Chickpea is something my hubby and daughter loves. When my daughter was small she used to call it Indian balls curry.
Soak the chickpeas overnight in 3 cups water. Pressure cook and keep aside.
Use 1/2 cup warm water and blend the tamarind with your hand to extract the juice. If using paste, dilute it in 1/2 cup water.
Heat a pan and bring the tamarind water to boil. Add turmeric powder, hing, salt and the sambar powder to this. Sim and let it boil for 10 minutes.
Add the cooked chickpeas and boil for another 10 mins.
Remove from fire, add coconut milk and mix well. Adjust salt.
Heat some oil in a small saucepan and temper some mustard seeds and then onions and the curryleaves.While hot, pour on the above gravy and mix well.
This is a another chickpea dish I am going to make over and over. As all of us loved it.
I have not made any new recipe with legumes other than this, so I am going to send this to Sra to her wonderful event Legume Love Affair.
Now this weekend my daughter played violin solo in the orchesra ( she does it as a hobby) so here are few pictures from the concert.
Have to admitt with my unprofessional camera the pictures are not food at all.
Congratulations to your daughter HC. That is indeed an accomplishment. As for the chickpeas they taste great in any type of curry.
Wow kozhambu looks awesome. Concert also looks awesome. Cheers to your daughter.
That was so good abt ur daughter. Convey my regards to her. Concert seems to be so good.
I love chickpeas in anything so this soup looks wonderful! What a talented daughter you have.
Indosungod thanyou. Yeah it is and here we too love chickpeas.
Congratulations to your daughter :) It must've been a proud moment for you.
Your daughter is very talented.. Congrats to her and all the best.. Kozhambu looks delicious.
Congrats to your daughter HC, Real achievement playing solo !
The chickpeas is just creamy and tasty!
The soup looks really delicious. Congrats to your daughter!
We love chickpeas too but I've never seen this kozhambu before. It looks good.
You must have felt very proud to see your daughter play on stage. Hope she continues with this hobby.
Thank you for your comment on the Dulce de Leche..I had no idea that there was a chance of exploding. I suppose I got lucky this time. I might have to try the oven approach next time.
Congrats to ur daughter..You musst feel proud rite??And lovely kuzhambu.
Congrats to your daughter and the curry looks very appetizing.
The soup looks really delicious. Congrats to your daughter!!!!!
Congrats on your daughter...how very cool!
congratulations to your daughter. that is indeed special.
kozhumbu looks yumm..and cheers to your daughter..I truly admire such talented youngsters..
I like eating toasted chickpeas. When my parents went back home from Mecca, they brought many ready-bought roast chickpeas. I tasted a bit strange firstly, but then I could not stop! I haven't cooked anything with chickpeas yet. Would love to try them sometimes in a curry or salad like this. Looks yummy!
Wow, you must tbe so proud of your daughter. Kozhambu looks great, have never had this kind of dish with it before.
Kozhambu tastes great - we add other veggies to it along with chickpea!
Congrats to your daughter HC! And that chickpeas gravy looks so different, I gotta check out this recipe at Nags' place..
I know how you feel - I'm more nervous than my daughter too when she has her Academie performances!
Congrats to ur daughter, H.C. I haven´t try chickpeas so I don´t know how its taste.
I love chickpeas too! The soup looks amazing! And Congrats to your daughter!
Hi all thankyou for all the best wishes. My daughter was really pleased to read about all the wishes.
kuzhambu looks superb...that was soo gud abt ur daughter...cheers..
Congrats to your talented daughter, you must be very proud of her. The chickpeas sound delicious.
Huge congratulations to your daughter H.C.
kozhambu is a new dish to me but it looks really delicious!
Congratulations to your daughter! You must be so proud.
The chickpea kozhambu is a double whammy, I suppose, with twice the amount of spices and onions. Thanks for the entry!
Two thumbs up to her for a great concert.
I bet the concert was nice - but that kozhambu looks great!
Hello happy cook. Thank you so much for visiting my blog. I love coming to yours because you are so honest and real, and I really do like your recipes. You must be so proud of your daughter. How wonderful it must have been to see her on stage.
That sounds like a nice way to enjoy some chickpeas.
Congratulations to your daughter. It is a great accomplishment indeed! You must be a proud mama.
Wow! You have such an awesome daughter. She's so cool! Congratulations!
I'm surprised that you have such a big daughter.
It must be a proud Moment for you.Playing Violin needs lots of dedication and practice.
My hearty congratulation to the young and promising talent.
Curry looks so creamy , a very different stuff from the usual red color curry for chickpea curry , I am sure it tastes great also,will check out at nag's place .
Oh My!I totally missed this post of your daughter playing solo..Sorry dear!..SPECIAL and HEARTY Congrats to her..
and wishing her many more to perform!!!..
Gosh, this looks delicious. One of my New Year's resolutions is to cook more ethnic food...Indian being at the top of my list! I just may have to try this.


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Xavier Elliot Starts a Charity Sewing Clothes for Homeless Kids
23 Jun, 2015 by	McGuire
A 10-year-old boy has figured out how to take the lemons life has handed him and make lemonade. The grade schooler, son of a Iraq veteran, was homeless after his father returned from service. Now that his struggle has ended, Xavier is reaching out to help others who are in the same shoes he once wore – in a very unique way:
Xavier Elliott, from Phoenix, Arizona, lived in about six shelters after his dad came back from Iraq; the US Army veteran came home from war with PTSD, and the family struggled to stay on its feet.
Now that they are no longer homeless, Xavier is incredibly grateful for everything he has, and he wants to use his good fortune to help kids whose families are still struggling. So he's using his own money and free time to sew new clothes, which he plans to donate to shelters around Arizona.
One day, Xavier watched as his mother, Stephanie, was making a purse on her sewing machine, and he asked her for a lesson.
'We have so much stuff I think we should give stuff [to] homeless kids,' he told KPNX of his project.
He soon started buying fabric with his own allowance, while his mom taught him the basics on her sewing machine, teaching him how to stitch together simple designs and patterns.
'I think the idea came to him from everything we have endured and wanting to make a difference for other people,' she told The Huffington Post.
One he's mastered the machine, Xavier plans to give the clothing he makes to organizations like the Salvation Army, Crisis Nursery, and an Arizona homeless shelter called UMOM New Day Center.
He's also using a Facebook page that his mother created to make his philanthropic project even bigger. There, the family is seeking donations of fabric, child-sized mannequins, and other equipment.
Xavier is soliciting monetary donations as well, so he can buy more fabric and his own sewing machine. So far, he has raised $546, and the fabric donations have been pouring in.
What a good-hearted kid. Kudos to him on his efforts, and I am hopeful that he will be successful in his efforts. If you wish to donate to Xavier, please go to Crowdrise.
Written by Katie McGuire. Follow Katie on Twitter @GOPKatie, or email the author at [email protected]
McGuire
Writer, Blogger. Political aficionado. Addicted to all levels of government campaigns.
More articles by McGuire

redpajama_set_name: RedPajamaCommonCrawl


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Your browser does not support Java, so nothing is displayed.
This was my very first java program (applet) back in the day. It is a game known as 'Paint By Numbers'. The goal is to reveal the hidden image on the board. The numbers on the columns and rows show the number of consecutive filled cells. For example, 3 5 means you have 2 blocks of filled cells of size 3 and 5 respectively. You can click on a single cell or select a block of cells. Initially, each cell is in undecided state. Single click turns the cell into white (empty) and clicking on a white cell turns it into blue (filled), clicking on a blue cell turns it into gray (undecided) again.
The numbers on the rows & columns are the constraints that you should respect when solving the puzzle. If any of these contraints are violated, you will see the numbers for the offending rows/columns turn red. A correct solution is found when no more gray cells left and no constraint is violated.
The solve button automatically solves the puzzle. The applet does not know what the final image looks like, all it knows are the values on the columns/rows just as it presents to you.


So, I've been patrolling GTA 5's city of Los Santos, wearing Tony Stark's Hulkbuster armor, which has been modded in thanks to master craftsman JulioNIB. I'm having a nice time. The armor looks great, I can fly, and I'm keeping the city safe.
The only problem is there's no Hulk. I'm all dressed up with no Hulk to bust. I look everywhere, but all I find are the city's usual non-gamma-radiated normals, and they're not the types who will pick up a truck and throw it into outer space or punch the ground so hard the earth's orbit will be altered. With no Hulk, I sort of don't know what to do with myself.
As I'm flying around, kinda bored, I get a sudden glimpse of something big and green. Is it the Hulk? Probably not—I don't hear any screaming or declarations of the intention to smash—but it's worth checking out.
I plunge down from the sky, landing dramatically, ready to do some busting, but it turns out to just be one of those green garbage trucks.
On the other hand, I did whoosh out of the sky heroically, so I should probably bust something. I give the truck a little dose of my repulsor beam, and it explodes, taking a nearby car with it.
Well, you can guess the rest. The police arrive and they are not pleased. While Iron Man isn't really the kind of misunderstood hero who constantly tangles with the good guys, I have to assume the Hulk won't magically appear. So, I start whomping on the cops.
The mod comes with a number of powers in addition to flying and looking cool. You can pick up, carry, and throw vehicles. You've got both single and double-handed repulsor blasts, as well as a chest repulsor. You can also fire homing missiles and darts, and you have a machine-gun, though I don't know why the Hulkbuster armor would fire bullets, which the Hulk is impervious to. Maybe just to get his attention?
While in the air, you can also target and attack enemies on the ground, which gives you a burst of speed and lets you land with a tremendous impact. That's a lot of fun, even with no Hulk to do it to.
I don't know if you've installed any OpenV mods or skins for GTA 5 yet, but if you haven't it's a bit exhausting. There are a number of steps, a bunch of different folders to manage and instructional videos to watch and different items to plop in very specific places.
JulioNIB has provided some pretty good instructions here, and links to everything you need to download, but keep in mind those instructions also link to a lot of other instructions and several videos. Also keep in mind: editing game files with OpenIV makes permanent changes to the files, so back up your game folder before you go messing with it.
Worth it? Hell, yeah. Getting to fly around Los Santos with Hulkbuster armor is fun, even if the Hulk never shows up.


