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Best Bar restaurants nearby. Discover the best Bar food in Shasta Lake. View Menus, Photos and Reviews for Bar restaurants near you.. The City of Shasta Lake (prior to incorporation it was known as Central Valley or CV) is a city in Shasta County, California, United States. It is the closest settlement to Shasta Dam and Shasta Lake reservoir, which are popular tourist destinations. The population was 10,164 at the 2010 census, up from 9,008 at the 2000 census.
Things to do in Shasta Lake
4 based on 74 reviews
While at dinner, a customer got into a fight with a employee, cops were called, later the thugs came back to throw rocks and break windows. Stay away
3 based on 14 reviews
Our prime rib was supposed to be medium, but was closer to well. Mashed potatoes and gravy tasted like packaged food. The ice cream served with our ice cream was nearly completely melted. $85.00 for two mediocre prime rib dinners...
4 based on 58 reviews
This is always a packed house with friendly faces and great drinks. Their food is amazing also! Really a fun place.
4 based on 51 reviews
Located in downtown Redding, CA this eatery has been planted here for several years. Lots of menu options from appetizers, salads, burgers, sandwiches and pizzas. Also many local beers on tap and in bottles. Tuesday is 'half off burger' day. Great management and staff.
3 based on 59 reviews
Shasta Lake Dining at Bridge Bay Marina Come enjoy a memorable, waterfront Shasta Lake dining experience at Bridge Bay, offering savory meals, welcoming staff and panoramic views. If you are in Redding, California, drive up for one of the best meals you w
amazing locale - excellent staff! food & kitchen not so much. an aching need for an actual chef to take over what is an outdated miserable kitchen. bar is excellent, and i even hope with a for real chef, the wine selection will also be upgraded. well, one can hope. i am a local and keep my boat here. bottom line, good bar but food sux.
4 based on 48 reviews
Vintage Public House it is a gathering place for people to get together and enjoy excellent food, drink, and live music. We offer Modern American Fare, that branches across many different cuisines, signature cocktails, 8 taps with rotating craft beer, and
My wife and I were visiting her family in Redding and they had not been here so off we went. Everything we had was terrific! Maple brown sugar sirloin, Risotta and prawns (way too much risotta!), salmon, fish&chips, and pork chops! Great value and portions. The desert Churro Apple Pie bowl, suggested by the waitress, was good. Perhaps another choice might have been better. The waitress seemed new, good thing it was not very busy.Worth the visit, make it busy!
4 based on 342 reviews
C.R Gibbs is located in the heart of Redding and is an open exhibition kitchen with a full bar, banquet facilities and American Grille Style Cuisine.
Our large tour group had made arrangements for lunch at CR Gibbs. We had our food pre-ordered and dined in the private dining room. We had our food quickly and my salad was a very generous size and so delicious. Appreciated the friendly service and inviting decor.
4 based on 80 reviews
We were staying at the Sheraton Sundial Bridge and this restaurant is right next door. Very convenient. Our server was awesome! The food was very good, and fresh. Beautiful atmosphere, lovely decor.
4 based on 508 reviews
Having visited Cattleman's twice when we were in Redding a year ago, since deciding to return in 2019 we have been looking forward to visiting Cattleman's on this visit.So, how was it? Well, I have to confess better than ever!My wife repeat-ordered the Steak and Shrimp Scampi, whilst I went for the Rustler Rib Eye on this occasion - delightful all round! The steak was sweet to the palate, cut like salmon, and tasted of the stuff of dreams!!I know of no other place which has steak like this place!
4 based on 256 reviews
Stopped in there for the first time and was quite impressed. Had a very good beer selection. Tried 2 different types and enjoyed them both. Had the large pretzel with beer cheese and it tasted great. When I'm in the area again and will definitely stop in again!
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These two rather tall styrofoam dragons were created to introduce the idea of outdoor sculpture to Burlington, Massachusetts. Hence, they are the godparents of the Burlington Sculpture Park.
They come out to play from time to time when there is a dragon-friendly event in town.
Boris and Natasha have each been outfitted with a dragon-wagon with wheels, which allows draggin’ the dragon wagons in parades. Check with us for availability in your next parade.

domain: artistic


text:
Researchers at the University of Sheffield have identified a new pathway that is disrupted in motor neurone disease (MND), a discovery which could pave the way for new treatments.
The ground breaking findings were discovered by converting skin cells to brain cells called astrocytes, and comparing the energy metabolism of cells taken from MND patients versus healthy controls, using a new metabolic phenotyping technology developed by Biolog, Inc, a Californian biotech company.
The scientists from the University of Sheffield’s Institute for Translational Neuroscience (SITraN) discovered the loss of a key enzyme that could have major consequences for how the central nervous system copes with ageing, stress and loss of energy metabolism.
MND, also known as amyotrophic lateral sclerosis (ALS), is an adult onset disorder involving loss of motor neurones that control skeletal muscles. At some point in a patient’s life, their motor neurones start to die, leading to muscle wasting and eventually death – typically within two years of diagnosis.
Disruption in the ability of central nervous system to create energy is thought to be a major contributing factor to the disease and could influence disease progression rates. Six people every day die of the disease in the UK and there is currently no cure.
The Sheffield scientists led by Dr. Scott Allen, Dr. Laura Ferraiuolo and Professor Dame Pamela Shaw from SITraN, used a method previously developed by Drs Ferraiuolo and Meyer in the USA to take skin cells from patients, reprogramme them into brain cells and observe them to identify novel pathways of metabolic dysfunction, which has never been done before.
Working in collaboration with the US-based company Biolog, Inc., a world leader in cell analysis, they employed a metabolic scanning technology to look for and find differences in the metabolism of astrocytes from MND patients. Astrocytes are star-shaped brain cells that play a key role in supporting motor neurones by acting as a crucial source of energy in the central nervous system.
Dr. Allen, who is a Motor Neurone Disease Association funded Senior Research Fellow, found that cells from MND patients had reduced ability to turn a metabolic substrate called adenosine into energy due to loss of a key enzyme called adenosine deaminase.
The consequence of this loss could be a toxic build-up of adenosine in the central nervous system and subsequent loss of inosine production, a metabolic intermediate that is generally protective. Until now, the link between MND and inosine production through the loss of adenosine deaminase had not been made. When Dr. Allen fed the brain astrocytes inosine, energy production increased and the patient’s astrocytes became more supportive towards motor neurones, helping them live longer.
“We are really excited about this set of results as no one has implicated adenosine deaminase in MND before,” said Dr. Allen.
“Our results indicate that the higher the level of adenosine deaminase, the greater the protection against adenosine mediated toxicity and the greater support towards motor neurones when given inosine.
“Although we are at an early stage, I think increasing adenosine deaminase levels, combined with inosine supplementation has the potential to slow down disease progression in MND patients. But a lot of further testing in the laboratory has to be performed.”
He added: “We have had fantastic support from Biolog and the work would not have been possible with charitable funding from Neurocare and the Motor Neurone Disease Association. I am looking forward to following up on these exciting results.”
Altering the level of adenosine deaminase by gene therapy has previously been shown to be beneficial and safe in patients suffering from severe combined immunodeficiency disease. Inosine is a safe and readily-available nutritional supplement, which has been successfully tested in Parkinson’s disease patients. There is therefore hope that, in the future, this combination of treatment could work in people with MND, improving their quality of life and helping them live longer.
Dr. Barry Bochner, Chief Executive Officer of Biolog, said: “The Biolog cell analysis technology was developed with the intent of providing scientists with a new approach for comparing normal versus disease cells to look for differences that could underlie the deficiencies.
Source: Read Full Article

