleaner kill, smaller wound areas and deeper penetration in large game.


text:
Holy Fuck took the world by surprise around 2005 because there was just nothing like them-a hardcore thrift-store found-object punk band with a relentless commitment to rhythm and a sense for atmosphere better matched to a close encounter of the third kind than a simple rock concert. Think Einstürzende Neubauten re-inspired by Fela Kuti with Brian Eno working as keyboard tech and every channel on the mixer set to max power. It was the best ride out there while it lasted, up to and including their 2010 full-length Latin, recorded largely in too-brief breaks while on the road. That album cemented Holy Fuck's sound and reputation for unapologetic instrumental noise but at the end of yet another insane touring cycle, it was time to take a break ... which turned into a hiatus ... which turned into a chance to explore other projects and production work. (Like the bands Lids, Dusted and Etiquette, or production for Metz, Alvvays and Viet Cong.)
The Toronto-based noise band released a new 4 song EP Bird Brains via LA record label Innovative Leisure. It was recorded live at various studios and is the first Holy F release since 2016's much-lauded Congrats LP.
The EP represents the ongoing creative process of a band whose members live in different cities (in both Canada and the US) yet insist their music should be a collaborative process. These four songs aim to capture the raw energy of four people, not apart as bedroom producers, but together as a spirited exchange of ideas, a dynamic interaction that comes from years of friendship.


text:
Children in Key Stage 2 were some of the first in the world to try a new technology that brings virtual reality into the classroom. The Google Expeditions experience enabled the classes to visit the Borneo rainforest, the bottom of the Caribbean sea and even Mars, all without leaving their seats. We had great fun testing it out and can't wait to use this kind of technology again to further our learning.
To learn more about Google Expeditions take a look at the video below.


text:
Strategic Planning
We value our client's thoughts, opinions, and ideas. Listening to visions and bringing them to life is what we do best.
Custom Design
We don't expect you to choose from a list of floor plans—we deliver one-of-a-kind dream homes through a seamless process centered around personalized, attentive client service.
Home Construction
We build according to our customers' plans and follow through with the highest quality of work offered in Central Florida.