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(CBS News) Maroon 5 frontman/"The Voice" judge Adam Levine and his girlfriend, model Anne Vyalitsyna, have ended their two-year relationship.
According to the Victoria's Secret and Sports Illustrated swimsuit model, the split was amicable.
"Adam and I have decided to separate in an amicable and supportive manner. We still love and respect each other as friends. I wish him all the best," Vyalitsyna (who is also known as Anne V) said in a statement, according to People.
Levine, 33, performed with Maroon 5 at last year's Victoria's Secret fashion show, where he walked down the runway with Vyalitsyna, 26. They reportedly met at a Sports Illustrated swimsuit issue release party in 2010.


text: Hello, here are some (really crap!) pics of my new pochette. Thanks to John for authenticating it for me x It is is fantastic condition inside and out, almost new, and I got it for £170. No dustbag or anything though. Excuse the pics - i only have a camera phone. The thing is, now that I have it, I don't think I actually like it! Does it look silly? I just don't know. I'm not sure about the colours either.

subset: commoncrawl


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Ahmedabad, April 29: The National Institute of Design in Ahmedabad is redesigning its future for greater interaction with industry and, consequently, getting to grips with real-life design demands.
No wonder, several top guns of Indian industry including Infosys chairman Narayana Murthy, Adi Godrej, Ratan Tata, Philips Indias Ramachandran, Hewlett-Packards Warren Greving and Jindal Groups director Deepika Jindal visited the institute in the last one year to see things for themselves.
Industry has realised that the right design makes all the difference in a technology-driven world and ensures competitive advantage. As a result, both good design and design institutes such as NID are becoming indispensable to industry.
The institute, too, is keen to cash in on the industrys interest and develop an interactive nexus with industry to sell its core competence of industrial design.
Till recently, most of the countrys automobile designs were imported despite the existence of a vast pool of indigenous talent.
Though design imports are continuing, there is a visible change with the countrys automobile industry seeking indigenous inputs.
Maruti, Mahindra and Telco are all beginning to dip into the pool of local talent to develop and redesign certain components of their vehicles.
Along with initiatives such as the Confederation of Indian Industrys design summit with NID in Delhi in December, the industrys interest has saved it some money and boosted local designers confidence.
The summit on Competitive Advantage through Design was the second in a series undertaken by the institute. The basic idea of holding such summits was to bring industry and top design academicians together on a common platform, an institute official said.
Encouraging the NID-industry nexus, students Ankur Sharma and Charles Tirkey were conferred the first and first runners-up prizes at the first K.C. Mahindra Award for Excellence in Automotive Design in Mumbai on April 23.
The summit was a give-and-take between industry and NID, with both gaining from the exercise.
While industry learnt about various design concepts and their conversion into products, the academicians gained in experience and came face to face with the real requirements of industry.
As many as 22 senior product-design students and two faculty members participated in a workshop with Telco engineers in Pune in December. The aim was to develop internal and external styling and designs for a range of buses that Telco will launch shortly.
The engineers were so impressed that Telco chief Ratan Tata came calling at the institutes Ahmedabad campus with a high-power delegation on January 16.
The Tata group then decided to work in partnership with the institute and establish a user interface design research chair at NID.
The institute proposed an NID-Tata three-week interdesign workshop in Ahmedabad, where multidisciplinary groups of designers from the country and abroad would generate design ideas in keeping with global trends.
Ratan Tatas visit generated such a buzz that the next month, a high-level team from General Motors followed, looking for local design talent.


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When I was younger (like 32 instead of 39), my mother and I would often get into the same squabble.
She would tell me to pray about something– like that I would suddenly be given a raise- and I would try to explain that that wasn't how raises were given where I worked and when that didn't work, I would just tell her my truth: that I didn't think it was appropriate to personally pray for favors from God. Because I am fully incapable of lying (this is far less of a gift than you think. Especially back then, it was absolutely impossible for me to just nod politely and move on if I had a different viewpoint. I had to explain myself. The good news is that I am not so stringent about all my truth telling now because I've realized a multitude of things and so now I can just say 'Yes, ma'am' and not feel wracked with guilt that I am a liar. I would tell you all these details but this post isn't about the nuances of truth telling so I'll save that for another day), I would explain to my mom that there were just two types of personal prayers I was comfortable offering up to the heavens, to the universe, to the God of my understanding: prayers for strength (or as I later learned, what Anne Lamott described as Help Me, Help Help Me) and prayers of thanks (in Anne Lamott speak: Thank You, Thank You, Thank You. As an additional aside, Lamott- whose nonfiction I adore-just released a new book called Help Thanks Wow: The Three Essential Prayers). Needless to say, my personal prayer philosophy didn't go over all that well with my mom. She wanted me to ask for specific stuff. As a person who had very thoughtfully etched out her faith, I wanted to know that I was given what I was given for a reason and it was my job in life to navigate it thoughtfully. Sometimes, I needed help and when I did, I would pray for guidance and grace but never for God to give me, let's say, a C in Calculus instead of the F I was currently earning. Sometimes, I was just bowled over with amazement at what I had been given and, in those instances, I didn't want to be accused of not noticing. Sometimes, I just said Thanks out loud as I wandered through life.
And today's spark is about the importance of saying that thanks.
There is something about gratitude that just makes our hearts happy, don't you think? Gratitude fill us up, shifts our perspective, makes us shine brighter. Gratitude is good stuff.
Years ago, I received a random, unsigned thank you note in the mail- not for something I specifically did but for how I moved through the world. That thank you note, as you might imagine because this is how these things work, came at the end of one of the crappiest weeks I had had as a teacher. Exhausted and defeated, I read that note and felt something in me shift. Maybe I am not so bad, I thought. Maybe I do have plenty to offer, despite the evidence I had been trying to mount against myself from that week. That thank you note? It saved me from myself at a moment when I felt like throwing it all in. That's powerful.
This year, one of my goals for my birthday list is to write 39 thank you notes. Now, in general, I LOVE writing thank you notes. I get a gift and even if the gifter says, "Now, no thank you notes!", I still write one. So thank you notes are kinda my thing. But my 39 thank you notes are going to be different. I am not going to thank people for things they have given me or done for me. I am going to thank them for how they are in the world and how that affected me.
I don't expect you to write 39 notes, but, this week, I do want you to write one note. A handwritten note on actual paper and then I want you to mail it. Send a little gratitude out there because, at the very least, it will make you feel better. But the fact is that it will likely do much more.


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- Here Kitty Said:
It it were me, I wouldn't worry about measuring. In the layout in the link, it doesn't look like the strips are even. I did a similar layout to this about 4 years ago, and I just started layering strips that I cut with my paper cutter. I didn't measure, just eyeballed it.
Little Miss Sunshine By Here Kitty
Little Miss Sunshine By Here Kitty
YES! That is exactly what I meant. Just a few scattered "rays" out from my center picture!
Thanks everyone so much for the ideas! I can't wait to try them!


text: What do you think about READE PM vernis in new stunning red? I try to find some pieces of vernis line in new red but nothing catch my eyes. Only this one that I think it should fit my style. As asking for the opinion about agenda and many members love new red vernis so I need to have a matching. As the top of bag is open so it may not be a safe bag? I also think that roxbury is nice and I may adjust the extra strap that provided with the bag to use with my little pouchette . What do you think? Anyway, I like READE mor than roxbury. What do you think about it?


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The book of Ruth tells of a young Moabite widow who, out of love for her widowed Israelite mother-in-law, abandoned her own culture, declaring, "Your people shall be my people, and your God my God" (1:16). Though she was destitute and needing to rely on the kindness of others, Ruth's disposition and character captured the attention of Boaz, a close relative of her deceased husband. Boaz fulfilled the role of kinsman-redeemer and took Ruth as his wife. Ruth serves as a wonderful example of God's providential care of his people, and of his willingness to accept Gentiles who seek him. Ruth was an ancestor of Christ. The author is unknown, but the genealogy at the end suggests that it was written during or after the time of David. (Ruth, 2001. The Holy Bible: English Standard Version, Wheaton: Standard Bible Society.)
Ruth 1.1-5; Story; Naomi Widowed
Ruth 1.6-18; Story; Ruth's Loyalty to Naomi
Ruth 1.19-22; Story; Naomi and Ruth Return
Ruth 2.1-23; Story; Ruth Meets Boaz
Ruth 3.1-18; Story; Ruth and Boaz at the Threshing Floor
Ruth 4.1-12; Story; Boaz Redeems Ruth
Ruth 4.13-17; Story; Ruth and Boaz Marry
Ruth 4.18-22; Story; The Genealogy of David