domain: biology


text: This paper presents a rear obstacle detection system based on depth information obtained from Kinect sensor. The proposed system can be used for parking assistance applications and backup aid. It improves the false detection rate in single view based algorithms by using depth information. In addition, real distance to obstacles can be calculated by depth information. It is possible to alarm to driver effectively according to distance. Although the proposed system is based on conventional edge detection algorithm, we confirm the feasibility of combining depth map with color image by improving the accuracy of detection. Experimental results show that depth fusion based obstacle detection algorithm is more accurate than single view based algorithms in terms of false detection rate.

domain: literature


Sex. Everyone knows what it is and how it works. But ask most people how they learned about it and few will probably tell you that they learned from their parents. That just seems awful! But actor and director Anthony Anderson, he learned about sex from his mother...in extreme detail!
Recently while appearing on Conan, Anderson told the incredible story about how, of all people, his MOTHER taught him how to perform...oral sex. What the hell? Eating the cookie. Going downtown. Knocking the man out of the boat. Call it what you want, but Anderson's mother taught him all about it. Listen to his incredible story below and think about that next time you're sitting down to have a heart-to-heart with your mom!


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The impact of COVID-19 has left pupils with Education, Health and Care Plans (EHCPs) attending special schools and colleges, around four months behind in academic development and five months behind with their wider development. This is according to new findings from ASK Research, supported by the National Foundation for Educational Research (NFER), a report of which Research Partner’s Claire Tyers is a key author.
The policy briefing, funded by the Nuffield Foundation, highlights the results of a survey of 192 headteachers as well as in-depth interviews with 40 specialist headteachers and 40 parents and carers of children and young people who attend specialist settings. The interviews, conducted in April-June 2021, aim to understand the continuing implications of the COVID-19 pandemic on pupils in special schools and colleges in England.
The report makes several key recommendations for government, locally and nationally. An effective recovery for special schools and pupils should:
- Focus on more than educational attainment.
- Specifically address emotional wellbeing and mental health – of pupils and staff.
- Increase health and care input for pupils with EHCPs.
- Extend support to families – ensuring they also recover and are able to support their children.
- Be informed by experts – trusting headteachers to decide what their setting needs and how best to allocate funding.
- Allow sufficient time for real recovery – not just offering a ‘one off’ or short-term solution.
- Address pre-existing funding shortfalls in SEND, which will have been exacerbated by the changes brought about by the pandemic.
Find out more on Nuffield’s website.

domain: sociology


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Iowa has witnessed a steady rise in drug and alcohol abuse over the years, mirroring a broader national crisis. As a community, it’s crucial to address this problem head-on, and one of the most effective ways to do that is through targeted interventions. If you’re seeking an addiction interventionist Iowa has various options available.
Understanding Iowa’s Substance Abuse Landscape
In Iowa, the rates of opioid and methamphetamine abuse have been particularly concerning. This problem isn’t just confined to urban areas; rural communities are equally affected.
Local experts and services, such as Addiction Interventionist Iowa, understand the unique factors contributing to substance abuse in Iowa, making them an invaluable resource when considering an intervention.
How an Addiction Interventionist Can Help
An interventionist acts as a mediator, guiding families through the emotionally charged process of confronting a loved one about their addiction. In Iowa, where family values often run deep, interventions can be a delicate matter.
A certified addiction interventionist Iowa offers can be the key to turning a confrontational, unproductive family meeting into a lifeline for someone you love.
Preparing for an Intervention in Iowa: Essential Steps
A successful intervention requires planning. This includes everything from the venue to the list of attendees and what each person will say.
Addiction Interventionist Iowa can assist you in the preparation phase, ensuring that everything is set for the intervention to be as effective as possible.
The Role of Family and Friends in an Intervention
Family and friends play an indispensable role in interventions. As the people closest to the individual suffering from addiction, their support can be crucial.
However, it’s essential to approach the intervention with a unified message, something an addiction interventionist Iowa specializes in.
Intervention Strategies: What Works in Iowa
Every culture and community have specific taboos and social norms surrounding addiction. Being aware of these can influence the effectiveness of different intervention strategies.
In Iowa, for instance, an open and non-confrontational approach may be more effective, something the experienced teams at Addiction Interventionist Iowa are well-versed in.
After the Intervention: What Comes Next?
The intervention is only the first step in the long road to recovery. After this, your loved one will need continuous support and possibly professional treatment.
Fortunately, in Iowa, there are many post-intervention services, making the transition smoother for both the addicted individual and their family.
Overcoming the Stigma of Addiction in Iowa
In many communities across Iowa, there exists a stigma surrounding addiction, which often prevents people from seeking help. This cultural aspect should not be overlooked when considering an intervention.
It’s important to approach the situation with empathy and understanding, making it clear that the aim is to help rather than criticize or ostracize. Breaking down these barriers of shame and silence is another area where the expert guidance from Addiction Interventionist Iowa can make a significant difference.
Resources and Support Networks in Iowa
Iowa boasts a robust network of support systems and resources for those struggling with addiction and their families. From state-funded programs to non-profit organizations, there’s a wealth of options available to assist in the recovery process.
Addiction Interventionist Iowa can help identify the most appropriate resources tailored to your loved one’s specific needs, offering that essential local touch. Our comprehensive approach ensures that you are never alone in this journey, from intervention planning to post-treatment support.
Final Thoughts: Making the Right Choice
When facing the arduous task of planning an intervention, don’t underestimate the value of local expertise. An addiction interventionist Iowa has to offer can provide invaluable insight into the state’s unique challenges and solutions when it comes to substance abuse.
Reach out to Addiction Interventionist Iowa today, and let us guide you through this challenging but rewarding journey.
Newport Beach CA 92660 US