So today we're going to have a look at best 10 Git GUI clients which you can install on Ubuntu and use them as version control tool for software development.
SmartGit is a cross-platform graphical Git client with seamless support for SVN, GitHub and Bitbucket. Apart from Windows and macOS, SmartGit can easily be used on Linux and its distros like Ubuntu. Good thing about SmartGit is it includes everything you need in one single package which includes graphical merge, commit history, Git-Flow, SSH-Client, file compare and file merge.
During the testing I have tried it for the first time and I have to admit that it doesn't take me long to get used to it thanks to its simple and easy to use user interface.
SmartGit is a highly customizable graphical Git client with options at your hand such as set preferences for merging and rebasing, layout control, built-in Conflict Solver tool, keyboard shortcuts, toolbars, syntax coloring and you can choose between pair of light and dark themes.
Software developers will find this Git client very useful because it focuses on the difficulties faced by developers and tries to make workflow as simple as possible for developers.
Written in Python, Git Cola is completely free but very powerful graphical Git client for Ubuntu and other Linux distros. It is the one of the best and fast version control tools available out there for software developers. It is simple but powerful client with features like clone, merge, push, pull and many other useful features.
Git Cola is also a cross-platform graphical client for Git which supports Windows, macOS, Linux and its popular distros such as Ubuntu and LinuxMint. It is an open-source GUI client for Git and it is highly customizable Git client out there. I have tried few tweaks during testing and it has responded as expected.
You can customize its window settings, language settings and many other features to enhance the work experience and make it hassle free.
GitEye from CollabNet is an easy-to-use Git GUI client which comes bundled with some useful tools which helps you improve development productivity. It offers push-button cloning for repositories of GitHub, CloudForge and TeamForge.
GitEye offers seamless integration with other popular tools such as Bugzilla, Jira, Hudson, Jenkins and many others. GitEye has clean and very powerful graphical user interface which is very easy-to-use with everything placed perfectly so that user finds whatever he needs easily.
It comes bundled with integrated issue tracker client that works both in online and offline mode, also it is equipped with Gerrit Code Review that provides notifications if there is any change or update in code. Overall it is any all-rounder package and many developers find this Git client very useful.
GitKraken is another graphical Git client which is powerful and reliable, which supports both Git and GitHub to work with. GitKraken ships-in with very modern looking user interface which is eye-catching and easy to navigate. I'll recommend GitKraken to the developers who daily engage in tedious development workflow because it offers some really useful features that make this task quite easier and hassle free.
Some of the notable features in GitKraken are built-in code editor, split view, syntax highlighting and file minimap. It also offers seamless integration with popular Git hosting services such as GitHub, GitLab and BitBucket. If you opt for GitKraken Pro then you can connect GitHub Enterprise, GitLab Self-Hosted, BitBucket Server and Azure DevOps.
As compared to other Git clients, GitKraken has really fast and reliable workflow. Well that is what I have noticed during all the Git clients listed here in this article.
GitForce is another cross-platform visual front-end to Git command line tool and it works on Windows and Linux and its distros like Ubuntu. It is very simple and easy to use but very powerful and reliable version control tool available for Ubuntu.
Written in C# and .NET 3.5 framework, the main objective of GitForce is to provide simple and easy-to-use graphical front-end to perform most common operations and software development tasks. Despite limited features, you can still rely on GitForce to perform task than getting engaged in Git command line tool.
Some of the features its offers are such as drag and drop, support for multiple repositories, scans local repositories easily and many basic features.
Gitg is a GNOME front-end for Git command line and it is a default graphical Git client on GNOME desktop environment. It offers many features such as staging, commit, open repository, clone repository and many more. It also enables GNOME shell integration which opens the way for various in app integrations and features for GNOME desktop users.
GNOMEs simple design doesn't restrict Gitg from offering full-fledged features as it is one for the best Git GUI clients listed here. At first I have found its user interface quite confusing but once I got used to it, it was very satisfying experience.
Developed in 2007 as a part of hackathon, Giggle is free and easy to use Git GUI client which works exclusively on Linux and its various popular distributions. It is simple front-end tool which allows developers to browse and view there repositories in graphical interface.
It offers all the basic features you can expect in Git clients such as stag, commit, browse and many more. User interface is simple and you will get used to it in a fly.
UnGit is not an application or client but it runs as a website in any web browser installed on your computer. It offers simple but very useful graphical user interface to replace Git command line tool. It comes with integration support for various plugins which enhance its workflow and performance.
UnGit can also be installed as a plugin in popular text editors such as Atom and Sublime Text.
Magit is a graphical interface for version control tool Git command line. It doesn't offer separate interface but it is implemented as GNU Emacs package instead. It is not up to the ranks of various graphical Git clients listed here but it allows software developers to perform almost all the version control tasks within Emacs window.
Despite being a plugin it offers features you can expect in a separate application such as visualization, smooth workflow, rebasing and many more. It is a platform independent plugin and can be integrated with Emacs installed on Windows, macOS and Linux.
Just like Magit, Egit is a graphical Git version control plugin for Eclipse. It is implemented on the top JGit Java Implementation of Git. It is simple but very useful and reliable Git tool which offers all the basic features to accomplish almost all software development version control tasks.


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* Our Quality Policy Statement is also available upon request.


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Haywards Heath Ceramics Group
Future Programme
"Josiah Wedgwood, Potter, Pioneer and Philanthropist" by Gaye Blake-Roberts
On Thursday 9 April 2015, Gaye Blake-Roberts, Curator & Director of the Wedgwood Museum, who lectures on Wedgwood worldwide, gave members a thrilling and exhaustive talk on "Josiah Wedgwood" as Potter, Pioneer and Philanthropist.
Gaye commenced with his heritage and continued into his working life – Josiah Wedgwood was born in Burslem, Staffordshire, was the last child of Thomas Wedgwood and Mary Wedgwood. By the age of nine, he was proving himself to be a skilled potter. He survived a childhood bout of smallpox to serve as an apprentice potter under his eldest brother Thomas Wedgwood IV. Smallpox left Josiah with a permanently weakened knee, which made him unable to work the foot pedal of a potter's wheel. As a result, he concentrated from an early age on designing pottery and then making it.
In his early twenties, Wedgwood began working with the most renowned English pottery-maker of his day, Thomas Whieldon, who eventually became his business partner in 1754. He began experimenting with a wide variety of techniques, an experimentation that coincided with the burgeoning of the nearby industrial city of Manchester. Inspired, Wedgwood leased the Ivy Works in the town of Burslem. Over the course of the next decade, his experimentation (and a considerable injection of capital from his marriage to a richly-endowed distant cousin) transformed the sleepy artisan works into the first true pottery factory
Wedgwood was keenly interested in the scientific advances of his day and it was this interest that underpinned his adoption of its approach and methods to revolutionize the quality of his pottery. His unique glazes began to distinguish his wares from anything else on the market. He was perhaps the most famous potter of all time.
By 1763, he was receiving orders from the highest levels of the British nobility, including Queen Charlotte. Wedgwood convinced her to let him name the line of pottery she had purchased "Queen's Ware", and trumpeted the royal association in his paperwork and stationery. In 1773, Catherine the Great of Russia ordered the Green Frog Service from Wedgwood; it can still be seen in the Hermitage Museum.
As a leading industrialist, Wedgwood was a major backer of the Trent and Mersey Canal dug between the River Trent and River Mersey, during which time he became friends with Erasmus Darwin. Later that decade, his burgeoning business caused him to move from the smaller Ivy Works to the newly built Etruria Works, which would run for 180 years. The factory was so-named after the Etruria district of Italy, where black porcelain dating to Etruscan times was being excavated. Wedgwood found this porcelain inspiring, and his first major commercial success was its duplication with what he called "Black Basalt". He combined experiments in his art and in the technique of mass production with an interest in improved roads, canals, schools and living conditions. At Etruria, he even built a village for his workers.
Not long after the new works opened, continuing trouble with his smallpox-afflicted knee made necessary the amputation of his right leg. In 1780, his long-time business partner Thomas Bentley died, and Wedgwood turned to Darwin for help in running the business. As a result of the close association that grew up between the Wedgwood and Darwin families, Josiah's eldest daughter would later marry Erasmus' son. One of the children of that marriage, Charles Darwin, would also marry a Wedgwood — Emma, Josiah's granddaughter. This double-barreled inheritance of Wedgwood's money gave Charles Darwin the leisure time to formulate his theory of evolution.
He belonged to the fourth generation of a family of potters whose traditional occupation continued through another five generations. Wedgwood's company is still a famous name in pottery today (as part of Waterford Wedgwood; see Waterford Crystal), and "Wedgwood China" is sometimes used as a term for his Jasperware, the coloured stoneware with applied relief decoration (usually white), still common throughout the world.
Gaye's lecture covered so much, the audience was left wanting to know more and probably wishing to hear the lecture again to fully appreciate the diversity of interests in Josiah Wedgwood's life.
This entry was posted in Uncategorized on 18/04/2015 by HHCG Admin.
"Rivets, Repairs and Replacement Handles" by DR MICHAEL SIGNY
February 13 @ 09:45 - 12:00
"Collecting Rockingham – Problems and Opportunities" by JIM SEWELL
CERAMICS STUDY DAY 2019 – Continental of the 16th/17th &18th Centuries & English Glass of the 17th & 18th Century by Jane Gardener
Italian Renaissance Maiolica by Susanne Higgott
"London Saltglazed Stoneware, mostly Brown" by PHILIP MERNICK
"The Art of Cameo Glass – from Ancient Rome to Emile " by Scott Anderson
"Wanli to Kangxi, Chinese Blue and White Porcelain from 1572 to 1722" by Nicholas Panes. Hon Vice President of the English Ceramic Group.
Categories Select Category Andy McConnell Anne Haworth Anniversary Anton Gabszewicz Belleek Clarice Cliff Discovery Day Eric Knowles Field Trips General Information Glass Goss Iznik Jane Gardiner Japanese Ceramics Jill Fanshawe Kato Jonathan Gray Kathy Nibblet Lectures Lindfield Pottery Maiolica Melanie Gibson Nick Panes Portmeirion Raymond Notley Rosemary Pemberton Royal Doulton Rye Pottery Sauceboats Show & Tell Swansea and America Uncategorized Wedgwood Will Farmer