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Claim: Earthquakes are most likely to happen when the weather is hot and dry.
Origins: Earthquakes are among the most fearsome natural disasters known to the planet. In a matter of seconds, they can bring thousands of lives to an end, cause devastating amounts of destruction, and even temporarily render the ground that is normally solid beneath our feet into a liquid, goopy mass. Unlike other natural killer phenomena such as hurricanes, tornadoes, and tsunamis, they strike without advance notice.
That earthquakes occur without warning makes them especially terrifying, because it's impossible to safeguard oneself and one's loved ones from harm that is randomly
Some regard hot and dry weather as "earthquake weather," a supposed precursor to a large quake. Yet there's not clear cut agreement on this combination, because others deem "earthquake weather" to be when it's hot and humid. This
belief about the weather conditions above the surface's affecting what's going on beneath is an old one, dating to the days of the ancient Greeks. Posited Aristotle, who believed quakes were caused by winds trapped underground, less wind above the surface must mean more below, hence earthquakes were more likely when the air was still.
While a number of major quakes have occurred when the weather was hot and dry (as have many when it was hot and sultry), the sorry truth is there's no correlation between weather and the occurrence of these devastating geologic events. Earthquakes begin miles below ground level, where the weather above is too far away to play any part in their occurrence. Moreover, they're caused by tectonic plates rubbing against one another, something that happens when the plates shift. Said shifting has been going on for millions of years, including during ice ages, periods when the Earth was a hot, humid swamp, and everything in between.
Another widespread belief is that earthquakes are far more likely to occur during the early morning, specifically within an hour of dawn. Proponents of that theory hold that at
that time of the day, when the ground is the most cool, activity along fault lines increases, likely as a result of contraction brought about by the drop in temperature.
As mentioned in relation to the "earthquake weather" belief, whatever is happening on the surface of the Earth is too far away from where quakes take place to have any effect on when or if temblors occur. While there have been some memorable quakes that fit the dawn timeframe (e.g. the 1994 Northridge quake, a
Some folks place their faith in the unusual reactions of animals, recalling that before at least a few major quakes, strange behavior was observed in critters local to the event. However, that animals have occasionally acted strangely just prior to the onset of a devastating earthquake doesn't mean they always do. A great many quakes have happened where everyone's cats, dogs, and even the wild critters in the field continued behaving in quite normal fashion right up until the moment that they hit.
As to why folks tend to believe animals can sense impending quakes, or the weather or time of day having something to do with an increase in seismic activity, humans look to make sense of their world and the sometimes terrifying forces at work within it through application of patterns. They examine tragedies in search of elements common to them, then look to apply whatever patterns they think they've found to future events, in hopes of seeing trouble coming before it arrives and thus of having a chance of getting out of its way.
Earthquakes, however, are not predictable, neither by lore nor through science. The Southern California Earthquake Center says of the notion that scientists have come up with a mechanism for determining when and where an earthquake will occur, "scientists cannot yet make precise predictions of their date, time, and place." So also says the
Other beliefs having nothing to do with their prediction have attached to earthquakes. There is the widespread notion that small earthquakes keep big ones from happening. By the lights of its theory, little shakers release pressure building along fault lines by dissipating it
While the theory has the ring of plausibility to it, it fails on the math. Each magnitude level on the Richter scale represents about
Another odd earthquake myth is the conviction that a major quake could cause California to split off from the rest of the continent and fall into the sea. The state is riddled with fault lines, after all, and its
From time to time, rumors will arise that the brilliant folks at Caltech (or just "scientists" in general) have discerned that a major quake is about to strike, but are striving to keep this news from the public to stave off widespread panic. This tale is a perennial, and while years may pass between its appearances, it never truly goes away. For instance, the April 2010 scare spread via Facebook, Twitter,
Often, the "Caltech knew this was coming" whisper follows a major quake, surfacing a few days after devastation has been wreaked. When told that way, the story becomes one of mistrust of government, who (if the story had been true) placed its own interest in maintaining the peace ahead of the safety of the public by deliberately keeping from it a warning that should have been heard.
Each time the gossip about foreknowledge surfaces, Caltech denies it. And rightly so, because no group of scientists or research organization anywhere has ever succeeded in correctly predicting a major earthquake by a matter of days. (Indeed, one fellow we know at Caltech said the closest he'd ever seen them come is seven years.)
In another earthquake tale that employs the "mistrust of government" theme, wags were known to assert that the government of California had deliberately underreported the magnitude of the 1994 Northridge quake so as to get out of forking over the rightful emergency relief funds to those whose homes or businesses were damaged by the event's ravages. That too was not the case, but to many to whom the
The best known of the quake legends, however, is the yarn about a crushed car found in the wake of the 1989 Loma Prieta quake. In it, said the whispers, was discovered the body of the car thief that had made off with it.
Barbara "seismic justice" Mikkelson
Last updated: 10 May 2010
Hutton, Kate. "Is There Any Such Thing As 'Earthquake Weather?'" Caltech [press release]. 1 October 1998. LaFee, Scott. "Quake Myths Rely on Cloudy Facts." The San Diego Union-Tribune. 9 April 2010 (p. A1). Muckenfuss, Mark. "Heard the One About Quake Weather?" [Riverside] Press Enterprise. 5 August 2000 (p. B1). Inside Bay Area. "Myth or Reality?" 14 April 2006. [Riverside] Press Enterprise. "Heed the Truth About Quakes." 21 April 2004 (The D; p. A4).


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Era of Argentine Subsidies Ending
President Kirchner Starts to Pare Back State's Largess Amid Strained Finances, a Slowing Economy
BUENOS AIRES—Amid sweltering summer heat, long lines of people jostled outside subway sales windows here Thursday to stock up on tickets before the government raises the price.
That 127% increase on Friday to 2.50 pesos (58 cents) is just the start of public-service cost increases facing Argentines this year as the government trims subsidies to confront a slowing world economy and its own strained finances.
Starting this...


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5 tips for Celebratory Eating
As a Health & Psychology of Eating coach, you might think I'd have some pithy advice about making your celebratory eating healthy.
You might have expected me to say:
- Start your meal with a big green salad
- Fill 2/3 of your plate with veggies
- For every glass of wine, drink 2 glasses of water
Now even though all those ideas are worthwhile, here's what I really want to say to you about celebratory eating…
It happens!
We are social beings and we love to celebrate.
We have foods that come around once a year, they remind us of holidays, people, history, traditions and more!
I'm NOT about dieting and depriving yourself, I'm about enjoying yourself fully…AND…I have 5 very specific tips that will help you navigate your celebratory or holiday eating
#1: Listen to your body when it says
Now that sounds so simple, but you and I know it's not.
The reality is – the food at your feast is luscious looking; the company is lively; the family dynamics are flying – and it's really easy to skip the moment your body says "stop, I'm done for now."
Frankly, most of us would rather not hear that first gentle "I"m full" whisper from our body. We don't want to stop eating.
Of course we don't want to stop!! It's fun to eat! It's delicious! And for goodness sakes, it's a holiday or a celebration!
So we ignore the "I'm full" message until our body starts talking really loud. Often we don't listen until it screams "I'm stuffed!" – and then unfortunately what often goes hand in hand with "I'm stuffed" is – "Oh, I wish I hadn't eaten so much."
So these tips are ways to help you listen to "I'm full" and to respect what it needs – which is to stop eating.
2. Pause every now and then during your feast.
Not just a simple lay-down of your fork, but really PAUSE. Have a great conversation with someone, lean back in your chair and take in the scene. Pause. It takes 20 minutes for the "I'm full" message to go from the belly to the brain. So give it some time to get there.
3. Promise yourself you can have more…later.
Many of us don't want to hear "I"m full" because it means we don't get any more of the delicious food in front of us. So promise yourself you can have more. You may even want to set your food aside, so that you're sure it doesn't get eaten by someone else. Do whatever it takes to assure yourself that there will be more available to you later.
4. Let yourself be sad that your tummy can only fit so much.
Being sad about having a stomach the size of your fist (which is how big our stomachs really are), may sound melodramatic. But honestly, I've punctuated many a meal with "I'm so sad."
- I get sad because I'm full and I love eating, and I do wish I had more room.
- I feel a bit sad because I'm having a good time, and I don't want to stop having fun.
I'm not lay-down-and-sob sad, but there's a moment of "bummer, the party is over" and "thanks so much for the great time, I'll see ya later dear food."
It's so important to acknowledge that moment of loss, because it brings us to the honesty of the moment. And in that honesty, we can really listen to our body. If we can hear our body's message, we can respect its needs, and stop eating when it says stop.
We're not always perfect (I'm not, for sure!), and sometimes we don't listen to what our body is saying. In those moments, deep self-compassion is what's necessary.
5. Take Bitters!
Bitters will help your digestion and help your body cope with the extra special foods you might be enjoying.
Here's more about bitters and the ones that I use the most.
I encourage you during holiday time and celebratory to not only enjoy the special relationships you have with your friends and family, but also to take the time to deepen your relationship with yourself, and listen deeply to what your body is saying to you.
~ ~ ~ ~ ~
Nina Manolson, MA, CHC, certified Health Coach and Psychology of Eating Coach believes that every woman deserves to feel good in her own body. She helps women create a healthy and positive relationship with their food and body so they can love their body and life! She's the founder of NinaManolson.com and NourishedWomanNation.com
She helps busy women look and feel their best. She specializes in working with women over 40 who have tried other diets and approaches but are still struggling with their body and food. She helps women step into a new wellness paradigm that makes self-care real and do-able and turns it into sustainable healthy habits that
She's the author of "Feed Your Kids Well In A World That Doesn't: an everyday guide to make healthy food happen in your home and beyond". She's also the recipient of the prestigious Health Leadership Award from the Institute for Integrative Nutrition.
To get your F.R.E.E. Video Series "What to do now, when everything you've done hasn't worked" by mail and receive her healthy recipes and wellness tips click here.


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Genevieve Martin's life feels like it's falling apart as her marriage crumbles. Wanting to escape her soon-to-be ex-husband's infidelity, she agrees to travel to Paris after her Uncle Dave's passing. Her cousin tries to convince Genevieve that she should take over Dave's Locksmith Shop, but Genevieve is unsure of what she wants for her future.
As Genevieve arrives in Paris and to Dave's shop, she begins to remember the summer she spent as a teenager with her uncle and his family, after the death of her mother, Angela. She finds that she is still a natural with locks, though she is hesitant to admit it. As Genevieve begins to recall her own past, she learns disturbing new information about the time Angela spent in Paris before Genevieve was born.
In The Paris Key, author Juliet Blackwell spins an interesting tale told from three perspectives; that of present-day Genevieve, teenage Genevieve and her mother Angela. Modern-day Genevieve encounters Uncle Dave's unique and endearing neighbors as she tries to convince herself not to take over his business. Teenage Genevieve navigates the deep and tragic loss of her mother while in a foreign city, with extended family members that feel like strangers. And Angela makes a decision that alters her life, and that of her family forever. Somehow, all of these narratives come together to create a deeply emotional tale you won't soon forget.
Will Genevieve stay in Paris? Or are the secrets of her mother's past far too painful?
Meg lives in Pennsylvania with her husband, Ryan. Library professional by day, freelance writer by night, Meg writes about life, entertainment and everything in between.
Review copy was provided free of any obligation by NAL. No monetary or any other form of compensation was received.