domain: psychology


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15 May 2003We are pleased to announce that Eric Clapton has agreed to write the foreword for our forthcoming book on Bob Marley with Kate Simon. He will also be signing the 350 Deluxe copies. In 1974 Eric Clapton took a cover version of 'I Shot The Sheriff' to the top of charts worldwide, boosting Bob Marley's already fast-rising star into the stratosphere.
Since 1974 Genesis has created signed limited edition books on behalf of authors and artists ranging from the Beatles to Buckingham Palace.
Genesis news and special offers direct to inbox.

domain: entertainment


text: A concise, pocket-sized guide to physical examination and companion to Seidel et al, Mosby's Guide to Physical Examination, 5th Edition, this beautifully illustrated, full-color clinical manual provides convenient, portable access to comprehensive guidance in physical examination. A dynamic author team gives the Handbook an uncommonly strong multidisciplinary perspective, emphasis on differential diagnosis, and expert coverage of both pediatrics and adult health. Color-coded type is used to make examination steps, expected findings, and unexpected findings easy to spot quickly. And now, a new appendix puts a variety of guides to special patient histories at readers' fingertips.Each chapter begins with a concise, clearly identified equipment list.Easy-to-follow, color-coded tables summarize the core examination content in each chapter. "Examination Techniques" appear in red, "Expected Findings" appear in blue, and "Unexpected Findings" appear in green."Pediatric Variations" tables, also color coded for easy reference, highlight differences in younger patients' disease presentations."Sample Documentation" sections at the end of each chapter provide straightforward examples of good documentation practice.A removable, pocket-sized quick-reference card inside the back cover presents a comprehensive at-a-glance reminder list of key elements of assessment for every body system and region. One side covers "Physical Examination-Objective Data," and the other covers "History-Subjective Data."Pages for Clinical and Reference Notes are interspersed throughout the book for personal clinical note-taking.Handy, two-column "Aids to Differential Diagnosis" tables summarize abnormalities and their distinguishing characteristics.

domain: library


text:
My cock throbbed so heavily that it was almost painful. I hold her wrists overhead as we do it, or tug her hair or flip her from one position to another and she loves all this. Enter your mobile number or email address below and we'll send you a link to download the free Kindle App. I can tell this is a good orgasm by the way the first two loads shoot out. I published the six novels that I had written in my spare time over the years to see if this was going to be something that I wanted to pursue. At one point, the woman made a reference to using a strap on on him and he had a low-level meltdown. So I keep this to myself.
Posts Tagged ‘erotic short stories’
Prepaid codes are delivered to you via email as soon as payment has been approved. She did not believe that she was being ignored, but rather that the man was unable to think of anything to say for himself. Audible Download Audio Books. Best friend wants me to pick wedding dress. In a last, desperate attempt to flatter, the man stuttered and forced a smile.
Lawrence Schimel : Water Taxi. Short Story (Gay Erotica)
Erotic novels For non-narrative erotic fiction see also Category: Fab, so glad you enjoyed, thanks for commenting sorry for the crazy long time in replying! I lift her skirt up and rip her knickers off. Shouldering my bag, I grabbed the glass of wine, turned and stepped forwards, slamming straight into a solid wall of man in front of me. If we say In Stock , we dispatch within the time frame of the shipping plan you selected. I can see she wants to use her hands to get to me. Ethan set my bag on the bed and turned towards me.
Createspace Independent Pub; 1 edition 22 October Language: Thank you for your hard work! Online Erotic Fiction Please note that I put my fiction online, for free, in order to initiate a dialogue with readers. Rick glided his hands over the tops of my thighs under the table. Mouth to mouth -rescued from the sea by two hunky Californian lifeguards, Amy gets a lot more than she bargained for She wants me to know that she belongs to me, that she submits to my sexual desires.