Pink Himalayan salt is all the rage recently, but though it might be pretty, is it really that much healthier for you than regular table salt? Below are a few facts about Himalayan pink salt to keep in mind.
Himalayan pink salt is believed to be one of the purest salts in the world – hence its comparatively hefty price tag. The salt gets its characteristic pink color from minerals deep beneath the mountains where it is mined. It is found in the foothills of the Himalayas in Pakistan, and is thought to be the remnants of a shallow sea which dried up several million years ago before the Himalayan mountains were formed.
Pink Himalayan salt is a naturally-occurring sea salt that is estimated to contain over 80 different trace minerals, including iron, magnesium, calcium, and potassium. Many people enjoy its delicate flavor and beautiful color, and while it's not a good idea to eat too much salt, Himalayan pink salt may be a healthier replacement for regular table salt.
While I would take this extensive list with a grain of salt (pun intended), replacing artificially made and highly processed table salt with this mineral-rich substitute shouldn't hurt – and may well prove to be a much healthier alternative.
Another Good Reason to Eat Chocolate!


I'm a full time Mum who runs a gardening and design business focused on helping other people to getting the most out of their gardens be it through advice, practical help or planting/design ideas.
Watering plants can seem overwhelming in the height of Summer when there's so much else to do. As part of the #TapChat that we're starting about daily water habits, we've asked Surrey-based gardener and designer Renée Canter aka 'The Girl who Gardens' to share her top tips for making sure the plants in your garden are getting the drink they need to grow and thrive – without needing too much time or water.
1. Get to know your soil so you can tell whether there is actually a need for watering.
Different soil types will look and feel different when moist and when dry. Put your spade in and have a look at what it looks and feels like at the surface and 25cm down to decide whether you need to water or not. Generally we need to water some plants during May-September and too much water can be just as damaging as not enough!
2. Ideally water plants early in the morning.
Watering plants in the early morning helps avoid evaporation loss in the heat of the day, or on a Summer evening where the dry soil will take in the water quickly.
Apply the water to the soil at the base of the plant rather than onto the leaves. It's the roots of the plant that 'drink' up the water and then they transport it to the stems and leaves for photosynthesis! Watering the leaves can increase the risk of disease. Watering this way also means you're not watering the weeds in between plants!
Even when it's dry, avoid the temptation to water everyday. Water in the early morning, as described above, and only once or twice a week. Enjoy the time spent watering and use it to keep an eye on how your plants are looking whilst taking in the changes.
6. Water the plants that really need it.
Established plants shouldn't need watering except in a drought. Focus your watering on newly planted plants, pots, containers, hanging baskets and the vegetable patch.
I hope these tips make it easier for you to water your plants. Look out for my blog posts on drought tolerant plants and a guide to making your garden more effective at needing and using water.
Click here to find more simple tips for saving water, time and money. Live in Sutton? Join the #TapChat Sutton to get free water saving products and tips.
Thank you for those tips. They sound great! I will adapt my watering from this moment on.


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Multi location attendance integration is one of the biggest challenge. Different locations have different requirements and local shades.
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The solution is integrated with Biometric devices to capture biometric punches on real time basis. If a Bangalore staff of a factory or store arrives at 8.00 AM then not only local but central management sitting in Calcutta will be able to see the T&A of that staff immediately.
SmartOffice T&A platform makes data integration seamless. It is an answer to all challenges faced by modern practices in growing organisations.


Enter your start date, end date and number of people and we will check availability and prices for you. The recommended duration for this activity is between 1 and 2 days. However, you can book as many days as suits your requirements. If your group size is more than 5 please contact us for a bespoke quote. Special prices for families and children: please contact us.
In the Finale rock climbing area, due to the nature of the terrain with lots of caves and ancient trails that cross the rock faces, we have created several "Adventure Trails". A mix of hiking on trails and exposed ledges, easy rock climbing, abseiling, easy caving. Your Guide will take care of all the rope sections and safety aspects.
There are several Via Ferrata in the Liguria region, ranging from 30 minute to 2 hours driving from Finale Ligure. The nearest is called Ferrata degli Artisti and is 30 minutes drive from Finale. There are other 2 Via Ferrata that are just over one hour drive from Finale. Other 4 Via Ferrata are just under 2 hours driving from Finale.
Finale Ligure is very accessible being only a 30 minute drive from Genova airport, 1 hour and 30 minutes from Nice airport, along with plenty of accommodation that you would normally find in a popular seaside area.
This is a really fun day and a real insight to the Finale backcountry. We meet for a coffee and briefing in the old town of Finalborgo. We then drive for 10 minutes to the Pora Valley, so close but seems so far from the hustle and bustle of the village. A 40 minute hike takes us to the top of and amazing cave without a roof. We do a 30 meter abseil into the cave and then exit though a little tunnel that leads us to a larger cave (need head torches for this section) and then back to the trail.
This Via Ferrata is well know for the painting on the rock walls by the artist Mario Nebiolo. It is a 700 metre climb with stunning landscape and exciting suspension bridges that leads to the summit of Bric Agnellino. From here, you can enjoy a 360° view of the Mediterranean the Alps. 30 min drive from Finale.
For the "adventure trails" and Via Ferrata, no previous experience is required. Obviously, some previous Via Ferrata, rock climbing or mountaineering experience is useful.
Technical equipment: harness, via ferrata set, helmet.
This activity is available to be booked for any date/dates within the availablity periods shown below. Because we tailor your booking to suit your exact requirements you can book this activity for as many days as you like. The recommended duration for this activity is between 1 and 2 days. If your group size is more than 5 please contact us for a bespoke quote. Special prices for families and children: please contact us.
"We spend a week in Finale with our family and experienced rock climbing, via ferrata and mountain biking. It is an amazing location for all these activities, considering that we also spent every afternoon on the beach. We are planning to be back every Spring. Thank you for the great guiding!"


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Eurosong kan syfta på:

 Eurosong – den nationella uttagning till Eurovision Song Contest i Belgien, se Belgiens bidrag i Eurovision Song Contest
 Eurosong – den nationella uttagning till Eurovision Song Contest i Bosnien och Hercegovina, se Bosnien och Hercegovinas bidrag i Eurovision Song Contest
 Eurosong – den nationella uttagning till Eurovision Song Contest i Irland, se Irlands bidrag i Eurovision Song Contest

redpajama_set_name: RedPajamaWikipedia


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Home / News / Amazon and Netflix are the first platforms of video streaming to win an Oscar
Amazon and Netflix are the first platforms of video streaming to win an Oscar
If you like cinema, likely which is that you've already taken due account which have been the big winners of the gala that was held last night in Los Angeles. Do not go to rating which has been the best film, or the mistake veteran Warren Beatty has made the time to announce the winner. This edition of the Academy Awards has been the emergence of the productions of video streaming between the nominees, and such, and as we have seen, has been a success, that over time could be extended.
Amazon, Google and Netflix competed in different categories with films and documentaries, but have only been Amazon and Netflix who have been the cat to the water. The film produced by Amazon Studios, Manchester by the Sea, has allowed to the actor Casey Affleck win the Oscar as best actor main, a film that had a cost of 10 million of dollars and that in the last Sundance film festival, already obtained the price to the best script original. Today it has raised 46.8 million dollars in cinemas and reach Amazon Prime Video service before the end of the year.
Netflix, meanwhile, has won the Oscar of the Academy for best documentary with the title The White Helmets, which gives us the work of a group of rescue volunteers who do their work in the war from Syria. This documentary was directed by Orlando von Eisiedel. If Apple takes seriously the audiovisual production and is set aside the reality shows as The Planet of the Apps, some analysts say that the Cupertino-based company might choose to these awards in about five years, as did Amazon, Netflix and Google, but the latter has not obtained any reward.
Hollywood Oscars streaming services winner of an Oscar	2017-02-27
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13 Brougham Place
North Adelaide SA Australia 5006
What do you want in life?
Contact Dr Darryl
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April 21, 2017 by eneesolutions
Ask anyone in the street what they'd like in life and generally it's to be happy.
Of course some may say that they'd like things like their health or to win the lottery, but almost without exception it's to be happy. It's a universal thing too regardless of culture or creed. It just seems that everyone wants to be happy. So what is this happiness thing and how do you get it?
I'm not going to pretend at the outset though that the message about the journey towards happiness and success is all good news. Neither am I going to suggest that it's easy. There are no silver bullets here.
Let's get the bad news over first.
In short, happiness and success is ongoing and you never really arrive. Part of me wishes that I could tell you that there was an end point and that there was a spot that you could aim for and you would some day get there and say "I've made it," but it "ain't necessarily so." It's a journey; it's a process. Having said that though, there are certainly things that you can do to promote more happiness in your life and more fulfilment. No doubt about it.
Someone once wrote, "Most people live in survival, not in fulfillment."
Striving for fulfillment
What we're looking for as a society today are tips about that journey, tips about how we can be more happy than not, more successful than not. Let me say it again in case you missed it! Most people live in survival, not in fulfilment. So the challenge today is how we actually move towards fulfilment; how do we get that? How do we strive for that? What does it mean?
This book shows you how and details the scientific and psychological principles that we now know bring about happiness and bring about success.
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Pursuit of Success & Happiness
You might surprise yourself at who you can become and what you are capable of in your journey called life.
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He has done it again! Dr Cross has delivered another of his highly successful and usable books. This book is the perfect tool in this high pressure world, be it in business or life in general.
About Dr. Darryl
is a clinical and organizational psychologist who is a Fellow of the Australian Psychological Society and the Australian Institute of Management
Dr Darryl Cross
Crossways Consulting
Copyright © 2020 Dr Darryl Cross