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Before discussing eczema treatments, it is important to fully understand this aggravating skin condition. Eczema can take on various forms and is caused by several different factors. In general, eczema is categorized into two groups: internally generated eczema and externally generated eczema.
This type of eczema is triggered when your skin gets in direct contact with a substance that causes a reaction from your skin. The eczema outbreak or rashes are only isolated on the surface of the skin.
This type of eczema occurs when a person's immune system reacts to certain entities that cause a skin reaction at the cellular level. Internally generated eczema is usually a result of two internal triggers occurring simultaneously. For example: a hormonal imbalance combines with an allergen.
In treating eczema, the goal is to prevent itching, inflammation and overall worsening of the skin condition. It is best to consult a skin doctor for diagnosis and treatment of this skin condition. There are prescribed medications and creams to relieve the uncomfortable symptoms of eczema.
The goal of treating eczema is to prevent inflammation, itching and the overall aggravation of the skin condition. A doctor or dermatologist should be consulted regarding the diagnosis and treatment of eczema. There are several creams, prescribed medication and natural alternatives on the market designed to relieve the symptoms of eczema.
Argan oil is known to be quite effective in reducing red rashes and itchiness on the skin. The oil can minimize flare-ups and is also a great natural remedy. Since moisturising the skin is essential for the treatment of eczema and many other skin conditions, Argan oil can be a great ally in the fight against eczema. Argan oil hydrates the skin in order for it to better perform its barrier function and is loaded with moisturising properties such as, Vitamin E (an antioxidant and anti-inflammatory), Omega 6 fatty acids (a potent skin moisturiser), Sterols (an anti-inflammatory which locks moisture in the skin) and Polyphenols (provides skin relief from allergen and nourishes the skin).


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Les Gusta Coger en la Cocina
Full video only available to VIP members, login or click here to register then upgrade your membership to VIP!
Download: Low Quality Video - High Quality Video - Original Video
Amateur couple having hot sex in various positions in the kitchen
By: amateurporn
Feb 20, 2011
Time: 06:52
Votes: 4
Favorited: 4
Views: 7580
- Posted by dutchiexxx on 24 Feb 11
So Lovely and hot....
- Posted by roadie46 on 20 Feb 11
Seen this in full months ago ,on google ,much better from the start of video though.
- Posted by MRDICK247 on 20 Feb 11
hope dont mind, this feels like spying on you love hearing the action and reactions i know we are ging to be veiwing this several times each a plus is you dont try to 4xzoom everyhing it makes this pleasure to watch both so natural loving each other bravo encore please
- Posted by macho on 20 Feb 11


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Breeding Objectives
The Urrbrae Suffolk Stud breeding program focusses on a sound combination of genetic principles and practical experience with the aim of breeding high quality, profit-making sires and ewes for commercial lamb production.
Urrbrae selects and breeds for the following key traits:
- Growth rate
- Easy lambing
- Moderate leanness
- High muscle depth
- Worm resistance
- Carcase quality
- Fertility
- Structural soundness utilising independent assessment
Performance Recording to Maximise Breeding Outcomes
Urrbrae Suffolk Stud is a strong advocate of performance recording. At birth lambs are electronically tagged and recorded for birth weight, birth type, sex, sire, dam, lambing ease, distance of dam from lamb(s) and comments relating to mothering ability.
Our Suffolks are not removed from the flock until all animals are weighed and scanned by an accredited LAMBPLAN operator at the post-weaning age of 5-6 months. This enables the most accurate assessment of each lambs genetic potential and allows us to select further breeding and sale stock with informed confidence.
Urrbrae utilises LAMBPLAN to provide accurate and timely details on the genetic merit of individuals in the stud flock. LAMBPLAN combines performance and pedigree information to provide Australian Sheep Breeding Values (ASBV's). The ASBV's are calculated from systematic combinations of performance information on individuals and their relatives. They are expressed as the difference between an individual's genetic merit and the genetic base to which the animal is compared.
The LAMBPLAN ASBV's obtained form the basis for which animals are retained in the flock or offered for sale. The most critical ASBV's are:
Birth Weight (BWT): estimates the genetic difference between animals in lamb birth weight.
Weaning Weight (WWT): estimates the genetic difference between animals in live weight at 100 days of age.
Post Weaning Weight (PWWT): estimates the genetic difference between animals in live weight at 225 days of age.
Post Weaning Fat (PFAT): estimates the genetic difference in GR fat depth at 45 Kilograms live weight (average).
Post Weaning Eye Muscle Depth (PEMD): estimates the genetic difference in eye muscle depth at the c site at 45 Kilograms live weight (average).
Post Weaning Worm Egg Count (PFEC): estimates the genetic difference in worm burden at 225 Days of age.


text:
Hard Question but fun to vote
mine is Tumblr 💙
- Vote A
- TumblrVote B
- Vote C
- Vote D
- YouTubeVote E
- SnapchatVote F
- Other?Vote G
Most Helpful Guy
well i dont use much social media, and youtube has been around for a long time and people never called it "social media" back in the day, thanks a lot society, if social media is defined as "websites and applications that enable users to create and share content or to participate in social networking." then by that definition, Second Life is considered social media lol.
I'll just take youtube for now11|00|0
Most Helpful Girl
I guess YouTube. I don't post comments on videos, but I'll thumbs up if I like it. Lots of variety on there; some good music, sometimes funny skits, or good tutorials.
I don't use much social media, like I don't have FaceBook or anything.11|00|0
Thank you!


text:
So for those who've been missing out, From Comics to Cinema is a weekly article that began on Reddit, focusing on the transition of popular comics characters from the page to the screen. You can view the original gallery here - http://imgur.com/a/99Cxt - or read on below!
So while I know the Marvel Cinematic Universe has picked up a lot of popularity, there may still be a lot of people out there wondering...
We're still three weeks away from [Captain America: The Winter Soldier](movie:254973) and anticipation is already high for what may be the first Blockbuster of 2014. As we begin the countdown to Winter Soldier, I thought it best to look back at Captain America: The First Avenger.
Captain America
Unlike the vast majority of Marvel Comics characters who debuted in the 60's and 70's, Captain America was originally created in March 1941 - a number of months before the Japanese attack on Pearl Harbor prompted America's involvement in the War. Captain America remained a popular character through the 40's and 50's; though it wasn't until Avengers #4 (1964) that the characters history was revised to see him falling into suspended animation at the end of WWII and subsequent reawakening in the modern world.
Project Rebirth
After the loss of project leader Abraham Erskine and the original formula, the Super Soldier program (codenamed Project: Rebirth) would continue to try and recreate the success they found in Steve Rogers. The subsequent experiments - in which 300 African American soldiers would be given an earlier version of the serum - proved fatal to all subjects except one: Isaiah Bradley, the 'Black Captain America'. Years later, Project: Rebirth would be revealed as just one - Weapon I - in an entire series of experiments codenamed Weapon Plus. Weapon Plus would play an essential role in the 'creation' of one other Marvel staple: Weapon X, aka Wolverine of the X-Men.
Red Skull
For as long as there has been a Captain America, there has been a Red Skull, debuting alongside the star-spangled avenger in 1941. This original Red Skull was named George Maxon - an American interested in overthrowing the US Government. Maxon would eventually be revealed as simply a 'pawn' for Johann Scmidt, the 'true' Red Skull. Schmidt originally wore a mask, but would later have his face transformed through exposure to his own 'Dust of Death' - a chemical weapon which would kill his victims by rapidly drying and shrinking the skin around their face and head, resulting in a 'red skull'. Though he seemingly died at the end of 'The First Avenger', the Red Skull has died numerous times in the comics, and has subsequently resurrected through a number of means.
Margaret 'Peggy' Carter
Peggy Carter was a member of the French Resistance, meeting and fighting alongside Captain America during World War II. Unlike her film equivalent, in the comics Peggy is not involved in Project: Rebirth, but rather meets Cap on the battlefield. After Cap's apparent death, Peggy would go into a deep state of mourning that would eventually lead her into the care of villain Doctor Faustus. Cap would save her from the evil Doctor, and she would go on to serve for both SHIELD and the Avengers before retiring to a nursing home.
Arnim Zola
In the comics, Arnim Zola is a skilled biochemist, who creates a means through which the human mind might be copied and imprinted upon a new subject. This imprinting can be done on other living creatures, or on mechanical constructs of his own creation. He's used this technology to create a series of robotic bodies which have allowed him to outlive his natural lifespan, and can transfer his consciousness to a new 'body' whenever his current one is destroyed. On film, there are a couple of allusions to his comic origin, including the image of his face projected through a digital screen; and blueprints in his lab which seem to depict a robot body similar to his form in the comics.
The Howling Commandos
The Howling Commandos was an elite fighting group initially serving in WWII under the leadership of Sgt. Nicholas Fury, with many of the Howling Commandos later serving as Nick Fury's inner circle after becoming Director of SHIELD. On film, Nick Fury is otherwise absent from the Howling Commandos roster, with the group serving as Cap's hand-picked support team.
From left to right: Jim Morita, Jacques Dernier, Dum Dum Dugan, James Montgomery Falsworth (Union Jack), 'Bucky' Barnes, and Gabriel Jones. Originally, Union Jack and Bucky were not part of the Howling Commandos, but rather (alongside Captain America) members of the Invaders, an international coalition of superheroes fighting for the Allies.
Reading Recommendations!
Marvel Masterworks: Captain America, Vol.1 - This collection takes you back to Captain America's first solo series, revisiting his origins, early encounters with the Red Skull, and acclimating to the 'modern' world (of the 1960's). Some may find these classic comics to be in harsh contrast to their more modern equivalent (it was simpler, sillier time); but they are a fun, easy introduction to the character.
Captain America: Man Out of Time - A modern retelling of the events surrounding Captain America's reawakening after his suspended animation, Man Out of Time is a uniquely introspective look at Cap's return and the journey he must undergo to find his place in the world. Man Out of Time isn't just a great Captain America primer, but also gives a nice peek into the early days of the Avengers.
Until next time...
I hope you enjoyed! Tune in next week as From Comics to Cinema continues its countdown with coverage on Nick Fury, SHIELD, and the supporting cast of Captain America: The Winter Soldier.