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Shenandoah National Park
10 Great Hikes in Shenandoah National Park
9 Amazing Things to Do in Shenandoah National Park
How to Hike Old Rag in Shenandoah National Park
Places to Visit Near Virginia
20 Amazing Things to Do in New River Gorge National Park
A Weekend Getaway to Asheville, North Carolina
A Weekend in Gatlinburg and Great Smoky Mountains National Park
USA Travel Inspiration
Best National Parks in the USA: 15 Amazing Parks for Your Bucket List
Best USA Road Trips: 18 Ideas for Your Next Big Adventure
Ultimate US National Parks List for 2023 (+ Printable Checklist)
Top Experiences in Virginia
Shenandoah National Park. Shenandoah National Park holds a special place in our hearts. Just two hours from where we live, we have spent more time here than any other national park in the United States. Shenandoah contains 500 miles of hiking trails with ranging difficulties. You can choose between short summit hikes, gorgeous walks to waterfalls, or you can tackle challenging climbs up the tallest mountains. You also have the option to walk a few miles of the legendary Appalachian Trail.
Hike Old Rag in Shenandoah. Old Rag is the toughest but the most rewarding hike in Shenandoah National Park. Some people say it’s the best hike in the mid-Atlantic region.
Blue Ridge Parkway. Virginia’s Blue Ridge Parkway is a scenic drive along the Appalachian and Blue Ridge Mountains. The drive is rated one of America’s most beautiful drives. Along the Parkway there are spots to hike, bike, camp, and fish in the gorgeous mountains.
Hike McAfee Knob. McAfee Knob is one of the most popular hikes in Virginia. It’s also the most photographed spot along the Appalachian Trail. For hikers, this is a great one to add your list.
Roanoke. Set in the Blue Ridge Mountains, Roanoke is a charming city that bustles with outdoor enthusiasts. There are great places to hike, bike, and rock-climb in the mountains that lay just outside your door. Nearby lays Smith Mountain Lake which is a popular spot to go fishing, boating, and water-sporting.
Colonial Williamsburg. Colonial Williamsburg was once one of the most politically active cities that fought for American independence. Patrick Henry gave his famous speech here, and many Founding Fathers walked the city streets. Today, 18th century buildings have been restored and actors play roles of farmers and slaves to portray what life was like at the time of the Revolution.
Arlington National Cemetery. The Arlington National Cemetery holds over 600 acres of land in which the fallen soldiers of the nation’s conflicts are buried. The Tomb of the Unknown Soldier stands in this cemetery, and the tomb contains the remains of unidentifiable soldiers from both World Wars, the Korean War, and the Vietnam War. The grave of President John F. Kennedy also lays in the Arlington National Cemetery.
Alexandria. Alexandria is an idyllic city with preserved colonial architecture and much history to experience. Stroll through the Old Town, shop at boutiques, and dine on the waterfront. Neighboring Alexandria is Mount Vernon, George Washington’s estate.
Mount Vernon. Mount Vernon was George Washington’s home from 1754 to 1799. Walk the grounds of the historic home and come inside to see portraits, family belongings, and Washington’s vivid design choices. You can also see George Washington and his family’s tomb at his Mount Vernon estate.
Virginia Beach. Come relax at Virginia Beach, a 14-mile golden expanse bustling with families, friends, and excited kids. Amusement parks, mini golf, seafood restaurants, creameries, and shopping centers line the beach. Additionally, you can visit neighboring parks and natural areas to go hiking and kayaking.
Richmond. Visit Virginia’s capital, a bustling hub for history and the arts. Drive Monument Avenue, tour the American Civil War Museum, take a haunted ghost tour, and bar-hop Richmond’s acclaimed pubs.
Manassas National Battlefield. Manassas was the site of the first major land battle in the Civil War. 13 months later, an even bloodier battle was fought here. Taking a tour of the battlefield and learning about the history of these two battles is an eye-opening experience and a must-see for anyone interested in American History.
Luray Caverns. Enter the eastern United States’ largest cave system. Walk along layers of platforms past dripping stalactites, rising stalagmites, and vibrant rock formations. You can hear the world’s only Stalactite Organ create its symphonic music as you travel through the deep caverns.
Natural Bridge State Park. Virginia’s Natural Bridge is a massive 215-foot limestone archway carved out by Cedar Creek. You can walk along a flat trail beneath the towering bridge and continue onwards a short way to reach a Monacan Indian Exhibition.
Chincoteague Island. Home to the famous Chincoteague ponies that roam free on the beach, Chincoteague Island is a beautiful and fun get-away destination. The island offers biking, kayaking, surfing, and swimming locations. You can also cross over to Assateague which is on the Maryland side of the island.
Chesapeake Bay Bridge Tunnel. This 17.6-mile-long bridge connects the Virginia mainland with the Virginia’s Eastern Shore. The bridge even goes underwater in two spots, making this bridge an engineering marvel.

domain: geography


Hinton on the Green & Aston Somerville is inviting applications for a part time lengthsman.
The post involves a maximum of 14 hours per month at £12.00 per hour. All work undertaken is to be agreed by the Parish Council.
For an application form and job description, please contact:
Clerk to the Parish Council


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How does one choose a perfect New Year’s Eve destination?
It all comes down to taste and personal preference. Whether you’re in for beach parties, crowded venues and spectacular fireworks, public gatherings and music concerts or in running away to a secluded place where hardly anyone can bother you, the planet we live on offers great choices for everyone.
Unlike Sir Thomas More who tried to appeal to everyone’s tastes in his Utopia – and found that tastes differ so widely that one would do far better to relax and enjoy life that trying to instruct or entertain a public – we would, for a change, like to suggest a sundry assortment of places that you probably won’t find in any of popular CNN’s lists.
London, Dubai, New York and other prominent places definitely are everyone’s favourite choice but a mere scratch beneath surface would help you unearth some hidden gems that will potentially make your New Year’s Eve 2019 one to remember.
There’s hardly a better place to start the new calendar year than in Tonga. Why? The reason is quite simple as this South Pacific’s only remaining Kingdom is actually one of the first places on the planet to welcome the New Year. Whereas for many people around the globe New Year is associated with snow and low temperatures, the Polynesian island of Tonga offers a unique chance to ring in the new calendar year barefoot at white sand beaches, crystal-clear waters and tropical temperatures. It’s also a perfect place for to hide from the busy everyday life and relax in an almost intact world. There are a number of activities to choose from in Tonga, starting from swimming with whales, diving, snorkelling, to bird watching and exploring the island’s unique history and culture.
Punta Del Este
For those who are just not ready to give up the summery feeling, there is a hidden gem on the opposite side of the globe – a place that has become one of the preferred destinations for thrill seekers from Argentina, Brazil, America and Europe as well. Uruguay’s Punta del Este is packed with a lively variety of options, starting from restaurants, nightclubs and serene scenery to admire. Punta del Este is a city where you can find anything that crosses your mind. One of the favourite New Year’s Eve attractions is the spectacle of fireworks on the promenade at midnight and the increasingly popular practice of staying up to watch the very first sunrise of the New Year.
Bulky Balkans might not be the first choice on your travel list but the buzzing Belgrade has been widely hailed as one of the liveliest cities in this part of the world. Steeped in a bloody history of constant occupation and oppression, Serbia’s capital is a unique mix of different style – a sensual combination of east and west and Europe’s hottest new destinations. Belgrade’s bursting nightlife offers a diversity of clubs, taverns, cafes and particularly popular river raft discotheques. The city’s center will host a music concert and it would be almost impossible to get away from the New Year’s holiday feeling. Belgrade is also home to a lively gaming scene with plenty of establishments to visit across the city. If you’re more of an online type of a gamer, you can always head over to a Live Casino straight from the comfort of your Belgrade accommodation and try your luck in a table game streamed live from one of European top-class tables.
The charming capital of Iceland has become quite a reputable place for travelers who are on the lookout for a unique atmosphere. There’s something raw and Viking-like about the Nordic nation – perhaps it has something to do with community bonfires which are a traditional Icelandic New Year’s Eve event. The rural feeling will quickly disappear as you step into the post-midnight party scene with various bars and nightclubs serving a perfect experience to follow the midnight fireworks. A lot of them. Reykjavik is called the party capital of the world and is definitely worth checking for yourself.
Arguably one of the most surprising places on the list, Niagara Falls is a fantastic place to celebrate your New Year’s Eve. This wonder of the world throws multiple events around the festive season. The outdoor celebration programme kicks off at Queen Victoria Park at 8:00 p.m. and includes a magnificent fireworks show launched from the Niagara Gorge at midnight. The show also includes a New Year’s concert and a New Year’s Eve Gala, making the big celebration such an action-packed event where all different tastes will be satisfied.