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Q: what is the difference between didselectrowindexpath and willselectrowindexpath in iphone? Give an explanation about difference between UITableView delegate methods:
didDeselectRowAtIndexPath:
-(void)tableView:(UITableView *)tableView didDeselectRowAtIndexPath:(NSIndexPath *)indexPath

and
willSelectRowAtIndexPath
-(NSIndexPath *)tableView:(UITableView *)tableView willSelectRowAtIndexPath:(NSIndexPath *)indexPath

A: willSelectRowAtIndexPath message is sent to the UITableView Delegate after the user lifts their finger from a touch of a particular row and before didSelectRowAtIndexPath.
willSelectRowAtIndexPath allows you to either confirm that the particular row can be selected, by returning the indexPath, or select a different row by providing an alternate indexPath.
Good luck
T

A: the code written in
-(void)tableView:(UITableView *)tableView didDeselectRowAtIndexPath(NSIndexPath*)indexPath

method is run after selected the row and the code is written in
-(NSIndexPath *)tableView:(UITableView *)tableView willSelectRowAtIndexPath(NSIndexPath*)indexPath

run just before the row selected.
they are same as
- (void)viewDidAppear:(BOOL)animated and - (void)viewWillAppear:(BOOL)animated

let me know if you have any confusion now.

redpajama_set_name: RedPajamaStackExchange


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IRAN: At least 24 journalists arrested since disputed election
By Alexandra Zaviswriter
June 21, 2009 1:55 PM PT
At least 24 journalists have been arrested in Iran since the start of protests last week against disputed election results, according to news organizations and a media watchdog.
The Paris-based Reporters Without Borders today released the names of 23 arrested journalists and bloggers, including the head of the Assn. of Iranian Journalists, Ali Mazroui.
Newsweek magazine later posted a statement saying that its correspondent, Maziar Bahari, a Canadian, was detained without charge this morning and has not been heard from since. Newsweek defended his coverage of Iran as 'fair and nuanced' and called for his release.
BBC says its correspondent, Jon Leyne, has been ordered to leave within 24 hours. But the British broadcaster says its Tehran bureau will remain open.
Iran's leadership has repeatedly accused Western journalists and their governments of fomenting the unrest over the reelection of President Mahmoud Ahmadinejad in a vote marred by allegations of fraud.
Since the June 12 poll, authorities have revoked the credentials of foreign journalists and barred reporters from the streets. Many websites have also been blocked.
Much of the news now coming out of Iran is being relayed by so-called citizen journalists who post pictures and videos on Internet sites such as Twitter, Facebook and YouTube.
-- Alexandra Zavis in Los Angeles
Alexandra Zavis
Alexandra Zavis is a former writer and editor on the Los Angeles Times' Foreign Desk who has reported from more than 40 countries. She spent a decade with the Associated Press, covering Liberia, Sierra Leone, Congo, Sudan, Somalia, Iraq and Afghanistan, among other war-torn places. After joining The Times in 2006, she has served as a Baghdad correspondent and as a California reporter covering poverty and veterans issues. She is a recipient of the American Academy of Diplomacy's Arthur Ross Award for distinguished reporting on foreign affairs and was part of teams of reporters awarded the Society of Professional Journalists' Sigma Delta Chi Award for foreign correspondence and APME's International Perspective Award. She is a graduate of Oxford University and City University in London.