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We've waited long enough and now temperatures are finally back in the 80s. But to some, today's heat may have felt more intense than what the thermometer read and there's a few reasons for that.
Most of us have heard of the "heat index". It's a tool used by meteorologists to help convey the effect that humidity has on our bodies. Basically the more humid it is, the warmer it will feel at any given temperature. When the heat index is in the 90s, even well-conditioned athletes need to be careful if they're outside.
Today's heat index was in the mid 80s. Certainly warm, but not dangerous. But there's one very important variable that the heat index doesn't account for: the sun. An area in direct sunlight can have a heat index 10-15 degrees higher than what's officially measured in the shade.
Another thing to consider is our own physiology. Sweating of course is our bodies' way of keeping us cool, but after months of shivering through winter it can take anywhere from 2 to 6 weeks for our system to become its most efficient. If you notice that you start sweating more easily in the later months of summer, that means you're acclimating to the heat.
Even once we are accustomed to the new season, staying hydrated and out of the sun during the middle of the day will go a long way in staying safe this summer.


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My Nonprofit Reviews
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1 reviews
Review for The Heart Connection, Des Moines, IA, USA
I started with The Heart Connection first as a Sibling Camper more than 20 years ago. What a wonderful experience it was to spend time with other kids that understood what it was like to go through the experience. The Heart Connection provides that support to the entire family and that is what makes them such a great organziation.
More Feedback
I've personally experienced the results of this organization in...
the families they serve and it does make a difference
What I've enjoyed the most about my experience with this nonprofit is...
the time with people that understand what the experience is
When was your last experience with this nonprofit?
Role: Volunteer & Camper, Volunteer, Staff, Volunteer.


text:
Over at Shutter Sisters today, Kristin write on her secret to really capturing those "in the moment" images. Her trick is a very good one. I've been known to do it, but I admit, I don't have much practice in that type of shooting. I'm very bad at getting my camera at the right angle to actually capture something in that way.
My trick is similar with my girls. In this picture, I started her talking on her favorite subject. You can see her forming the words, it is literally on the tip of her tongue. Sssshhhhhaaaarks. I asked her questions about them that were open ended – no "yes or no" questions, or else the "shoot" will be over before it starts. I asked her things like, "what is your favorite sea creature?" "what colors do sharks come in?" "where do sharks play?" and "what do they play?" on and on. While this is going on, I didn't really make eye contact or get too excited. I fiddled with my camera like I was trying to change the settings, very passive about the whole thing. I would lift it up, shoot a frame or two, and again act like I was changing a setting. Talking the entire time to her.
She won't look at the camera. EVER. Well, unless it is some total stranger then she'll go right up to them and give them the most gorgeous smile. But anyway, this is my trick and it is all I can do. It is how I got these shots, and these shots. And, I know they aren't perfect in a technical sense, but to me they really don't get much better than this. They really capture little bits of her personality.
P.S. – I want to make this: http://ashleyannphotography.com/blog/2010/08/05/diy-chicken-wire-frame-memory-card-clips/


text:
Time: 9:00am PDT / Noon EDT
Call-In Info: 1-641-715-3660, access code 651025
Moderator: Andrew Myers
Notetaker: Michael J. Giarlo
- Collin Brittle (Virginia Tech)
- Lynette Rayle (Cornell)
- Esmé Cowles (Princeton)
- Josh Gum (Oregon State)
- Anna Headley (Chemical Heritage Foundation)
- Jeremy Friesen (Notre Dame)
- Jennifer Lindner (Art Institute of Chicago)
- Justin Coyne (Stanford)
- Carolyn Cole (Penn State)
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- David Tolmie (University of Washington)
- Bess Sadler (Data Curation Experts)
- Lakeisha Robinson (Yale)
- Greg Luis (Oregon State)
- Roll call by timezone per following order - ensure notetaker is present
- folks outside North and South America
- Eastern timezone
- Central timezone
- Mountain timezone
- Pacific timezone
- folks who were missed or who dialed in during roll call
- Fedora Performance and You (Michael J. Giarlo)
- Performance metrics and tests
- Next Fedora Tech call is tomorrow, Thursday 1/24 at 11am ET (agenda)
- Sufia 7.3.0 release forthcoming (Michael J. Giarlo)
- Calling for testers (needs QA & testing of upgrade path from 7.2.0)!
- Community expectations around GitHub code review (Justin Coyne)
- Self-merges after 1 or more approvals are OK as long as there are 0 dissenting reviews and no unresolved comments.
- Jeremy to make a change to CONTRIBUTING.md and roll out that change once the PR is approved
- New version of hydra-head imminent (Justin Coyne)
- https://github.com/projecthydra/hydra-head/releases/tag/v10.4.0.rc1
- Still needs https://github.com/projecthydra/hydra-head/pull/398
- Backport https://github.com/projecthydra/hydra-head/commit/06a1bfc17ad3326f3a9f8002688ec0c9a97cbaab? Fixes https://github.com/projecthydra/sufia/issues/2820
- Anyone interested in a backport, thinking of CC users? Unclear.
- Justin to write the hydra-tech list about these changes
- Release of hydra 11.0.0. (Bess Sadler)
- Bess will include the new version of hydra-head and re-tag the release
- Bess will update Dive Into Hydra and Dive Into Hydra Works tutorials accordingly
- Bug when configuring as part of the include block (Jeremy Friesen)
- Please review the bug for the heads up. The short of it is gem configuration should not happen as part of a included block of an ActiveSupport::Concern module.
- CanCan and curation concerns (Joe Atzberger)
- Remove from agenda
- (STANDING) Update on Sufia/CurationConcerns Consolidation Plan (Justin Coyne)
- (STANDING) Sufia 7.3 Blockers - University of Cincinnati local sprint. (Thomas Scherz)
- Moderator/notetaker for next time:
- Moderator: Jeremy Friesen
- Notetaker: Carolyn Cole


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Toulouse track Delporte
By Patrick Haond
Last Updated: 01/01/70 1:00am
Toulouse are contemplating a move to sign Osasuna midfielder Ludovic Delporte.Toulouse are contemplating an audacious move to bring Osasuna midfielder Ludovic Delporte back to France.
The Ligue 1 side are seeking reinforcements to their midfield following the long-term injury suffered by Stephane Dalmat.
Delporte has emerged as a target for Toulouse and the club are keeping tabs on his performances in La Liga.
But Toulouse would have to delve deep into their transfer kitty if they are to hold out any hope of luring the 24-year-old back to his homeland.
He only joined Osasuna in the summer on a free transfer after leaving Albacete and he penned a three-year contract with the club.
Osasuna, though, set Delporte's release clause at é¼6 million and that may dissuade Toulouse from firming up their interest.