domain: travel


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This excellent article appeared in the Newsletter for the Mundabiddi Trail and is reprinted here with permission from the author.
AVOIDING AND TREATING MOUNTAIN BIKE INJURIES
By Stuart Gray, Munda Biddi Trail Foundation Member
THAT EXTRA 10%.....or SHOULD BLOKES WEAR TIGHTS??
Anyone with even the most remote interest in cycling would have been glued to the TV during coverage of the Tour de France. Who could fail to be impressed by the examples of extreme endurance and bravery of the athletes pushing the pedals for hundreds of kilometres each day, up mountains, down mountains at 90 kph, in heat, cold, wet, and then picking themselves up only to cycle on after horrific prangs.
Can you imagine picking the scabs off your wounds each day to allow for more joint and muscle extensibility? Can you visualise cycling eight stages of the tour with a fractured coccyx, or completing the entire distance with broken ribs or clavicle? Some might say that is giving 110%!! How awe-inspiring is Lance Armstrong? Winning seven Tours and overcoming metastatic cancer twice must be a good example of 110% effort.
It is Armstrong’s brush with cancer that provides the clue to another extra 10% that is rarely mentioned by name. Cancer usually spreads through the lymphatic vessels. The lymphatics tend to get the same consideration as our roof gutters and down-pipes, that is, we tend to forget them until water starts seeping through the ceiling. Armstrong’s cardiac output, which is twice the volume of the average male, pumps blood to his working muscles and after giving up oxygen and nutrients, 90% of blood and fluid is drained from the limbs by the veins whilst the other important 10% of high protein fluid is drained by the lymphatics. The lymphatic vessels filter the fluid (lymph) and return it to the blood to become plasma again. Without this occurring, blood pressure and blood volume would rapidly decrease, leading to serious health threats.
After a day in the saddle, fluid tends to pool in the lower limbs and even Armstrong’s exceptional physiology needs some assistance in order to recover enough to do the same riding the next day. This assistance comes in the form of a warm down on the wind trainer, rehydration by drinking water and electrolyte drinks, a shower, a high carbohydrate meal, manual lymph drainage massage (gentle effleurage and pettrisage) in elevation, and a compression garment. The latter could be in the form of stretch material cycling long-johns or Sport Skins which were initially made popular by Steve Waugh who was guarding against Deep Vein Thrombosis. Today most league football teams and our national cricket team wear these garments to assist venous and lymphatic return and thereby hasten recovery from competition and training.
For the rest of us mere mortals it would be beneficial to wear some compression during and after exercise. Those of us with some deficiency of the veins or lymphatics could suffer swelling (oedema) without this support.
When cycling the Munda Biddi Trail end-to-end with full panniers and having to push a heavily laden bike up numerous rocky hills, the legs will often feel tired, heavy and congested. The campsites do not have the luxury of showers or a resident masseur so the recovery routine should be to rehydrate, have a wash while your pasta is cooking, put on your compressions skins and elevate your legs while doing some gentle pumping and stretching exercises.
After a week in the saddle you might be happy to wear tights!

domain: health


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Sunday Chicken Stew
Total TimePrep: 30 min. Cook: 6-1/2 hours
- 1/2 cup all-purpose flour
- 1 teaspoon salt
- 1/2 teaspoon white pepper
- 1 broiler/fryer chicken (3 pounds), cut up and skin removed
- 2 tablespoons canola oil
- 3 cups chicken broth
- 6 large carrots, cut into 1-inch pieces
- 2 celery ribs, cut into 1/2-inch pieces
- 1 large sweet onion, thinly sliced
- 1 teaspoon dried rosemary, crushed
- 1-1/2 cups frozen peas
- 1 cup all-purpose flour
- 2 teaspoons baking powder
- 1/2 teaspoon salt
- 1/2 teaspoon dried rosemary, crushed
- 1 large egg, lightly beaten
- 1/2 cup 2% milk
- In a large shallow dish, combine the flour, salt and pepper; add chicken, a few pieces at a time, and turn to coat. In a large skillet, brown chicken in oil; remove and keep warm. Gradually add broth to the skillet; bring to a boil.
- In a 5-qt. slow cooker, layer the carrots, celery and onion; sprinkle with rosemary. Add the chicken and hot broth. Cover and cook on low until chicken and vegetables are tender and stew is bubbling, 6-8 hours.
- Remove chicken; when cool enough to handle, remove meat from the bones and discard bones. Cut meat into bite-size pieces and return to the slow cooker. Stir in peas.
- For dumplings, in a small bowl, combine the flour, baking powder, salt and rosemary. Combine the egg and milk; stir into dry ingredients. Drop by heaping teaspoonfuls onto simmering chicken mixture. Cover and cook on high (do not lift the cover while simmering) for 25-30 minutes or until a toothpick inserted in a dumpling comes out clean.
Nutrition Facts1 each: 420 calories, 13g fat (3g saturated fat), 113mg cholesterol, 1403mg sodium, 42g carbohydrate (10g sugars, 5g fiber), 33g protein.
Sep 27, 2017
This was a hearty dish but found that the Rosemary was pretty overpowering. It was not a favorite in our household.
Jul 2, 2017
The stew smells so good while it's cooking. It has a wonderful flavor!!
Apr 4, 2014
I have been making this one for several years. The only thing I do differently is double the recipe and leave out the pea's. Just personal preference. Wonderful.
Sep 18, 2013
Perfect, homey, comfort food, chicken stew. I used chicken thighs (it's what I had on hand) and the chicken turned out so moist and the dumplings were awesome. The smell while it was cooking was also divine. We will be making this one again.
Aug 20, 2012
This is a very easy recipe..I have made it twice and the chicken falls off the bone. My family loves the dumplings. I added sweet potatoes.
Jul 15, 2012
I found this recipe in the magazine years ago and I love it. It is a stew and is NOT meant to be creamy. I always make it with chicken thighs and have cooked it with bone in and bone out, depending on what I have around. Do not expect it to be overly flavorful, as the rosemary is the star of this show.
Mar 19, 2012
This was just ok. Even with all of the seasonings, (even adding poultry seasoning, seasoning the chicken, adding the pan juices, etc.), it was STILL bland. I was really suprised. The veggies and chicken cooked well, but not very flavorful. Won't make this one again.....too time consuming for just an average dish.
Feb 18, 2012
My husband and I thought this was pretty good, but not great. I think I'll go back to my standard chicken and dumplings although I may make these dumplings instead of my regular. This recipe was filling, but my personal preference is for a creamier, soupier chicken and dumplings.
Jan 19, 2012
It was good, and flavorful, but didn't make enough. Of course my husband made it and followed the recipe to a "T", so unfortunately all the leftovers I was looking forward to are just a pipe dream. If I was to make it again I would double or triple the recipe. I have a family of six, 2 adults, 2 adult sized teen boys, and a 10 and 8 yr old. I need a LOT of food to feed the whole gang.
Jan 13, 2012
I have been preparing this recipe for my family for a couple years now. It is one of our favorite winter comfort dishes.I do make a couple small changes. First of all, I buy boneless, skinless chicken thighs. This saves time in having to do the deboning process. 2nd, I put all the vegies in my crockpot and poor the broth right on top. Brown the chicken and put on top of everything else. I have found no difference in heating the broth before pouring it over all ingredient. Last, my family doesn't like peas so I substitue fresh green beans.Yum Yum