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A bivalent recombinant protein inactivates HIV-1 by targeting the gp41 prehairpin fusion intermediate induced by CD4 D1D2 domains
Lu Lu1,2,
Chungen Pan2,3,
Yuan Li1,2,
Hong Lu2,
Wu He2 &
Shibo Jiang1,2
Most currently approved anti-HIV drugs (e.g., reverse transcriptase inhibitors, protease inhibitors and fusion/entry inhibitors) must act inside or on surface of the target cell to inhibit HIV infection, but none can directly inactivate virions away from cells. Although soluble CD4 (sCD4) can inactivate laboratory-adapted HIV-1 strains, it fails to reduce the viral loads in clinical trials because of its low potency against primary isolates and tendency to enhance HIV-1 infection at low concentration. Thus, it is essential to design a better HIV inactivator with improved potency for developing new anti-HIV therapeutics that can actively attack the virus in the circulation before it attaches to and enter into the target cell.
We engineered a bivalent HIV-1 inactivator, designated 2DLT, by linking the D1D2 domain of CD4 to T1144, the next generation HIV fusion inhibitor, with a 35-mer linker. The D1D2 domain in this soluble 2DLT protein could bind to the CD4-binding site and induce the formation of the gp41 prehairpin fusion-intermediate (PFI), but showed no sCD4-mediated enhancement of HIV-1 infection. The T1144 domain in 2DLT then bound to the exposed PFI, resulting in rapid inactivation of HIV-1 virions in the absence of the target cell. Beside, 2DLT could also inhibit fusion of the virus with the target cell if the virion escapes the first attack of 2DLT.
This bivalent molecule can serve as a dual barrier against HIV infection by first inactivating HIV-1 virions away from cells and then blocking HIV-1 entry on the target cell surface, indicating its potential for development as a new class of anti-HIV drug.
Thus far, thirty-two anti-HIV drugs (including five fixed-dose combinations) have been licensed by the United States Food and Drug Administration (FDA) for treatment of HIV infection/AIDS (http://www.hivandhepatitis.com/hiv_and_aids/hiv_treat.html). Most of these drugs act inside the host cell to inhibit viral replication by targeting HIV reverse transcriptase, protease, or integrase. Two of them, enfuvirtide (also known as T20) [1] and Marviroc [2], act on surface of the target cell to block viral fusion and entry by interacting with the HIV-1 gp41 N-terminal heptad repeat (NHR) and the coreceptor, CCR5, respectively. However, none of these anti-HIV drugs can inactivate virions in the absence of the target cell.
Some of the non-ionic surfactants, such as Nonoxynol-9 (N-9), could effectively inactivate HIV-1 virions by lysing the viral envelope membrane [3]. However, because of its high cytotoxicity, it cannot be used as an anti-HIV drug in clinics since it can also damage cellular membranes [4]. Therefore, it is essential to design an HIV inactivator using a protein or peptide that can actively attack the virion before it attaches to and enters into the host cells, with low or no toxic effect on the host cells.
To infect a target cell, HIV-1 first binds to the cellular receptor CD4 and then a co-receptor, CXCR4 or CCR5 through its envelope glycoprotein (Env) surface subunit gp120. Subsequently, the HIV-1 Env transmembrane subunit gp41 changes its conformation from native to prehairpin fusion intermediate (PFI) state by inserting the fusion peptide (FP) into the target cell membrane. Three molecules of the gp41 N-terminal heptad repeats (NHR) interact with each other to form a N-trimer, a PFI conformation, which is then associated with the gp41 C-terminal heptad repeats (CHR) to form a hairpin-like six-helix bundle (6-HB) (Figure 1A), bringing the viral and cellular membranes into close proximity necessary for fusion [5–7]. Mutations in the multiple highly conserved tyrosine and dileucine motifs in gp41 cytoplasmic domain lead to a loss of HIV-1 Env-mediated membrane fusion [8], and several specific amino acid changes in gp120 V3 region and gp41 are associated together with CXCR4 and/or CCR5 usage [9], suggesting that both gp120 and gp41 play important roles in HIV-1 entry and are attractive targets for developing an HIV-1 inactivator and/or entry inhibitors [10].
Design, expression and characterization of 2DLT. A) The schematic view of the interactions between the NHR and CHR of gp41 and C-peptides. The dashed lines between NHR and CHR indicate the interaction between the residues located at the e and g positions in the NHR and the a and d positions in the CHR. The sequences of peptide T20, C34 and T1144 are shown, and the pocket-forming sequence (aa 565–581) in the NHR and pocket-binding domain (aa 628–635) in the CHR are colored in red and green, respectively. B) Schematic view of the 2DLT and D1D2 molecules (not drawn to scale). The D1D2 of CD4 (aa 1–185) and T1144 are connected by a 35-mer of linker (GGGGS)7. C) A diagram showing the predicted structure of 2DLT interacting with gp120. The complex containing the gp120 core (yellow), the two-domain sCD4 (D1D2) (red) and the T1144 (green), was derived from the coordinates of the published X-ray crystallographic complex [11] using the Pymol program (http://pymol.sourceforge.net). D) The model of CD4-induced gp41 PFI. Soluble CD4 (sCD4) or CD4 D1D2 domains bind to HIV-1 Env surface subunit gp120, resulting in the formation of the gp41 PFI with the exposed grooves on the NHR-trimer, which is a target for HIV-1 inactivator. E) Characterization of 2DLT. The soluble recombinant proteins 2DLT and D1D2 were expressed in E. coli using the PDI-chaperone expression system and analyzed by SDS-PAGE (a); Western blot using anti-CD4 polyclonal antibody (b); anti-T1144 polyclonal antibody (c); and by ELISA using anti-CD4 pAb T4-4, a conformation-dependent mAb Sim.4 and anti-T1144 pAbs F). The data are representative of results from three similar experiments performed in triplicate (means ± SD).
Monomeric soluble CD4 (sCD4) that can specifically binds to the HIV-1 gp120 and then inactivate the virion was one of the first anti-HIV-1 agents tested in clinical trial. Unfortunately, it failed to reduce the viral loads in HIV-1-infected individuals [12, 13]. However, sCD4 and CD4-mimetics could efficiently induce the formation of the gp41 PFI with the exposed grooves on the NHR-trimer [14], which is the target of peptidic HIV fusion inhibitors, such as SJ-2176 [15], T20 [16], C34 [17, 18] and T1144 [19, 20]. These results suggest that a molecule containing a CD4 or CD4-mimetic and a gp41 PFI-binding domain (such as T1144) can inactivate HIV-1 more efficiently than sCD4 or CD4-mimetic since T1144 can bind to the exposed gp41 grooves induced by binding of sCD4 or CD4-mimetic to gp120 to speed the virus inactivation. Based on this hypothesis, we engineered a bivalent protein, designated 2DLT, in which the D1D2 domains of CD4 were linked to T1144 by a 35-mer flexible linker to allow the free movement of the two functional domains in the bivalent molecule (Figure 1B). The D1D2 fragment in this bivalent protein is expected to bind specifically with gp120 on the surface of HIV virions or HIV-infected cells (Figure 1C) and trigger formation of the gp41 PFI with the exposed hydrophobic grooves (Figure 1D), while the T1144 domain can bind to the exposed grooves on the gp41 NHR-trimer, resulting in rapid inactivation of the cell-free HIV-1 before its attachment to the target cell. Indeed, the 2DLT protein could effectively bind to both gp120 and gp41, block gp41 6-HB formation, inactivate cell-free HIV-1 and inhibit HIV-1 Env-mediated cell-cell fusion, but without the sCD4-mediated enhancing effects on HIV-1 infection. Therefore, this engineered bivalent molecule has substantial potential for development as an anti-HIV therapeutic for treatment of patients who fail to respond to the current anti-HIV drugs and as a topical microbicide for preventing sexual transmission of HIV.
Construction, expression and characterization of the bivalent fusion protein 2DLT
The expression plasmids pD1D2-PDI and p2DLT-PDI were constructed by linking the DNA fragment encoding D1D2 with those coding the 35-mer linker (GGGGS)7 and T1144 sequentially by three-step overlapping PCR using the corresponding primer pairs. The nucleotide sequences of the vectors were confirmed by DNA sequencing. The recombinant bivalent protein 2DLT and the control protein D1D2 (Figure 1B) were expressed in E. coli. To avoid the formation of inclusion bodies, we used the protein disulfide isomerase (PDI) chaperone-expression system since we and others have shown that PDI, as a fusion partner, could significantly increase the soluble expression of recombinant proteins in the cytoplasm of E. coli[21, 22], and we successfully obtained soluble D1D2 and 2DLT proteins. After purification, we analyzed these proteins with SDS-PAGE and Western blot. On the gels, two proteins migrated near the expected position of the proteins (Figure 1Ea, D1D2: ~23KD; 2DLT: ~29KD). Both displayed specific interaction with anti-CD4 polyclonal antibodies (pAb) (Figure 1Eb). While the anti-T1144 pAb bound with 2DLT at the predicted size, no band was revealed in the D1D2 lane (Figure 1Ec). Similarly, anti-CD4 pAb (T4-4) could react with both D1D2 and 2DLT, while anti-T1144 pAb was able to recognize 2DLT only in the ELISA. Notably, both D1D2 and 2DLT could react with the CD4-specific and conformation-dependent monoclonal antibody (mAb) Sim.4 (Figure 1F), suggesting that the soluble D1D2 and 2DLT may have a correctly folded conformation.
2DLT inactivated cell-free HIV-1 virions
To determine whether 2DLT could inactivate HIV-1 virions, we used PEG-6000 method, as previously described [23–26], to separate HIV-1 from the recombinant proteins 2DLT and D1D2, as well as the control peptides T1144 and T20, and then measured the residual infectivity of the treated HIV-1 particles. As shown in Figure 2 and Table 1, 2DLT inactivated cell-free HIV-1 Bal (laboratory-adapted R5 virus), HIV-1 IIIB (laboratory-adapted X4 virus), and primary R5 isolates with different subtypes (92US657, 93 MW959, and 92TH009) in a dose-dependent manner with 50% effective concentration (EC50) ranging from 17.3 to 78.6 nM, which is about 2- to 6-fold more potent than D1D2 against the corresponding viral strains. In contrast, T20 and T1144 at a concentration up to 500 nM did not show significant effects on virus inactivation. Different from the peptide HIV-1 fusion/entry inhibitors T20 and T1144, the bivalent protein 2DLT is capable of directly inactivating HIV-1, making it a good potential candidate for developing novel anti-HIV drugs.