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One of the most formidable impediments to clinical translation of RNA interference (RNAi) is safe and effective delivery of the siRNAs to the desired target tissue at therapeutic doses. We previously described in vivo cell type-specific delivery of anti-HIV small-interfering RNAs (siRNAs) through covalent conjugation to an anti-gp120 aptamer. In order to improve the utility of aptamers as siRNA delivery vehicles, we chemically synthesized the gp120 aptamer with a 3′ 7-carbon linker (7C3), which in turn is attached to a 16-nucleotide 2′ OMe/2′ Fl GC-rich bridge sequence. This bridge facilitates the noncovalent binding and interchange of various siRNAs with the same aptamer. We show here that this aptamer-bridge-construct complexed with three different Dicer substrate siRNAs (DsiRNAs) results in effective delivery of the cocktail of DsiRNAs in vivo, resulting in knockdown of target mRNAs and potent inhibition of HIV-1 replication. Following cessation of the aptamer-siRNA cocktail treatment, HIV levels rebounded facilitating a follow-up treatment with the aptamer cocktail of DsiRNAs. This follow-up injection resulted in complete suppression of HIV-1 viral loads that extended several weeks beyond the final injection. Collectively, these data demonstrate a facile, targeted approach for combinatorial delivery of antiviral and host DsiRNAs for HIV-1 therapy in vivo.
Combinatorial antiretroviral therapy which combines two or more antiretroviral drugs that act on different stages of the HIV life cycle is currently used to suppress viral replication. Although highly effective, there are often undesirable side effects and compliance problems as well as the emergence of drug resistance that complicate the use of this therapy.1 Because of these issues, a number of investigators have been exploring other approaches for inhibiting HIV-1 infection. Nucleic acid-based therapeutics have been considered as a promising alternative to the small molecule antiviral to overcome these limitations. One of the most potent of these is the use of RNA interference (RNAi).2,3 In the case of HIV-1, either the entire genome of the virus or a large number of HIV-1 host-dependency factors (HDFs) are potential targets for RNAi.4,5,6 Numerous RNAi applications targeting HIV-1 or cellular factors have demonstrated efficient suppression of viral replication both in cell culture and in animal models.7,8 The entire repertoire of HIV-1–encoded genes (tat, rev, gag, pol, nef, vif, env, vpr, and the long terminal repeat) are susceptible to RNAi-induced gene silencing in cell lines.9,10,11 Moreover, host genes such as the chemokine receptor CCR5 can be targeted alone or in combination with viral sequences in a tailored fashion to reduce the emergence of viral escape mutants.12,13,14
Although siRNA-based therapeutic strategies for combating HIV/AIDS hold considerable promise, the actual clinical applications of RNAi for treatment of HIV/AIDS has been limited due to concerns about inefficient systemic delivery of siRNAs to the appropriate target cells or tissues; and viral escape from the RNAi mechanism.15,16 Efforts to improve the therapeutic performance of siRNAs for the treatment of HIV/AIDS should include delivery formulations with targeting moieties as well as combinatorial approaches with other therapeutics. Recent targeted delivery approaches have provided encouragement that this obstacle may be surmountable. For example, single chain antibody-siRNA chimeras targeting either the T-cell CD7 (Cluster of Differentiation 7) receptor17 or HIV-1 glycoprotein gp12018 have been demonstrated to functionally deliver anti-HIV siRNAs in vivo. By using the lymphocyte function-associated antigen-1 (LFA-1) integrin-targeted immunoliposome system, selective targeting and delivery of an anti-CCR5 siRNA to lymphocytes in vivo has been demonstrated.19 As mentioned above, the rapid emergence of viral escape mutants often abrogates RNAi efficacy. A single nucleotide mutation in a critical position within the HIV target sequence relative to the siRNA site of interaction could diminish and even eliminate RNAi activity.20,21,22,23 Therefore, a dual functioning aptamer-mediated cell type-specific delivery of combinatorial RNAi therapeutics also has been developed. For example, HIV-neutralizing aptamers specific to either the CD4 (cluster of differentiation 4) receptor24 or HIV-1 glycoprotein gp12025,26,27 have been harnessed for targeting delivery of multiple anti-HIV siRNAs in vitro as well as in humanized mice.
We previously described a noncovalent strategy to assemble a single HIV-1 gp120 aptamer with three different Dicer substrate siRNAs (DsiRNAs) targeting HIV-1 tat/rev or the HDFs (CD4 and Transportin-3 (TNPO3)).26 The resulting aptamer-siRNA conjugates were internalized into cells expressing the HIV-1 envelope and the combination of aptamer-DsiRNAs effectively inhibited viral replication in HIV-1–infected cells. In this design format, both the aptamer and siRNA portions are chemically synthesized and subsequently conjugated via a "sticky bridge", which is composed of a 16-base 2′ OMe, 2′ Fl-modified GC-rich sequence attached to the aptamer via a polycarbon linker (Figure 1). The physical combination of the HIV-neutralizing aptamer and combination of anti-HIV siRNAs has the potential for large-scale chemical production along with the facile delivery of combinations of siRNAs to mitigate the emergence of viral escape mutants.
In the present study, we utilized a humanized mouse model25,28,29,30,31 to evaluate the in vivo antiviral efficacy of the chemically synthesized anti-gp120 aptamer-sticky bridge-DsiRNA conjugates. Humanized Rag2−/−γc−/− mice (RAG-hu) were infected with HIV-1 and subsequently given weekly intravenous injections of the anti-gp120 aptamer-sticky bridge-siRNA conjugates targeting both the virus and cellular transcripts. The results obtained demonstrate functional delivery of all three siRNAs and potent suppression of HIV-1 infection as well as protection against viral-induced CD4+ T-cell depletion. A follow-up treatment of the aptamer-DsiRNAs conjugates following viral rebound resulted in complete, long-term suppression of HIV-1 viral loads. Collectively, these data demonstrate a facile, cell type-specific aptamer-mediated delivery of DsiRNAs in a combinatorial fashion for targeting both viral and cellular transcripts in vivo.
As we previously described, a GC-rich "sticky bridge" facilitates the interchange of different DsiRNAs with a given aptamer.26 We have used this approach to target three different transcripts (HIV-1 tat/rev and two HIV-1 HDFs12 CD4 and TNPO3) with an aptamer that targets HIV gp120. The 3′-end of the aptamer and one of the two siRNA strands were chemically conjugated with 16 bases of complementary GC-rich sequences, thereby, allowing the aptamer and siRNA portions to be noncovalently joined via Watson-Crick base-pairing by simple mixing of the two reactants (Figure 1). Two different design formats (named N-1 and N-2) were constructed. In the N-1 design, the 3′-end of the antisense strand of the DsiRNA was appended to a bridge complementary sequence (Figure 1), whereas the 3′-end of sense stand was joined with this sequence in the N-2 design.
Although both designs showed inhibition of HIV-1 infection in human T cell lymphoblast-like cell line (Supplementary Figure S1a), the N-1 design mediated more efficient knockdown of the target mRNA and exhibited better strand selectivity (Supplementary Figure S1b,c). The Tat/rev N-1 and TNPO3 N-1 conjugates were observed to have somewhat better efficacy in gene silencing than the corresponding Tat/rev N-2 and TNPO3 N-2 conjugates, consistent with the comparison of their HIV-1 p24 data. In vitro Dicer assays indicated that Dicer preferentially enters these substrates from the two base 3′-overhang.26 Since the orientation of the sense and antisense strands differs between N-1 and N-2 with respect to the two base 3′-overhang, Dicer cleavage results in predictably different 21-23 siRNA products from these two configurations which can have different potencies.26
In order to validate that the siRNAs released from the conjugates were actually triggering RNAi, we next investigated siRNA-directed mRNA cleavage using a modified 5′-RACE (Rapid amplification of cDNA ends) PCR assay (as described in Supplementary Materials and Methods). It has been established that Ago2 mediates cleavage between bases 10 and 11 relative to the 5′ end of each siRNA.32,33 Thus the RACE PCR product sequence analyses of the target should reveal a 3′ linker addition at the base positioned 11 nucleotides from the 5′ end of the siRNA. The experimental RNAs (Tat/rev 27-mer siRNA, Tat/rev N-1 or N-2) and a HIV pNL4-3 luc were first cotransfected into HEK293 cells with Lipofectamine 2000. PCR bands of the predicted lengths based upon Dicer cleavage of the DsiRNAs were detected in the total RNA samples from HEK293 cells transfected with the conjugates following three nested PCR reactions (Supplementary Figure S2a). The gel-purified bands of the predicted lengths were TA cloned and individual clones were characterized by DNA sequencing to verify the expected PCR products Tat/rev N-1 and N-2 treatment produced two distinct cleavage sites (Supplementary Figure S1a,b). For Tat/rev N-1, the cleavage takes place at the predicted position for the siRNA duplex between positions 10 and 11 from the 5′ end of the siRNA antisense strand. However, Tat/rev N-2 produced some non-predicted cleavage positions, suggesting a secondary Dicer cleavage of the siRNA portion might exist. Similarly, TNPO3 N-1 produced the predicted cleavage RACE product for the siRNA duplex between positions 10 and 11 from the 5′ end of the siRNA antisense strand. Two distinct non-expected cleavages took place in the treatment with the TNPO3 N-2 conjugate (Supplementary Figure S2c,d). Together, these results suggest that more predictable and potent Dicer cleavage of the DsiRNA species is generated from the N-1 design.
In the conjugates, all the RNA components were chemically synthesized with 2′-fluoropyrimidine modifications since the original aptamer selection was carried out using this backbone modification. To test the stability in serum, one aptamer-tat/rev siRNA conjugate (Tat/rev N-1) was incubated in 50 or 10% normal mouse serum for different lengths of times at 37 °C followed by denaturing polyacrylamide gel electrophoretic analyses (Supplementary Figure S3, experimental details were detailed in Supplementary Materials and Methods). After 8 hours of incubation, ~30% of the full-length 2′-fluoro–modified conjugate was detectable in 50% serum and more than 50% was detectable in 10% serum, validating the stabilizing effects of the 2′-Fl modification.
Given the positive results of anti-gp120 aptamer-mediated DsiRNA delivery in HIV-1–infected human T cell lymphoblast-like cell line and primary human peripheral blood mononuclear cells (PBMCs), we next sought to determine whether the aptamer-cocktailed DsiRNA conjugates (N-1 design format) with a combination of three siRNAs (targeting HIV-1 tat/rev and HIV-dependency factors CD4 and TNPO3) are functional in vivo. The ability of the conjugates to suppress HIV-1 viremia was evaluated in Rag2−/−γc−/− mice (RAG-hu) infected with HIV-1 NL4.3 via intraperitoneal injection. Three weeks post-infection, the mice became viremic and were subsequently injected via the tail vein with preparations of the aptamer-cocktailed siRNA conjugates as described in the Materials and Methods section. Control groups were administered with buffer or naked cocktailed DsiRNAs.
Five to eight animals in each treatment group were given five weekly injections of 0.25 nmol of aptamer-cocktailed DsiRNAs or 0.25 nmol total of a combination of the anti-tat/rev, anti-CD4, and anti-TNPO3 DsiRNAs. To determine whether the established viral loads could be impacted by the treatments, the mice were monitored by quantitative real-time PCR for plasma viral loads on a weekly basis (Figure 2a and Supplementary Table S1a). We observed a general pattern of decreased viral loads in the majority of aptamer-cocktailed DsiRNA conjugate-treated mice. The suppression of viral loads was on average three logs relative to the control groups and this difference reached statistical significance with a rank sum P = 0.0005 (Figure 2a and Supplementary Table S1c). The viral loads were suppressed to below detectable levels within 3 weeks in five of eight conjugate-treated mice (at week 9–11 of post-treatment, as shown in Supplementary Table S1a). This suppression persisted for as long as 3 weeks following the termination of the injections, indicating sustained efficacy of the conjugates. There was no suppression of viral loads in the animals treated with the DsiRNAs alone (Figure 2a and Supplementary Table S1a).
Once the aptamer-DsiRNA treatments were terminated, viral loads increased in the majority of the treated animals. To determine whether or not the treatment had enriched for viral mutants resistant to the aptamer-DsiRNA conjugates, we asked whether or not re-treatment with the conjugates could restore inhibition of viral levels to those seen with the first set of treatments. Experimental animals in which the viral loads had become elevated were re-treated with two separate injections of the aptamer-cocktailed DsiRNA conjugates at 12.5 and 13.5 weeks following the last administration from the previous treatment period (Figure 2b and Supplementary Table S1b). The re-treatment resulted in a statistically significant (P = 0.0143) suppression of HIV-1 levels (Supplementary Table S1d). Complete suppression persisted for 2 weeks beyond the re-treatment period in the aptamer-DsiRNA conjugate-treated mice. Three of the four treated mice had undetectable viral loads even up to 6 weeks following the re-treatment as shown in Supplementary Table S1b.
We validated the aptamer-mediated DsiRNA delivery to infected T lymphocytes using a TaqMan quantitative PCR (qPCR) assay on RNA isolated from peripheral blood cells collected following both the second treatment and 2 weeks subsequent to the final treatment in the first experimental set of animals (Figure 2). The results presented in Figure 3a reveal that the tat/rev siRNA was detectable in all of the aptamer-DsiRNA conjugate-treated mice at 2 weeks of treatment. Even at 2 weeks following the last injection, the siRNAs were still detectable in all the conjugate-treated mice (Figure 3b), indicating the stability and retention of the siRNAs delivered by the aptamer in vivo. In contrast, no siRNA was detected in the cellular fractions from mice treated with the naked DsiRNA.
We next asked whether the siRNAs triggered sequence-specific mRNA degradation in the HIV-1–infected, aptamer-DsiRNA–treated Rag-hu mice. Reduction in the mRNA levels of the three targeted transcripts (HIV tat/rev, CD4, and TNPO3) in blood cells of infected and treated mice were assessed by quantitative real-time PCR. The aptamer-DsiRNA conjugates substantially decreased the corresponding targeted mRNA levels in the treated mice following the secondary and last injection with the aptamer-DsiRNA conjugates (weeks 9 and 13) (Figure 3c,d).
HIV-1 infection is associated with the progressive loss of CD4+ T cells through apoptosis or decreased production.1,34 The CD4 cell count is a standard tool for monitoring the progression of HIV-1/AIDS. Significant loss of CD4+ helper T cells could accelerate immunodeficiency. Considering the significant suppression of HIV-1 viral replication, we next determined whether treatment of HIV-1–infected RAG-hu mice with the aptamer-DsiRNA conjugates could protect against HIV-1–mediated CD4+ T-cell depletion. We evaluated CD4+ T-cell levels in peripheral blood collected biweekly during and after treatment (Figure 4 and Supplementary Table S2a). For the HIV-negative mice, the level of CD4+ T cells remained stable within a 5% variation throughout the course of the experiments. In the animals that were infected with HIV-1 but not receiving treatment, viral infection lead to a steady decline in CD4+ T cells beginning at week 4 post-infection falling from initial levels of about 65% to below 35% at 18 weeks. When infected mice were treated with the aptamer-DsiRNA conjugates, the levels of CD4+ T cells remained near the levels of the uninfected mice and the CD4+ T cells remained stable well beyond the last treatment (~55%), indicating that the treatment also significantly impacted on CD4+ cell survival (P = 0.0348) (Supplementary Table S2b).
It had been reported earlier that synthetic siRNAs delivered via systemic administration can nonspecifically activate innate inflammatory cytokine production (tumor necrosis factor-α, interleukin-6, and interleukin-12) as well as interferon (IFN)-responsive genes, and this, in turn, can trigger undesirable cellular toxicity.35,36 Hence, we assessed the potential induction of type I IFN-mediated gene expression using a quantitative reverse transcription-PCR (qRT-PCR) expression assay on total RNAs derived from PBMCs isolated from the aptamer-siRNA conjugate treated and control mice. IFN-α treatment of PBMCs was used as a positive control to confirm upregulation of p56 and OAS1 gene expression. No significant differences in IFN-responsive gene expression were observed in RNAs isolated from treated or control animals at different times post-treatment (Figure 5a,b).
We also evaluated the innate IFN response within 2 and 24 hours after the injection of the conjugates by using an ELISA. IFN-α levels in HIV-1 uninfected and HIV-1–infected animals with or without treatment were monitored directly and no significant elevation of IFN-α was observed (Figure 5c). In contrast, IFN-α (>3 ng/ml) was induced in the control animals treated with poly I:C (polyinosine-polycytosine), a double-stranded RNA activator of innate IFN response.