domain: food


text:
Since 2003, H5N1 influenza viruses have caused over 400 known cases of human infection with a mortality rate greater than 60%. Most of these cases resulted from direct contact with virus-contaminated poultry or poultry products. Although only limited human-to-human transmission has been reported to date, it is feared that efficient human-to-human transmission of H5N1 viruses has the potential to cause a pandemic of disastrous proportions. The genetic basis for H5N1 viral transmission among humans is largely unknown. In this study, we used guinea pigs as a mammalian model to study the transmission of six different H5N1 avian influenza viruses. We found that two viruses, A/duck/Guangxi/35/2001 (DKGX/35) and A/bar-headed goose/Qinghai/3/2005(BHGQH/05), were transmitted from inoculated animals to naïve contact animals. Our mutagenesis analysis revealed that the amino acid asparagine (Asn) at position 701 in the PB2 protein was a prerequisite for DKGX/35 transmission in guinea pigs. In addition, an amino acid change in the hemagglutinin (HA) protein (Thr160Ala), resulting in the loss of glycosylation at 158–160, was responsible for HA binding to sialylated glycans and was critical for H5N1 virus transmission in guinea pigs. These amino acids changes in PB2 and HA could serve as important molecular markers for assessing the pandemic potential of H5N1 field isolates.
H5N1 influenza viruses have caused over 400 human infections in 15 countries and continue to circulate in poultry and wild birds. Most human infections resulted from direct contact with virus-contaminated poultry or poultry products. It would be disastrous if H5N1 viruses acquired the ability to efficiently transmit among humans, because the mortality rate may exceed 60%. However, the genetic basis for transmission of H5N1 influenza viruses is largely unknown. Here, we demonstrate that the amino acid residue at 701 of PB2 is a prerequisite for transmission of H5N1 viruses in a mammalian guinea pig model. Interestingly, we found that the absence of glycosylation at residues 158–160 of the HA gene is pivotal for the H5N1 virus to bind to human-like receptors and to transmit in a mammal host. These findings are important for assessing the pandemic potential of H5N1 field isolates.
Citation: Gao Y, Zhang Y, Shinya K, Deng G, Jiang Y, Li Z, et al. (2009) Identification of Amino Acids in HA and PB2 Critical for the Transmission of H5N1 Avian Influenza Viruses in a Mammalian Host. PLoS Pathog 5(12): e1000709. https://doi.org/10.1371/journal.ppat.1000709
Editor: Barbara Sherry, North Carolina State University, United States of America
Received: August 26, 2009; Accepted: November 24, 2009; Published: December 24, 2009
Copyright: © 2009 Gao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Chinese National Natural Science Foundation 30825032, the Chinese National Key Basic Research Program (973) 2010CB534000, 2005CB523005 and 2005CB523200, by grants from the Chinese National S&T Plan 2006BAD06A05 and 2006BAD06A04, by a contract research fund from the Ministry of Education, Culture, Sports, Science and Technology, Japan, for Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases and, in part, by Grants-in-Aid for Specially Promoted Research and for Scientific Research, by ERATO (Japan Science and Technology Agency), and by National Institute of Allergy and Infectious Diseases Public Health Service research grants, USA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The H5N1 avian influenza viruses (AIVs) have attracted extensive attention for their deadly impact on both animals and humans. The number of people who have been subclinically infected with H5N1 viruses is very limited , and H5N1 AIVs have a 60% fatality rate in humans (World Health Organization [WHO]; http://www.who.int). H5N1 AIVs first surfaced in China in 1996 , with chicken-lethal and avirulent strains being isolated from geese in Guangdong province . In 1997, a reassortant H5N1 AIV that carried the HA gene from an A/goose/Guangdong/1/96-like virus caused an outbreak of disease in poultry in Hong Kong and crossed over into humans, resulting in 18 cases of infection with six deaths ,. In 2003 and 2004, H5N1 AIVs infected poultry and humans in numerous countries of southeastern Asia . In 2005, several genotypes of H5N1 AIV caused outbreaks in wild migratory birds at Qinghai Lake in western China, and one genotype spread widely to different species across a wide geographic area that included Europe and Africa . To date, H5N1 AIVs have caused disease in more than 60 countries (Office International des Epizooties [OIE]; http://www.oie.int), with cases of human infection being reported in 15 countries (World Health Organization [WHO]; http://www.who.int). Despite substantial efforts to control these outbreaks, H5N1 AIVs have continued to evolve and spread, perpetuating the fear of an influenza pandemic if these viruses acquire the ability to transmit efficiently among humans. Understanding the genetic determinants that control H5N1 AIV transmission in mammalian hosts will help protect public health.
The transmissibility of influenza viruses is determined by the virus, environmental factors, and host factors –. The viral traits governing transmission efficiency have not been well characterized. The affinity of the viral HA protein for sialic acid α-2,6 linked glycan (α-2,6 glycan) is necessary for transmission of the 1918 H1N1 influenza virus between ferrets . The viral polymerase complex is also involved in determining viral host range, replication and pathogenicity – and plays a role in transmission ,. Cold and dry environmental conditions favor the transmission of human influenza virus in guinea pigs ,.
Host factors also affect replication and transmission of influenza viruses. Under experimental conditions, AIVs do not replicate efficiently in humans , and human viruses do not replicate efficiently in ducks . In one study, the human upper airway tract was reported to contain cells that predominantly express α-2,6 glycan , an environment that favors the replication and transmission of human influenza viruses, which preferentially bind to α-2,6 glycans. However, in another study, viruses with preferential binding to α-2,3 glycans were reported to bind to cells in the human upper respiratory tract . The first human isolates in the 1957 and 1968 pandemics preferentially bound to α-2,6 glycans even though their HAs were derived from avian viruses, which preferentially recognize α-2,3 glycans . These findings suggest that for viruses with HAs from AIVs to be efficiently transmitted among humans, they need to preferentially bind to α-2,6 glycans .