Inactivation of HIV-1 by 2DLT. Recombinant proteins and peptides were added to HIV-1 Bal (A), HIV-1 IIIB (B), and 92US657 (C), respectively, followed by incubation for 60 minutes at 4°C. The mixtures were then cooled on ice before addition of PEG-6000 solution at a final concentration of 3% for separation of HIV-1 particles as described in the Methods. The HIV-1 particles containing pellets were re-suspended in tissue culture medium and titered for infectivity. The percentage of residual infectivity is shown in a scale. The data are representative of results from three similar experiments performed in triplicate (means ± SD).
Table 1 Inactivation of cell-free HIV-1 R5 and X4 strains by 2DLT
Like sCD4 and D1D2, 2DLT could bind to gp120 on the cell surface
To investigate the inactivation mechanism of 2DLT, we first determined whether the bivalent protein 2DLT retains the biological activities of CD4. As expected, 2DLT could bind to gp120 protein as strongly as sCD4 and D1D2, while T1144 was unable to bind with gp120 as shown in ELISA (Figure 3A). We then used SPR assays to determine whether the 35-mer linker L35 and the C-peptide T1144 in the bivalent protein would affect the binding affinity of the D1D2 domain in 2DLT to gp120. The results confirmed that 2DLT could interact with gp120 in a dose-dependent manner with high binding affinity (KD = 1.9e-8) comparable to D1D2 (KD = 2.1e-8), while T1144 exhibited no binding to gp120 (Figure 3B). Using a flow cytometry assay, we demonstrated that 2DLT could bind to the native gp120/gp41 (Env) expressed on the surfaces of CHO-WT cells as effectively as sCD4 and D1D2, while T1144 was unable to bind to the cell-expressed Env without sCD4 stimulation. None of these peptides or proteins could bind to CHO-EE cells that express no Env of HIV-1 (Figure 3C).
Binding of the soluble 2DLT and D1D2 proteins to gp120 protein or to gp120 expressed on the cell surface. A) Binding of sCD4, D1D2, 2DLT, T1144 to rgp120 as measured by ELISA. The data are representative of results from three similar experiments performed in triplicate (means ± SD). B) The binding affinity of sCD4 (a), D1D2 (b), 2DLT (c), and T1144 (d) to gp120 as determined by SPR assay. The recombinant gp120 (rgp120) was immobilized onto the CM3 sensor chip. The proteins and peptide at various concentrations were injected onto the surface. The affinity constant of each sample was calculated in one-site binding modes by BIAcore evaluation software. C) Binding of sCD4, D1D2, 2DLT, and T1144 to the HIV-1 Env (gp120/gp41) expressed on CHO-WT cell surfaces as determined by flow cytometry. CHO-EE cells expressing no HIV-1 Env molecule were used as control cells.
Like T1144, 2DLT bound to the groove on the gp41 PFI N-trimer and subsequently blocked gp41 6-HB formation
To determine whether 2DLT, like T1144, could interact with the exposed grooves on the gp41 N-trimer, we tested the binding activity of 2DLT to 5-helix, a mimic of N-trimer with exposed groove, using an ELISA. As shown in Figure 4A, 2DLT could bind to 5-helix as effectively as T1144, while D1D2 and sCD4 exhibited no significant binding, suggesting that the soluble recombinant protein 2DLT can function like T1144 to interact with the grooves of gp41 N-trimer formed at the PFI state.
Inhibition of gp41 6-HB formation by 2DLT. A) Binding of D1D2, 2DLT, and T1144 to the grooves on the 5-helix as assessed by ELISA. B) The inhibition of D1D2, 2DLT, and T1144 to the 6-HB formation between N36 and C34-biotin was detected by ELISA. Each sample was tested in triplicate, and the data are presented as means ± SD. C) The inhibition of D1D2 and 2DLT at graded concentration to the 6-HB formation between N36 and C34-FAM was determined by FN-PAGE. The peptide N36 was incubated with 2DLT or D1D2 at 37°C for 30 minutes before addition of the peptide C34-FAM at graded concentrations. After incubation for another 30 minutes, the mixtures were analyzed by FN-PAGE (panels a and b). The gels in panels a and b were stained with Coomassie Blue (panels c and d).
The gp41 6-HB formation is a critical step during HIV-1 fusion with the target cell. The peptides derived from the gp41 CHR, e.g. C34 and T1144 are able to bind with viral gp41 N-trimer to block the 6-HB core formation [19, 27]. Here, we used a sandwich ELISA and fluorescence native polyacrylamide gel electrophoresis (FN-PAGE) to determine if 2DLT, like T1144, possessed inhibitory activity on gp41 6-HB formation in a model system mimicking the gp41 6-HB core formation by mixing the gp41 N36 and C34 (or FAM-labeled C34) peptides at equal molar concentration [17, 28]. In the ELISA, 2DLT, like T1144, inhibited the 6-HB formation in a dose-dependent manner with an IC50 of 0.5 ±0.06 μM, while D1D2 protein at 10 μM exhibited no significant inhibition (Figure 4B). Similarly, 2DLT could effectively block 6-HB formation in a dose-dependent manner when it was tested at 5, 10, and 20 μM as shown in the FN-PAGE (Figure 4Ca and Cc, lanes 5 to 7), whereas D1D2 protein at the same concentrations showed no significant inhibition (Figure 4Cb and Cd, lines 5 to 7). The D1D2 and 2DLT bands were not observable on the gels because they carry net positive charges, like the N-peptide N36 (lane 1 in Figure 4C) and run in a reversed direction under the native gel condition as previously described [27, 29]. These results indicate that 2DLT can interact with the gp41 N-trimer and block the 6-HB core formation between viral gp41 NHR and CHR domains.
2DLT could disrupt the function of the CD4-induced gp41 PFI, and caused no significant enhancement of HIV-1 infection in CD4-/CCR5+cells
Recently, Haim et al. have demonstrated that binding of sCD4 or CD4-mimetics to HIV-1 gp120 could induce a short-lived PFI N-trimer, resulting in increased binding of the C34-Ig protein to the groove on the N-trimer, using a cell-based ELISA as described in Methods. Using a similar approach, we examined whether 2DLT may act on this intermediate to rapidly inactive viruses by the interaction of T1144 domain on it and NHR of gp41 on Env. Indeed, binding of C34 to HIV-1 Env was rapidly enhanced after D1D2 pulse, and then gradually decreased as the time of D1D2 pulse was prolonged at 25°C (Figure 5Aa) or 4°C (Figure 5Ab), which are coincident with the report by Haim et al. There was no C34 binding immediately after T1144 pulse. Strikingly, the binding ability of C34 to the pulsed Env was lost more rapidly in the 2DLT group than the D1D2 group at 25°C (Figure 5Aa) and 4°C (Figure 5Ab). These results suggest that because of the presence of T1144 in the bivalent molecule, 2DLT can decay the N-trimer-exposed PFI at a faster rate than D1D2. Therefore, we believe that 2DLT may have a mechanism of action different from D1D2 molecule in inactivating the CD4-induced gp41 PFI. The D1D2 domain in 2DLT may destabilize the gp41 PFI triggered by its binding to gp120, while its T1144 domain may interact with the exposed N-trimer in the viral gp41 to form heterogonous 6-HB, resulting in the stabilization of the gp41 PFI. It has been reported that sCD4 can modestly enhance the infectivity of some HIV-1 strains at suboptimal concentrations in CD4-CCR5+ cells because sCD4 may efficiently replace CD4 on cell surface to drive infection of the CD4-negative and CCR5-positive cells [30]. Here, we investigated whether 2DLT could enhance infectivity of CCR5-using HIV-1 strain Bal in Cf2Th-CCR5 (CD4-/CCR5+) cells, using D1D2 and T1144 as controls. As shown in Figure 5B, D1D2 at the concentrations from 5 to 200 nM significantly enhanced the infection of the virus in the CD4-/CCR5+ cells, while 2DLT and T1144 at the same concentrations showed no enhancement of HIV-1 infection, suggesting that 2DLT exhibits no enhancing effect on HIV-1 infectivity in CD4-/CCR5+ cells.
2DLT-mediated rapid decay of the CD4-induced gp41 PFI and lack of D1D2-induced enhancement of HIV-1 infection in CD4-/CCR5+cells. A) Decay of the gp41 PFI at 25°C (a) and 4°C (b) after pulse activation with D1D2 (2.5 μM), 2DLT (2.5 μM) or T1144 (2.5 μM) was measured by a cell-based ELISA, as described in the Methods. B) The infectivity of HIV-1 Bal (100 TCID50/well) in Cf2Th-CCR5 (CD4-/CCR5+) cells in the presence of D1D2, 2DLT or T1144 was determined by ELISA for p24 antigen production as described in the Materials and Methods.
2DLT inhibited HIV-1 infection and HIV-1-mediated cell-cell fusion
Subsequently, we determined the inhibitory activities of 2DLT against HIV-1 IIIB-mediated cell-cell fusion and against infection by laboratory-adapted HIV-1 IIIB (X4) and Bal (R5) strains in MT-2 and TZM-b1 cells, respectively, as well as several primary HIV-1 isolates in PBMCs using D1D2, T1144 and T20 as controls. Like T1144 and T20, 2DLT also exhibited potent inhibitory activities against HIV-1 IIIB-mediated cell-cell fusion (IC50 = 19.03 nM) and infection by HIV-1 strains IIIB (IC50 = 5.64 nM) and Bal (IC50 = 10.78 nM), which are about 5- to 15-fold more potent than D1D2 (Figure 6). Similarly, 2DLT could also significantly inhibit infection by the primary HIV-1 isolates tested at a low nanomolar level (Table 2). Notably, 2DLT displayed much higher anti-HIV-1 activity than D1D2 in all assays. These results suggest that 2DLT, in addition to its HIV-1 inactivation function, can also serve as an HIV-1 fusion/entry inhibitor, like T20, against a broad spectrum of HIV-1 strains, irrespective of co-receptor usage.
Inhibition of 2DLT on HIV-1 infection. The infectivity of IIIB (subtype B, X4) in MT-2 cells (A) or HIV-1 Bal (subtype B, R5) in TZM-bl cells (B) in the presence or absence of the fusion protein (D1D2 or 2DLT) or peptide (T20 or T1144) was detected by using in-house p24 kits or Promega's luciferase kits, respectively.
Table 2 Inhibitory activity of the recombinant proteins and peptides on HIV-1-mediated cell-cell fusion and HIV-1 replication
To determine the effect of preincubation of the virus with an inhibitor on the inhibitor's anti-HIV-1 activity, we preincubated HIV-1 Bal virus with 2DLT, D1D2, T1144 or T20 for 0, 15, 60 and 240 minutes, respectively, before addition of the mixture to TZM-bl cells. Their inhibitory activity on HIV-1 Bal infection was then tested. We found that the potency of 2DLT remarkably increases with increasing preincubation times. After preincubation for 240 minutes, the inhibitory activity of 2DLT was increased about 4-fold, while that of T1144, D1D2 and T20 was increased only about 0.3- to 0.6-fold (Table 3). This result suggests that preincubation of 2DLT with HIV-1 may trigger the increased exposure of the gp41 PFI, because of the increased binding of D1D2 domain in 2DLT with gp120 on virions, resulting in the increased binding of its T1144 domain to the exposed gp41 PFI and enhanced inhibition of HIV-1 infection.