A major challenge in the treatment of HIV-1 infection is the rapid generation of drug-resistant mutations. It has been demonstrated that prolonged culturing of HIV-infected cells harboring expressed short hairpin RNAs often results in the evolution of escape variants that are resistant to the expressed short hairpin RNA.20,21,23,37 To determine whether or not treatment of HIV-1–infected animals with the aptamer-DsiRNA conjugates resulted in the evolution of escape mutants, we carried out deep sequencing analyses of the entire envelope gene as well as the HIV-1 tat/rev siRNA target sequence. Pooled viruses obtained from serum were collected from aptamer-DsiRNAs conjugate-treated animals at weeks 7 through 9 post-treatment. Illumina deep sequence analyses of viral RNAs (Supplementary Table S3) revealed multiple point mutations in the envelope sequences of the conjugate-treated mouse compared with the parental virus. The average frequency of unique point mutations compared with the parental virus was <3% (frequency is defined as the number of reads with mutations as a percentage of total reads). Supplementary Table S3 lists all the point mutations with a frequency above 1% in both the HIV-1 env-targeted domain and tat/rev-targeted domain, in which six and five mutations were detected in the gp120 and gp41 domain, respectively. Only four point mutations were found in the tat and rev shared exon, and no mutations were observed in the tat/rev siRNA target sequence (5982–6008 nt). These data suggest that the aptamer-multiplexed DsiRNA conjugate treatment did not select for viral escape mutants.
The efficacy and sequence specificity of RNAi make this an attractive approach for the therapeutic treatment of a wide variety of human maladies ranging from cancer to infectious diseases.38,39,40,41 RNAi-based therapeutics can be multiplexed to prevent viral escape and both viral and cellular sequences can be targeted. Another class of therapeutic nucleic acids, aptamers, show promise as potential for targeted siRNA delivery.42,43 Aptamers specific to either CD424 or HIV-1 gp12025,26,27 have been successfully combined with anti-HIV-1 siRNAs to obtain a dual-inhibitory drug that is able to functionally deliver anti-HIV-1 siRNAs for target mRNA knockdown and either neutralize the virus (anti-gp120 aptamer) or block viral entry (CD4 aptamer).
We previously demonstrated that systemic administration of a dual functioning anti-gp120 aptamer-tat/rev siRNA chimera efficiently suppressed HIV-1 infection prevented helper CD4+ T cell decline in humanized mice.25 This aptamer-siRNA chimera was produced by T7 transcription, the conventional method for RNA aptamer production. For each siRNA fused to the aptamer, a different DNA transcription template has to be synthesized. In the present study, we took advantage of a chemically synthesized gp120 aptamer with a 3′ polycarbon linker fused to a backbond modified "sticky" sequence that allows the annealing of any siRNA duplex with a complementary sequence on one of the siRNA strands. Although several preclinical studies of aptamer-mediated siRNA targeting have been successful in the animal models of cancer and other diseases, siRNA activity is often compromised when it is covalently fused to an aptamer. It is necessary to develop guiding rules for designing active conjugates. To find out a better design configuration, we simply constructed two different design formats (named N-1 and N-2) and assessed their antiviral and gene-silencing activities using qRT-PCR and 5′-RACE PCR to monitor target knockdown. We observe that the orientation of the siRNA conjugated with aptamer impacts Dicer processing and mRNA cleavage, thus resulting in different RNAi activity and antiviral efficiency. The N-1 design in which 3′-end of the antisense strand of siRNA is appended to the complementary strand of the sticky bridge sequence was more effectively processed into a functional siRNA product with better RNAi potency than the N-2 design. Thus, we used the N-1 format with better RNAi potency to evaluate the in vivo efficacy of the aptamer-mediated delivery of three different DsiRNAs in vivo in a humanized mouse model of HIV-1 infection.
In order to avoid the ineffectiveness in RNAi-based HIV-1 therapy due to the generation of viral escape mutants, one of the strategies is to mitigate viral escape from RNAi by using multiple RNAi effectors. Our recent study44 has demonstrated that a dendrimer-mediated cocktail of anti-host and -viral DsiRNAs nanoparticle delivery system provided more viral suppression and T-cell protection in vivo than the dendrimer-individual siRNA nanoparticle. Herein, in addition to targeting conserved viral target (HIV Tat/rev transcript), we therefore chose to transiently inhibit the expression of two HDFs (CD4 and TNPO3) in combinatorial mode since CD4 is absolutely required for HIV-1 entry, and TNPO3 is required for viral integration. We have demonstrated that intravenous injections with the anti-gp120 aptamer-DsiRNA conjugates consisting of a cocktail of these three DsiRNAs downregulated all three targeted transcripts in vivo, suggesting that a combination of DsiRNAs can be functionally delivered in vivo by the same chemically synthetic RNA aptamer. Furthermore, the inhibition of these targets resulted in a marked suppression of HIV-1 viral loads and protection of CD4+ T-cells from HIV-mediated depletion. Five of the eight mice tested had undetectable viral loads even up to 3 weeks after the final treatment (Supplementary Table S1a), indicating sustained efficacy of the conjugates. Moreover, we show that viral rebounds which took place 3 months after cessation of the first treatment was fully suppressed by re-injection of the aptamer-DsiRNA conjugates. Importantly, this suppression lasted for an additional 3 weeks beyond the re-treatment period. Taken together, these data demonstrate a functional in vivo delivery of multiplexes anti-HIV-1 DsiRNAs via a chemically synthesized aptamer with a sticky bridge. Compared to a time-consuming, covalent aptamer-single siRNA chimera, the chemically synthesized aptamer and this "sticky brigde" strategy, the advantage of the synthetic RNA aptamer offer many favorable advantages, such as (i) large-scale manufacture with a relative lower cost; (ii) facile, noncovalent interchange of various siRNAs with the same/different aptamer portions; (iii) combinatorial RNAi effectors providing a safe and effective antiviral efficiency in the absence of viral resistance.
Materials. Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich (St Louis, MO), all restriction enzymes were obtained from New England BioLabs (Ipswich, MA) and all cell culture products were purchased from GIBCO (Gibco BRL/Life Technologies, a division of Invitrogen, Grand Island, NY). Random primers (Invitrogen, Carlsbad, CA); Lipofectamine 2000 (Invitrogen); Random primers (Invitrogen); HEK 293 and CCRF-CEM (ATCC); CHO-Env Transfectants (CHO-WT and CHO-EE); the HIV-1 NL4.3 and HIV-1 IIIB viruses were obtained from the AIDS Research and Reference Reagent Program.
siRNAs. DsiRNAs were purchased from Integrated DNA Technologies (Coralville, IA).
Anti-tat/rev Site I 27-mer: Sense: 5′-GCG GAG ACA GCG ACG AAG AGC UCA UCA-3′ antisense: 5′-UGA UGA GCU CUU CGU CGC UGU CUC CGC dTdT-3′.
Anti-CD4 27-mer: Sense: 5′-GAU CAA GAG ACU CCU CAG UGA GAA G-3′ antisense: 5′-CUU CUC ACU GAG GAG UCU CUU GAU CUG-3′ (2′-OMe modified U was underlined.)
Anti-TNPO3 27-mer: Sense: 5′-CGA CAU UGC AGC UCG UGU ACC AG dGdC-3′ antisense: 5′-GCC UGG UAC ACG AGC UGC AAU GUC GUU-3′.
Aptamer-siRNA conjugates. A-1-stick, stick-sense, and stick-antisense were chemically synthesized by the Synthetic and Biopolymer Chemistry Core in the City of Hope. And the corresponding stick-siRNAs and aptamer-stick-siRNAs were formed as previously described.45
A-1-stick: 5′-GGGAGGACGAUGCGGAAUUGAGGGACCACGCGCUGCUUGUUGUGAUAAGCAGUUUGUCGUGAUGGCAGACGACUCGCCCGA XXXXXXXGUACAUUCUAGAUAGCC-3′
Tat/rev N-1:
Antisense-stick: 5′-UGAUGAGCUCUUCGUCGCUGUCUCCGCXXXXXGGCUAUCUAGAAUGUAC-3′
Sense strand: 5′-GCGGAGACAGCGACGAAGAGCUCAUCAUU-3′
Tat/rev N-2:
Sense-stick: 5′-GCGGAGACAGCGACGAAGAGCUCAUCAXXXXXAGGAAGUGGCGCACCU-3′
Antisense strand: 5′-UGAUGAGCUCUUCGUCGCUGUCUCCGC UdT-3′
Antisense-stick: 5′-UACACGAGCUGCAAUGUCGGCUUUGCU XXXXXGGCUAUCUAGAAUGUAC-3′
Sense strand: 5′-CAAAGCCGACAUUGCAGCUCGUGUAUU-3′
Sense-stick: 5′-CGACAUUGCAGCUCGUGUACCAGGCXXXXXGGCUAUCUAGAAUGUAC-3′
Antisense strand: 5′-GCCUGGUACACGAGCUGCAAUGUCG UU-3′
Stick-sense: 5′-UCAAGAGACUCCUCAGUGAGAAGAA XXXXXGGCUAUCUAGAAUGUAC-3′
Antisense strand: 5′-UUCUUCUCACUGAGGAGUCUCUUGAUU-3′
The underlined nucleotides corresponds to the stick sequence. The stick sequence contains 2′-OMe–modified A and G and 2′-F–modified U and C. The 2′-F–modified U and C are highlighted as bolded text. The italic X indicates the 3 carbon linker (C3) between the aptamer/siRNA and stick sequences.
Generation and HIV-1 infection of humanized Rag2−/−γc−/− mice (RAG-hu mice). Humanized BALB/c-Rag2−/−γc−/− mice were prepared as previously described using human fetal liver-derived CD34+ cells.28,46 Briefly, neonatal mice were conditioned by irradiating at 350 rads and then injected intrahepatically with 0.5–1 × 106 human CD34+ cells. Approximately 12 weeks post-reconstitution, mice were screened for human cell engraftment. Blood was collected by tail bleeds, and RBCs were lysed using the Whole Blood Erythrocyte Lysing Kit (R&D Systems, Minneapolis, MN). The white blood cell fraction was stained with antibodies against the human pan-leukocyte marker CD45 (Caltag, a division of Invitrogen, Grand Island, NY) and FACS analyzed as described. To infect human cell reconstituted RAG-hu mice, HIV-1 NL4-3 (1.2 × 105 viral load IU/ml) in a 100 µl volume was injected intraperitoneally at least 12 weeks after cell engraftment. Viral loads were examined weekly and viremia was established in all the mice by 4 weeks. Treatment was done by intravenous injection on the last day of week 4 with 0.25 nmol experimental RNAs (4.6 µg cocktailed DsiRNAs including equal amount of three DsiRNAs: tat/rev DsiRNA, TNPO3 DsiRNA, and CD4 DsiRNA, or 14.1 µg A-1-stick–cocktailed DsiRNAs conjugates with equal amount of three DsiRNA portions) in a 40 µl volume, followed by another the next day. Later, the injections were continued on a weekly basis for 4 weeks. In the second in vivo treatment experiment, 0.25 nmol conjugates in a 40 µl volume were administered at 23 and 24 weeks of post-infection like above.
Measurement of viral load in plasma. To quantify cell-free HIV-1 by qRT-PCR, RNA was extracted from 25 to 50 µl of EDTA-treated plasma using the QIAamp Viral RNA kit (QIAGEN, Valencia, CA). cDNAs were produced with Superscript III reverse transcriptase (Invitrogen) using a primer set specific for the HIV-1 long terminal repeat sequence, and qPCR was performed with the same primer set and a long terminal repeat-specific probe using Supermix UDG (Invitrogen) as described.
Flow cytometry. Whole blood was collected and RBCs were lysed as reported previously. Peripheral blood cells were stained for hCD3-PE and hCD4-PECy5 (Caltag) markers and analyzed using a Coulter EPICS XL-MCL FACS analyzer (Beckman Coulter, Brea, CA). CD4+ T-cell levels were calculated as a ratio of the entire CD3 population (CD4 + CD3+:CD4 − CD3+). To establish baseline CD4+ T-cell ratios, all mice were analyzed before infection.
Detection of tat/rev siRNA. At 1, 3, and 9 weeks post-injection, blood samples were collected and small RNAs were isolated with MirVana miRNA isolation Kit (Applied Biosystems, Foster City, CA) according to the manufacturer's instruction. The siRNA quantification was performed using TaqMan MicroRNA Assay according to manufacturer's recommended protocol (Applied Biosystems). Ten nanogram of small RNA, 0.2 µmol/l stem-loop RT primer, RT buffer, 0.25 mmol/l dNTPs, 3.33 units/ml MultiScribe RT, and 0.25 units/ml RNase inhibitor were used in 15 µl RT reactions for 30 minutes at 16 °C, 30 minutes at 42 °C, and 5 minutes at 85 °C, using the TaqMan MicroRNA reverse transcription Kit (Applied Biosystems). For real-time PCR, 1.33 µl of cDNA, 0.2 mmol/l TaqMan Probe, 1.5 mmol/l forward primer, 0.7 mmol/l reverse primer, and TaqMan Universal PCR Master Mix were added in 20 µl reactions for 10 minutes at 95 °C and 40 cycles of 15 seconds at 95 °C and 1 minute at 60 °C. All real-time PCR experiments were done using an iCycler iQ system (Bio-Rad, Hercules, CA). Primers were as follows: Site I Looped RT primer: 5′-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACA CAG CG-3′ Site I forward primer: 5′-GCT GAT GAG CTC TTC GTC G-3′ Site I reverse primer: 5′-GTG CAG GGT CCG AGG T-3′ Site I probe primer: 5′-6- FAM- TCG CAC TGG ATA CGA CAC AGC GAC GA –BHQ1-3′. In this case, a synthetic 27-mer duplex RNA was used as positive control.
Assays of targeted gene expression. Human PBMCs were obtained from transplanted mice at 1 and 3 weeks post-injection and total RNAs were isolated with STAT-60 (TEL-TEST "B") according to the manufacturer's instructions. Residual DNA was digested using the DNA-free kit per the manufacturer's instructions (Ambion, a division of Invitrogen, Grand Island, NY). cDNA was made using 2 µg of total RNA. RT was carried out using Moloney murine leukemia virus reverse transcriptase and random primers in a 15 µl reaction according to the manufacturer's instructions (Invitrogen). Expression of the tat/rev coding RNAs was analyzed by qRT-PCR using 2× iQ SyberGreen Mastermix (BIO-RAD) and specific primer sets at a final concentration of 400 nmol/l. Gapdh expression was used for normalization of the qPCR data. Primers were as follows: IIIB or NL4-3 tat/rev forward primer: 5′-GGC GTT ACT CGA CAG AGG AG -3′ IIIB or NL4-3 tat/rev reverse primer: 5′-TGC TTT GAT AGA GAA GCT TGA TG-3′ CD4 forward primer: 5′-GCT GGA ATC CAA CAT CAA GG-3′ CD4 reverse primer: 5′-CTT CTG AAA CCG GTG AGG AC-3′ TNPO3 forward primer: 5′-CCT GGA AGG GAT GTG TGC-3′ TNPO3 reverse primer: 5′-AAA AAG GCA AAG AAG TCA CAT CA-3′ gapdh forward primer 1: 5′-CAT TGA CCT CAA CTA CAT G-3′ gapdh reverse primer 2: 5′-TCT CCA TGG TGG TGA AGA C-3′.
Interferon assays. Total RNA was isolated from PBMCs of treated mice using STAT-60. Expression of mRNAs encoding p56 (CDKL2) and OAS1 were analyzed by qRT-PCR using 2× iQ SyberGreen Mastermix (BIO-RAD) as described above and specific primer sets for these genes at final concentrations of 400 nmol/l. Primers were as follows: P56 (CDKL2) forward, 5′-TCA AGT ATG GCA AGG CTG TG-3′ P56 (CDKL2) reverse, 5′-GAG GCT CTG CTT CTG CAT CT-3′ OAS1 forward, 5′-ACC GTC TTG GAA CTG GTC AC-3′ OAS1 reverse, 5′-ATG TTC CTT GTT GGG TCA GC-3′ gapdh expression was used for normalization of the qPCR data.
To measure any induced IFN-α directly, human IFN-α1 ELISA Ready-SET-Go! (eBioscience, San Diego, CA) was used. Mice were injected with the naked cocktailed DsiRNA or ptamer-cocktailed DsiRNA conjugates. At 2 and 24 hours post-treatment, 25–50 µl of EDTA-treated plasma was collected from three mice per treatment group and from three positive control mice which had been intravenously injected with 5 µg of poly (I:C) (Sigma-Aldrich) in a 50 µl volume. Plasma levels were evaluated as per instructions supplied in the kit.
Statistical methods. The mouse viral loads and CD4:CD3 T-cell ratios were plotted by using a Lowess smoother across values. Viral loads were first log-transformed before smoothing and then anti-transformed for plotting. Missing values were imputed with a last observation carried forward scheme. The calculations were conducted as previously described.25,47
Figure S1. HIV-1 suppression by chemically synthesized aptamer (A-1-stick) and aptamer-stick-siRNA conjugates.
Figure S2. Chemically synthesized aptamer-stick-siRNA conjugates are processed by Dicer and induce mRNA cleavage.
Figure S3. 2′-Fluoro–modified aptamer-stick-siRNA conjugate in mouse serum.
Table S1. HIV-1 viral loads in individual RAG-hu mice.
Table S2. CD4 T-cell levels in individual treated and control RAG-hu mice.
Table S3. Sequence analysis of mutant virus from animals treated with aptamer-stick–cocktailed DsiRNA conjugates.
Materials and Methods.
We thank National Institutes of Health AIDS Research and Reference Reagents Program for HIV-1 related reagents used in this work. The authors would like to thank City of Hope DNA sequencing core (Harry Gao and Jinhui Wang) for Solexa Deep Sequencing, City of Hope Bioinformatics Core facility (Xiwei Wu and Haiqing Li) for prompt data analyses, and John Burnett for critical reading of the manuscript. This work was supported by National Institutes of Health RO1 grants AI057066 and AI073255 to R.A., AI29329, AI42552, and HL07470 to J.J.R. This work has also been facilitated by the infrastructure and resources provided by the Colorado Center for AIDS Research grant P30 AI054907. J.J.R. and J.Z. have an issued patent on "Cell-type specific aptamer-siRNA delivery system for HIV-1 therapy". Patent no.: US8,222,116 B2, issued date: 17 July 2012. The other authors declared no conflict of interest.
HIV-1 suppression by chemically synthesized aptamer (A-1-stick) and aptamer-stick-siRNA conjugates.
Chemically synthesized aptamer-stick-siRNA conjugates are processed by Dicer and induce mRNA cleavage.
2′-Fluoro–modified aptamer-stick-siRNA conjugate in mouse serum.
HIV-1 viral loads in individual RAG-hu mice.
CD4 T-cell levels in individual treated and control RAG-hu mice.
Sequence analysis of mutant virus from animals treated with aptamer-stick–cocktailed DsiRNA conjugates.
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text:
Giant-Size Astonishing X-Men #1 (Preview)
in Comic Previews Comment
- Story by
- Joss Whedon
- Art by
- John Cassaday
- Colors by
- Laura Martin
- Cover by
- John Cassaday
- Publisher
- Marvel Comics
Too big to be contained in a normal issue, the grand finale of Joss Whedon (Buffy, Firefly) and John Cassaday's (CAPTAIN AMERICA, Planetary) landmark run is right here! The powerlords of Breakworld bring the fight to Earth. Can the X-Men stop them from destroying the whole planet? Will the Avengers join their fight? What we do know is this: one of the X-Men won't walk away from this fight. Plus spotlight interviews with Whedon and Cassaday!