Ferrets and guinea pigs have been successfully used as models to evaluate the transmissibility of AIVs and other species of influenza viruses in mammalian hosts , , , , , –. H5N1 AIVs exhibit varying levels of replication and virulence in mammalian mouse and ferret models ,,. In this study, we used guinea pigs as a mammalian model to examine the replication and transmission of six H5N1 AIVs that exhibit different replication and virulence phenotypes in mice. We also explored the genetic requirements for H5N1 AIV transmission in this mammalian host.
Replication of H5N1 AIVs in guinea pigs
The six H5N1 AIVs used in this study have previously been shown to be lethal for chickens ,, but to differ in their replication and lethality in mice (Table 1). To investigate the replication of these viruses in guinea pigs, two animals were intranasally inoculated with 106EID50 of virus and euthanized three days post-inoculation (p.i.). Nasal wash, trachea, lung, brain, kidney, spleen and colon were collected from each animal for virus titration in eggs. A/duck/Guangxi/22/01 (DKGX/22), which does not replicate in mice ,, was detected in the lung of one guinea pig on day 3 p.i., but not in the nasal wash or trachea of either of the other two inoculated animals (Table 1). The other five viruses were detected in the nasal washes, tracheas and lungs of both animals inoculated. Virus was not detected in the brains, kidneys, spleens, or colons of any of the animals inoculated. We also infected two animals for each virus and observed them for two weeks for signs of pathogenicity. After two weeks p.i., all of the animals seroconverted except for one of the two guinea pigs infected with the DKGX/22 virus (Table 1). None of the animals showed disease signs during the observation period. These results indicate that replication of H5N1 viruses in guinea pigs is restricted to the respiratory system.
Transmission of H5N1 AIVs in guinea pigs
Groups of three animals were inoculated with 106 EID50 of each virus intranasally and three naïve animals were introduced into the same cage 24 h p.i. Evidence of transmission was based on the detection of virus in the nasal wash and on seroconversion at the end of the two-week observation period. As shown in Figure 1A, virus was not detected in the nasal washes of any inoculated or contact guinea pigs in the DKGX/22-inoculated group. In the DKFJ/17-, DKSH/13- and DKGD/22- inoculated groups, virus was detected in the nasal washes of all three inoculated guinea pigs between days 2–10 p.i., but not in any of the contact guinea pigs (Figure 1B,C,E). In the DKGX/35- and BHGQH/3-inoculated groups, virus was detected in the nasal washes of all three inoculated guinea pigs between days 2–6 or days 2–10 p.i., respectively and was also detected in the nasal washes of all three contact animals between days 4–12 p.i. (Figure 1D, F). Seroconversion occurred in all inoculated groups; however, in the DKGX/22-inoculated group, only two of the three animals seroconverted (Table 2). In the contact animal groups, seroconversion was only observed among animals placed with the DKGX/35- and BHGQH/3-inoculated animals. These results indicate that the transmissibility of H5N1 AIVs in guinea pigs varies among viral strains, and of the six test viruses, only DKGX/35 and BHGQH/3 transmit efficiently in this mammalian host.
Groups of three guinea pigs were inoculated i.n. with 106EID50 of test virus and, 24 hours after the inoculation, three contact guinea pigs were placed in each cage. Nasal washes were collected every two days from all animals beginning 2 days p.i. for detection of virus shedding. (A) DKGX/22 virus; (B) DKGX/17 virus; (C) DKSH/13 virus; (D) DKGX/35 virus; (E) DKGD/22 virus; and (F) BHGQH/3 virus. Each color bar represents the virus titer from an individual animal. The dashed blue lines in these panels indicate the lower limit of detection.
PB2 amino acid 701N is required but not sufficient for H5N1 AIV transmission in guinea pigs
Of the six viruses tested, DKGX/22 and DKGX/35 have very similar genomes ,. Using DKGX/22 and DKGX/35, we previously determined that the amino acid at position 701 in the PB2 protein is important for H5N1 avian influenza virus replication in mice . To determine whether this amino acid also contributes to the replication and transmission of H5N1 viruses in guinea pigs, we tested two mutants, DKGX/35-PB2-N701D (35/PB2-701D) and DKGX/22-PB2-D701N (22/PB2-701N) in guinea pigs. We found that when the D701N mutation was introduced in the PB2 protein (22/PB2-701N), the replication of the DKGX/22 virus dramatically increased. This mutant virus replicated in the nose, trachea, and lung of the guinea pigs at titer levels comparable to those observed in the DKGX/35-inoculated animals (Figure 2A). In the transmission experiment, 22/PB2-701N was detected in the noses of the inoculated guinea pigs from days 2–8 p.i., but no virus was detected in the contact animals (Figure 2B). The mutant 35/PB2-701D virus also replicated in the nose, trachea, and lung of the inoculated animals, although virus titers were appreciably lower than those observed in animals inoculated with wild-type DKGX/35 (Figure 2A). Transmission of 35/PB2-701D in guinea pigs was not detected (Figure 2C). These results indicate that PB2 701N is required, but not sufficient, for transmission of these duck H5N1 AIVs in guinea pigs, suggesting that other genes also contribute to this function.
(A) Groups of two guinea pigs were inoculated i.n. with 106EID50 of test virus and then euthanized on day 3 p.i. Organs were collected for virus titration in eggs. (B) Transmission of the 22/PB2-701N virus in guinea pigs. (C) Transmission of the 35/PB2-701D virus in guinea pigs. The dashed blue lines in these panels indicate the lower limit of detection.