Table 3 Effect of pre-incubation of the virus with inactivators or inhibitors on inhibition of HIV-1 replication
Soluble CD4 (sCD4) was previously recognized as a potential HIV-1 inactivator since it can bind to gp120 for inducing inactivation of HIV-1 virions. However, a very high concentration of sCD4 is required to neutralize infection by primary HIV-1 isolates [14, 31, 32]. Even worse, sCD4 at low concentration can enhance HIV-1 infection of CD4-/CCR5+ cells [14]. To improve sCD4-mediated HIV-1 neutralizing activity, several groups have constructed hybrid proteins by fusing CD4 or D1D2 with human IgG (CD4-IgG2 or PRO 542) [33, 34] or with a monoclonal antibody (mAb) specific for the CD4-induced epitope, such as 17b (sCD4-17b) [35]. In a clinical trial, CD4-IgG2 treatment led to about 0.5 log10 reduction in viral load [34]. However, CD4-IgG fusion proteins have two limitations. First, the molecule is too large to be expressed in a large quantity. Second, the inactivator targeting only gp120 may not be very effective in rapid decay of the gp41 PFI, a critical step of inactivating HIV-1 [14]. Therefore, the CD4-induced gp41 PFI is expected to be a novel target for development of HIV inactivators with improved efficacy.
In this study, we engineered a bivalent HIV-1 inactivator, 2DLT by targeting CD4-induced gp41 PFI with exposed grooves on the NHR-trimer. It consists of a D1D2 fragment of CD4, a 35-mer linker, and a C-peptide T1144 (Figure 1B). As expected, 2DLT could effectively inactivate HIV-1, including both laboratory-adapted and primary strains with different subtypes and tropism (Figure 2 and Table 1). It bound to gp120 via its D1D2 domain (Figure 3A-C) and then interacted with the exposed grooves on the NHR-trimer via its T1144 domain, resulting in a rapid decay of the gp41 PFI (Figure 5A). Unlike sCD4 and D1D2, 2DLT did not enhance HIV-1 infection in CD4-/CCR5+ cells (Figure 5B). Like T1144, 2DLT could also bind to the exposed groove on the gp41 NHR-trimer (Figure 4A) and inhibit the gp41 6-HB core formation (Figure 4B and C), as well as block HIV-1 fusion and replication through its T1144 domain (Figure 6 and Table 2). The bivalent HIV-1 inactivator 2DLT is able permanently to inactivate HIV-1 at any time it comes into contact with the virions, while T20 can only inhibit HIV-1 fusion within several minutes when the virion or virus-infected cell attaches to the target cell (Figure 7). Compared with the non-ionic surfactant-based virus inactivators, such as N9 [36, 37] and C31G [38, 39], 2DLT protein has much lower cytotoxicity, with an in vitro CC50 (50% cytotoxicity concentration) value greater than 100 μM on the cells susceptible to HIV-1 infection and reproductive tract epithelial cells (data not shown), and better specificity since it specifically interacts with the HIV-1 Env, rather than other components that are also present in host cells [40, 41]. Compared with sCD4 or D1D2, 2DLT can decay the CD4-induced gp41 PFI much more rapidly and efficiently through the interaction of its T1144 domain with the exposed grooves on the NHR-trimer.
The putative mechanisms of action of 2DLT to inactivate virions and to inhibit viral fusion and entry. 2DLT binds to gp120 via its D1D2 domain and induces the exposure of the grooves of the gp41 trimer, which subsequently binds the T1144 domain in 2DLT, leading to irreversible inactivation of HIV-1 (model a). Like T1144, 2DLT may also directly bind to gp41 NHR via the T1144 domain to inhibit HIV-1 fusion with the target cell (model b).
Similar to several gp120-specific mAbs, sCD4 was shown to modestly enhance the infectivity of HIV-1 at suboptimal concentrations in CD4-/CCR5+ cells [30], which is more prominent in some simian immunodeficiency virus (SIV) and SIV-related HIV-2 strains [14, 42–45]. This phenomenon was explained by the hypothesis that sCD4 can efficiently replace cell-surface CD4 to drive virus infection in CD4-negative and CCR5-positive cells [30]. In the present study, we found that the soluble D1D2 molecule could enhance infectivity of a CCR5-using HIV-1 strain Bal in Cf2Th-CCR5 (CD4-/CCR5+) cells at the concentration from 5 to 200 nM, while 2DLT and T1144 exhibited no enhancement of HIV-1 infection at the same concentration range. This suggests that unlike the soluble D1D2 protein, the D1D2 domains in 2DLT may not be able to replace CD4 on cell surface to drive HIV-1 infection in the CD4-/CCR5+ cells or the rapid decay of the gp41 fusion-intermediate mediated by 2DLT results in the abolishment of the potential enhancement of HIV-1 infection.
We have demonstrated that like the isolated T1144 peptide, the T1144 domain in 2DLT could also effectively inhibit HIV-1 infection and HIV-1-mediated cell-cell fusion (Figure 6 and Table 2). The dual functions of 2DLT make it superior to the other kind of inactivators (such as N9 or C31G) since if the HIV-1 virions are not completely inactivated, 2DLT is able to block the viral fusion and entry with its T1144 domain.
We have not tested the ability of 2DLT to induce drug-resistant mutations in HIV-1 variants because it will be difficult to interpret the results from the HIV entry inhibitor-induced mutation studies. For example, people may generally believe that sCD4 should induce drug-resistant mutations in the CD4-binding site in viral gp120. However, the mutation sites in the sCD4-resistant HIV-1 variants were mainly located in the V1, V2, and V3 loops in gp120 and the NHR region in gp41 [46]. Similarly, the mutation sites in the HIV-1 variants resistant to T2635, an analogous peptide of T1144, were not located in the hydrophobic pocket of the gp41 NHR domain, which is the main target site of T2635, T1144 and other C-peptides with the pocket-binding domain, but rather in other regions in gp120 and gp41 [47]. This could be possibly explained by the fact that both the CD4 binding site in gp120 and the hydrophobic pocket in gp41 are highly conserved, and the viruses with mutations at these sites may not survive due to the loss of the critical viral functions. Therefore, both D1D2 and T1144 may possess high genetic barrier to resistance. We expect that 2DLT may have a higher genetic barrier to resistance than D1D2 and T1144 when each of them is used alone since 2DLT, like the combination of D1D2 and T1144, is able to simultaneously interact with two target sites, while D1D2 or T1144 can bind to only one target site.
We have designed and engineered a bivalent protein 2DLT, which can speed the T1144 domain-mediated decay of the gp41 PFI induced by binding of D1D2 domain to gp120, resulting in rapid inactivation of HIV-1 virions. It may also inhibit HIV-1 fusion and entry through its T1144 domain in case when the HIV-1 virion escapes from 2DLT-mediated inactivation. Both of its binding sites in gp120 and gp41 are highly conserved, and it can actively attack the virus in the absence of host cells, making it a promising candidate for further development as a therapeutic for the treatment of HIV/AIDS or as a topical microbicide for preventing sexual transmission of HIV.
CHO cells stably transfected with either the HIV-1HXB2 Env-expressing vector pEE14 (CHO-WT) or control pEE14 vector (CHO-EE) were cultured in Glutamine-deficient minimal essential medium (GMEM-S) containing 400 μM Methionine sulfoximine (Sigma, St. Louis, MO). Cf2Th/syn CCR5 cells stably expressing CCR5 receptors were cultured in DMEM complemented medium with 10% FBS, pen/strep, 500 μg/ml G418, 500 μg/ml zeocin and 3 μg/ml puromycin (Invitrogen, Carlsbad, CA). MT-2 and TZM-b1 cells, as well as laboratory-adapted HIV-1 strains IIIB and Bal, and primary HIV-1 isolates were obtained from the AIDS Research and Reference Reagent Program of NIH. The N-peptide N36 (aa 546–581), and C-peptides C34 (aa 628–661), T1144, and T20 (aa 638–673), used in this study were derived from the NHR and CHR, respectively, of the HIV-1HXB2 gp41 (Figure 1A). These peptides (> 95% purity) were synthesized by a standard solid-phase FMOC method using an Applied Biosystems model 433A peptide synthesizer.
Construction of vectors encoding D1D2 and 2DLT
To create the expression plasmid pD1D2-PDI and p2DLT-PDI, DNA fragments encoding D1D2 (aa 1–185 of CD4), the 35-mer linker (GGGGS)7, and T1144 were linked together by three-step overlapping PCR. We took p2DLT-PDI as an example. First, the D1D2 (FD1D2his: 5′-CGCGGATCCCATCACCATCACCATCATAAGAAAGTGGTGCTG-3′, RD1D2: 5′-CACTTCCTCCTCCTCTATGCTGGAGGCCTTCTGGAA-3′), L35 (FL35: 5′-GGAGGAGGA GGAAGTGGCGGCGGCGGCTCGGGTGGTGGTGGTTCTGGAGGTGGCGGTAGCGGAGGTGGAGGTAGTGGAGGC-3′, RL35: 5′-GCTACCTCCGCCTCCCGAACCTCCGCCTCCA CTACCTCCACCTCCGCTACCGCCACCTCCAGAACCACCACCACCCGAG-3′) and T1144 (FT1144: 5′-GAGGCGGAGGTAGCACGACCTGGGAAGCATGGGACAGAGCTATTGCTG AATACGCAGCTAGGATAGAAGCTTTACTCAGAGCTTTA-3′, RT1144: 5′-CGGAGAT CTCTATAATTCCCTTAAGGCTGCTTCATTCTTTTCTTGCTGTTCTTGTAAAGCTCTGAGTAAAGCTTCTATCC-3′) DNA fragments were genera-ted by overlapping PCR using the corresponding primer pairs. Second, the DNA fragments coding for L35 and T1144 were linked by overlapping PCR with the primers FL35 and RT1144. Third, the two DNA fragments encoding D1D2 and L35-T1144 were linked by overlapping PCR with the DNA fragment D1D2 and the primers FD1D2his and RT1144. Finally, the amplified DNA fragment coding for 2DLT was digested by BamHI and EcoR I and inserted into the expression vector pGEX-6p-1 to generate the p2DLT plasmid. In order to prevent the formation of the inclusion bodies in E. coli, we inserted a protein disulfide isomerase (PDI) [21, 22] DNA sequence (aa 18–508) with PreScission protease cutting site (called ppase site) in the N terminus into the EcoR I and Xho I sites located at the C terminus of His-2DLT gene in the plasmid p2DLT to extend the GST-his-2DLT reading frame, resulting in the generation of chimeric GST-his-2DLT-ppase-PDI. This plasmid is called p2DLT-PDI. The sequences were confirmed by DNA sequencing.
Protein expression and purification
To express D1D2 and 2DLT fusion proteins, Escherichia coli strain Rosetta 2 (DE3) pLysS (Novagen) was transformed with pD1D2-PDI and p2DLT-PDI, respectively, cultured at 37°C to OD600 = 0.4, then induced at 16-22°C for 8–12 h with 0.4 mM IPTG. The cells were harvested and lysed by sonication in the presence of protease inhibitor mixture (Roche). After centrifugation, supernatants containing the fusion protein were collected. The protein was purified with Glutathione-Sepharose 4B affinity columns and cleaved with PreScissionTM Protease (GE Healthcare). These fusion proteins were purified by His·Bind® Purification Kit (Novagen) and fast protein liquid chromatography (FPLC), and then analyzed by SDS-PAGE.