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ECA has 230 members from 54 associations
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Third edition of the ESSMA Summit successfully held in Lyon
Wednesday, 18 01 2017
The third annual ESSMA Summit took place at the newly built Parc Olympique Lyonnais from the 16th to the 18th of January. Organised by the European Stadium & Safety Management Association (ESSMA), the three-day event saw the participation of over 350 delegates, a 20% increase from last year's Summit in Bilbao.For three days, the venue, home of ECA Member Club Olympique Lyonnais, hosted not only many ECA Member Clubs representatives, but also professionals from the stadium industry and representatives from other clubs, leagues and national associations, as well as experts from a range of other sports.This year again, the European Club Association was actively involved in the organisation of the event as official partner, alongside the European Professional Football Leagues (EPFL) and the International Centre for Sport Security (ICSS). The main purpose of the Summit was to allow experts from the stadium business to gather and exchange best practices on the latest trends in the industry, notably including fan entertainment, safety & security, management & commercial exploitation as well as pitch management.The first day was entirely dedicated to a Stadium Development Seminar, with keynote speeches from stadium experts notably representing FC Bayern München, Feyenoord Rotterdam, FK Austria Wien and HNK Rijeka as well as the Italian Serie B. To end the day, an in-depth tour of the Parc Olympique Lyonnais was organised for all participants.The second day was opened by the welcome words of Olympique Lyonnais Chairman and ECA Executive Board Member Jean-Michel Aulas, following which a detailed presentation by UEFA on the EURO 2016 & 2020 tournaments was given. The day was completed by presentations from representatives of Olympique Lyonnais, Coca-Cola, US Bank Stadium in Minneapolis (USA) as well as a panel discussion on stadium development in France.As for the third and last day, twelve different workshops divided in four main categories such as Fan Entertainment, Stadium Management, Safety Management and Pitch Management, were organised. ECA Member Clubs actively contributed to the success of these workshops, notably thanks to the presentations of Arsenal FC, Olympique Lyonnais, Malmö FF, Ferencvárosi TC, as well as the Allianz Stadion in Vienna and the Amsterdam ArenA. A final visit of the Olympique Lyonnais training centre concluded the successful ESSMA Summit 2017.The next edition of the ESSMA Summit will be held at the iconic Aviva Stadium in Dublin, Rep. of Ireland, in January 2018. More information can be found on the ESSMA official website.
ECA celebrates its 10th anniversary
ESSMA Summit 2018 successfully held in Dublin
Second edition of the ECA CMP continues in Bilbao
Athletic Club to host second session of the ECA CMP
2016/17 Financial Fair Play fines redistributed to clubs
Leading the way for football clubs in Europe


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Portugal's debt credit rating is cut to junk by Fitch
PORTUGAL'S credit rating was cut to below investment grade by Fitch Ratings due to the country's rising debt level and weakening economy.
The long-term rating was lowered one level from BB+ to BBB- with a negative outlook, Fitch said yesterday. Portuguese 10-year bonds fell after the announcement, with the yield at 12.14pc.
"The country's large fiscal imbalances, high indebtedness across all sectors, and adverse macroeconomic outlook mean the sovereign's credit profile is no longer consistent with an investment-grade rating," Fitch said.
The ratings of utility EDP-Energias de Portugal and telecommunications company Portugal Telecom are unaffected, Fitch said.
Prime Minister Pedro Passos Coelho is cutting spending and raising taxes to meet the terms of a €78bn bailout plan from the European Union and the IMF.
As the country's borrowing costs surged, Portugal followed Greece and Ireland in April in seeking a bailout and now aims to return to bond markets in 2013.
Barclays Capital said it would cut Portugal's government bonds from its Euro Treasury Index by December after the Fitch announcement. The move makes the bonds "ineligible" for inclusion in the index, Huw Worthington, a fixed-income strategist in London, said.
Bond indexes
Citigroup said it would keep Portugal's bonds in its indexes after the downgrade. The company's bonds indexes use rankings from S&P and Moody's, said Nishay Patel, a fixed-income strategist at Citigroup in London. "A one-notch downgrade from S&P will take Portugal out of the index."
S&P cut the country's rating twice in March to BBB-, one level above junk status, and has a negative outlook. Portugal's rating was cut to below investment grade in July by Moody's. The long-term government bond ratings were lowered to Ba2 from Baa1.
The government aims to trim the budget deficit from 9.8pc of GDP in 2010 to 5.9pc in 2011, 4.5pc in 2012, and to the EU ceiling of 3pc in 2013. Debt will reach 100.8pc of GDP this year and peak at 106.8pc in 2013 before starting to decline, the government forecast. Debt was 93.3pc of GDP in 2010.
Fitch said its "base case" was that government debt would increase to around 110pc of GDP at the end of 2011 and peak at around 116pc at the end of 2013.
The rating company expects Portugal to meet its deficit targets for this year and next year.
"The 2012 budget contains significant expenditure reductions, mainly on pensions and civil-service pay," it said in the statement.
Portuguese Finance Minister Vitor Gaspar on November 16 said the second quarterly review of its financial-aid programme was "successful", allowing it to receive another rescue payment tranche of €8bn.
The so-called troika of the European Commission, ECB and IMF said that Portugal's plan was "off to a good start", adding that the 2012 budget includes "bold and welcome measures". (Additional reporting Bloomberg)
Irish Independent