SDS-PAGE and Western blot analysis
Purified fusion proteins were analyzed by SDS-PAGE as previously described [48]. Briefly, D1D2 or 2DLT was mixed with 4X SDS sample buffer (Novagen, Gibbstown, NJ) and boiled for 5 minutes or kept at room temperature (RT) before loading onto a 10-20% Tricine-Glycine gel (Invitrogen, Carlsbad, CA). The electrophoresis was conducted in SDS-PAGE running buffer with 125 V constant voltage at 4°C. In Western blot, the anti-human CD4 and anti-T1144 polyclonal antibodies were used.
Enzyme-linked immunosorbent assay (ELISA)
D1D2 and 2DLT fusion proteins were characterized by ELISA as previously described [49]. Briefly, they were coated onto a 96-well polystyrene plate (Costar, Corning Inc., Corning, NY) (10 μg/ml in 0.1 M Tris–HCl, pH 8.8), which was blocked with 2% non-fat milk in PBS. The polyclonal antibodies against CD4, conformation-dependent monoclonal antibody against CD4 (Sim.4) and anti-T1144 polyclonal antibody, respectively, were added to the plate. After incubation at 37°C for 60 min, horseradish peroxidase (HRP)-labeled antibodies (ZYMED Laboratories, S. San Francisco, CA) and the substrate TMB (Sigma) were added, sequentially. The binding of D1D2 and 2DLT to gp120 or gp41 NHR, and their inhibitory activity on gp41 6-HB formation were determined by ELISA as previously described [50].
The binding of D1D2 and 2DLT to gp120/gp41 expressed on the cell surface was detected by flow cytometry as previously described [49, 51]. Briefly, the cultured CHO-WT (with Env) and CHO-EE (with no Env) cells were detached from plate and washed with wash buffer (PBS containing 5% GBS) three times and incubated with the testing protein for 1 h at 4°C. After three washes, anti-CD4 or anti-T1144 polyclonal antibody was added for 1 h at 4°C. After three washes, FITC-conjugated anti-rabbit or mouse antibody was added and incubated for 1 h at 4°C. After three washes, the cells were examined by flow cytometry and the fluorescence intensity was recorded by FACSCalibur (Becton Dickinson).
Surface plasmon resonance (SPR) assay
The binding affinity of D1D2 and 2DLT to gp120 was measured by SPR using the BIAcore3000 system (Pharmacia, Piscataway, NJ), following the Manual of the Biomolecular Interaction Analysis (BIA) Technology as described previously [52]. Briefly, gp120 (100 μg/ml) was immobilized onto the CM3 sensor chip by amine coupling, and the unreacted sites were blocked with ethanolamine. The dissociation reaction was done by washing with running buffer (10 mM HEPES pH7.4 containing 0.15 M NaCl, 3.4 mM EDTA and 0.005% v/v surfactant) for at least 2 min.
Fluorescence native polyacrylamide gel electrophoresis (FN-PAGE)
FN-PAGE for detecting 6-HB formation as described before [27]. Briefly, a testing peptide or protein (100 μM) was pre-incubated with N36 (100 μM) at 37°C for 30 min, followed by addition of C34-FAM (100 μM) at 37°C for 30 minutes. The mixtures were added into Tris-glycine native sample buffer (Invitrogen, Carlsbad, CA). The samples (20 μl) were then loaded onto Tris-glycine gels (18%; Invitrogen, Carlsbad, CA), which were run under 120 V constant voltage at room temperature for 1 h. The gels were stained and visualized with the FluorChem 8800 Imaging System (Alpha Innotech Corp., San Leandro, CA) using a transillumination UV light source with excitation wavelength at 520 nm and then with Coomassie Blue.
Inhibition of HIV-1 infection
Inhibitory activities of D1D2 and 2DLT on HIV-1 infection were determined as previously described [53, 54]. For inhibition of HIV-1 IIIB (subtype B, X4) infection, 100 TCID50 of the virus was added to 1 × 104/ml MT-2 cells in RPMI medium 1640 containing 10% FBS in the presence or absence of the test peptide or protein overnight. The culture supernatants were removed, and fresh media were added. On the fourth day post-infection, culture supernatants were collected for detection of p24 antigen by ELISA. For inhibition of infection by the HIV-1 strain Bal (subtype B, R5), TZM-bl cells (1 × 105/ml) were pre-cultured overnight and infected with Bal at 100 TCID50 in the presence or absence of the test peptide or protein overnight. The cells were harvested and lysed on the fourth day post-infection with lysing reagent. The luciferase activity was analyzed using a luciferase kit (Promega, Madison, WI) and a luminometer (Ultra 386, Tecan, Durham, NC) according to the manufacturer's instructions. For testing the effect of pre-incubation times on the inhibitory activity of 2DLT, the TZM-b1 assay was performed after pre-incubation of the HIV-1 Bal virus with the inhibitors for 0, 15, 60 and 240 min. For inhibition of primary HIV-1 isolate infection, peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors. The PHA-stimulated cells were infected with a primary HIV-1 isolate at a multiplicity of infection (MOI) of 0.01 in the absence or presence of peptide or protein at graded concentrations. The supernatants were collected on the 7th day post-infection and tested for p24 antigen by ELISA as previously described [54, 55]. The percent inhibition of p24 production or luciferase activity was calculated.
Inactivation of HIV-1 virions
The virus inactivation by D1D2 and 2DLT was determined as previously described [25, 56, 57]. Briefly, 100 μl of the protein or peptide at graded concentration were added to 100 μl of an HIV-1 strain (200 TCID50/ml), followed by incubation at 4°C for 1 h. Then, PEG-6000 was added to the treated virus at final concentration of 3%, 4°C for 1 h. The mixture was centrifuged on a microfuge at 15,000 rpm for 30 min. The supernatants were removed and the pellet was washed with 3% PEG in PBS containing 10 mg/ml BSA. The viral pellet was then resuspended in 100 μl of PBS, before addition of 100 μl MT-2 or TZM-bl cells (1 × 105/ml). After cultur at 37°C for 3 days, p24 production in MT-2 cell culture or luciferase activity in TZM-bl cell culture was tested as previously described [58–60].
Cell-based ELISA
To measure the effects of D1D2 and 2DLT on induction of short-lived PFI of HIV-1 Env, we used a cell-based ELISA as previously described [14]. Briefly, CHO-WT cells steadily expressing HIV-1 Env were seeded in 96-well plates (5 × 104/well). Cells were then harvested and washed twice with blocking buffer (35 mg/ml BSA, 10 mg/ml non-fat dry milk, 1.8 mM CaCl2, 1 mM MgCl2, 25 mM Tris, pH 7.5 and 140 mM NaCl). For pulse activation experiments, the cells were incubated with D1D2 (2.5 μM) or 2DLT (2.5 μM) suspended in blocking buffer for three minutes, washed three times with blocking buffer and incubated with the C34-biotin (2 μM). To study the temperature dependence of NHR groove exposure, the D1D2- or 2DLT-pulsed cells were incubated at the requisite temperature for different lengths of time. The cells were subsequently returned to room temperature for incubation with C34-biotin. Cells were then washed four times with blocking buffer and four times with washing buffer (140 mM NaCl, 1.8 mM CaCl2, 1 mM MgCl2 and 20 mM Tris, pH 7.5). A horseradish peroxidase-conjugated streptavidin (ZYMED Laboratories, S. San Francisco, CA) was then incubated with the samples for 45 minutes at room temperature. Cells were washed 5 times with blocking buffer and 5 times with washing buffer. HRP enzyme activity was determined after the addition of 33 μl per well of a 1:1 mixture of Western Lightning oxidizing and luminal reagents (Perkin Elmer Life Sciences) supplemented with 150 mM NaCl. Light emission was measured.
HIV-1 infectivity in CD4-/CCR5+cells treated by D1D2 or 2DLT
The effects of D1D2 and 2DLT on HIV-1 infection of CD4-/CCR5+ target cells were evaluated as previously described [14]. Briefly, HIV-1 Bal (100 TCID50/well was cultured with Cf2Th-CCR5 cells (1 × 106 cells per well) at room temperature for 1 h, then was resuspended, followed by addition of D1D2 or 2DLT at different concentrations. After a short centrifugation, the mixture was incubated for 8–12 h at room temperature. The viral infectivity was measured three days later.
Inhibition of HIV-1-mediated cell-cell fusion
HIV-1 mediated cell-cell fusion was measured with a dye transfer assay as previously described [27, 29]. In brief, the HIV-1IIIB chronically infected H9 (H9/HIV-1IIIB) cells were labeled with Calcein-Am (Molecular Probes, Inc., Eugene, OR). After washes, the fluorescence-labeled H9/HIV-1IIIB cells were incubated with MT-2 cells at 37°C for 2 h in the absence or presence of an inhibitor at a graded concentration. The percentage of fused cells was counted under a fluorescence microscope (Zeiss, Germany), and the 50% inhibitory concentration of each drug was calculated with the Calcusyn software program [27, 29].
CHR:
C-terminal heptad repeat
NHR:
N-terminal heptad repeat
HR:
Heptad repeat
PFI:
Prehairpin fusion-intermediate
Native-PAGE:
Native polyacrylamide gel electrophoresis
6-HB:
Six-helix bundle
sCD4:
Soluble CD4
CC50:
50% cytotoxicity concentrations.
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MT-2, TZM-b1, CHO-WT, CHO-EE, and Cf2Th/syn CCR5 cells, the HIV-1IIIB chronically infected H9 cells, and HIV-1 strains were obtained from the AIDS Research and Reference Reagent Program, NIH, USA. This work was supported by grants from the National Natural Science Foundation of China (#81173098 to SJ and #81102476 to LL), 973 Programme of China (#2012CB519001 to SJ) and "Chen Guang" Project of SMEC and SEDF (11CG03) to LL.
Key Laboratory of Medical Molecular Virology of Ministries of Education and Health, Shanghai Medical College and Institute of Medical Microbiology, Fudan University, Shanghai, 200032, China
, Yuan Li
& Shibo Jiang
Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, 10065, USA
, Chungen Pan
, Hong Lu
, Wu He
The Institute of Human Virology, Key Laboratory of Tropical Disease Control of MOE, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, 510080, China
Chungen Pan
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Correspondence to Shibo Jiang.
SJ conceived the idea and designed research. LL, CP, YL, HL, and WH performed research. LL, and SJ analyzed the data and wrote the paper. All authors read and approved the final manuscript.
Lu, L., Pan, C., Li, Y. et al. A bivalent recombinant protein inactivates HIV-1 by targeting the gp41 prehairpin fusion intermediate induced by CD4 D1D2 domains. Retrovirology 9, 104 (2012) doi:10.1186/1742-4690-9-104
Six helix bundle
HIV-1 fusion inhibitor