DKGX/35 H5N1 AIV binds to both α-2,3- and α-2,6-linked glycans
HA receptor specificity plays an important role in the transmission of influenza viruses ,,,. The affinity of viral HA protein for α-2,6-glycan is required for the transmission of human influenza virus among ferrets ,. We examined the receptor-binding specificity of DKGX/35 by hemagglutination assays using resialyated cRBCs. DKGX/35 could bind to cRBCs resialylated with either α-2,3- or α-2,6-glycans, wheras the H1N1 human influenza virus BC/05 only bound to α-2,6 glycans (Figure 3).
The upper panel shows the hemagglutination by two HA units of each virus while the lower panel shows the HA titers of test viruses with 0.5% cRBCs treated as follows: cBRCs, untreated; Desial cRBCs, treated with VCNA; α-2,3 cRBC, VCNA treated and resialylated with α-2,3 glycans; α-2,6 cRBC,VCNA treated and resialylated with α-2,6 glycans.
Chandrasekaran et al reported that the sialylated glycans in the α-2,3 and α-2,6 linkages have different topologies, and the HAs of H1N1 and H3N2 influenza viruses that have adapted to humans specifically bind to the long α-2,6 glycan topology, whereas the HA of H5N1 viruses, A/Vienam/1203/04 and A/Hong Kong/486/97, does not . We tested the binding of the HAs of the DKGX/35 and BC/05 viruses with different glycans using a dose-dependent direct binding assay. As shown in Figure 4A, the human H1N1 virus BC/05 bound with high affinity to short and long α-2,6-linked glycans (6′SLN and 6′S-Di-LN) and showed minimal affinity for α-2,3-linked glycans (3′SLN and 3′S-Di-LN). DKGX/35 exhibited affinity for all four test glycans, although its affinity for the α-2-3-linked glycans was slightly higher than that for the α-2,6-linked glycans (Figure 4C). These results indicate that the HA of the avian H5N1 DKGX/35 virus binds to α-2,6-linked sialosides, including long α-2,6 glycans, as well as to α-2,3-linked sialosides.
Mutation of the human influenza virus-like amino acids 226L and 228S of the DKGX/35 HA protein affects receptor binding specificity and decreases viral transmission in guinea pigs
Several amino acids within the H5N1 influenza virus HA gene are associated with receptor-binding specificity –. The DKGX/35 virus HA protein contains eight avian influenza virus-like amino acids (Table 3). To investigate how changing these eight amino acids to those found in human virus HAs affect H5N1 virus receptor-binding preference, we generated a series of mutants in the DKGX/35 virus background that contained one or two amino acid changes at these critical positions in the HA protein. We tested their receptor binding specificities by using hemagglutination assays. As shown in Figure 3, the mutants with the single amino acid changes of 137A, 186K, 196R, 226L, 227N and 228S retained the same receptor binding preference as the wild-type DKGX/35 virus. The same result was obtained with mutant viruses containing the double amino acid changes of 133V/138V and 186K/196R. However, the mutant containing the double amino acid change of 226L/228S (35/HA-226L/228S) bound to only α-2,6 glycan resialylated cRBCs and not α-2,3 glycan resialylated cRBCs. This lack of binding affinity to α-2,3 glycans was confirmed by using dose-dependent direct binding assays (Figure 4C). Together, these data indicate that the amino acids 226L and 228S of the HA protein are important for the receptor binding preference of the H5N1 influenza virus, as has been reported elsewhere ,.
We then asked whether this change in receptor binding preference affected the replication and transmission of the mutant virus in guinea pigs. The 35/HA-226L/228S virus replicated as well as the wild-type DKGX/35 virus in the noses of guinea pigs, but did not replicate in their tracheas and lungs (Figure 5A). In the transmission study, the 35/HA-226L/228S virus was only detected in two of three contact animals (Figure 4C). These results indicate that the double amino acid mutations of Q226L and G228S in the HA protein abate the affinity of the DKGX/35 virus to bind α-2,3 glycans, and also slightly decrease its transmission among guinea pigs.
(A) Groups of two guinea pigs were inoculated i.n. with 106EID50 of test virus and euthanized on day 3 p.i. Organs were collected for virus titration in eggs. (B) Transmission of 35/HA-226L/228S in guinea pigs. (C) Transmission of 35/HA-160T in guinea pigs. The dashed blue lines in these panels indicate the lower limit of detection.
Mutation of the avian influenza virus-like amino acid 160T of the DKGX/35 virus affects its receptor binding specificity and abolishes viral transmission in guinea pigs
As shown in Table 3, the DKGX/35 HA has two amino acids, 160A and 192I, that are conserved in most human influenza viruses. To investigate how these two amino acids affect the receptor-binding specificity of the H5N1 virus, we generated two mutant viruses in the DKGX/35 background, 35/HA-160T and 35/HA-192T, and analyzed their receptor binding preferences. 35/HA-160T bounds to α-2,3 glycan resialylated cRBCs but completely lost the ability to bind to α-2,6 glycan resialylated cRBCs, as determined by hemagglutination assays (Figure 3). The mutation I192T did not change the receptor binding preference of the DKGX/35 virus (Figure 3). The loss of receptor-binding affinity of 35/HA-160T to α-2,6 glycans was further confirmed by dose-dependent direct binding assays (Figure 4D).
We then tested the effect of the mutation A160T on the replication and transmission of DKGX/35 in guinea pigs. 35/HA-160T replicated as well as the wild-type DKGX/35 virus in the respiratory system of guinea pigs (Figure 4A), but was not detected in any of the contact animals (Figure 4B). These results indicate that the amino acid 160A of the HA protein is required for the H5N1 virus to bind the human α-2,6 sialic acid cellular receptors and for its transmission among guinea pigs.
The amino acid mutation of A to T at position 160 of the DKGX/35 HA protein formed a new potential N-linked glycosylation site -NST- at amino acid positions 158–160. To investigate whether this potential N-linked glycosylation site is indeed glycosylated, we performed Western blot analysis of the HA polypeptides of DKGX/35 and 35/HA-160T treated with or without PNGase F enzyme. As shown in Figure 6, the HA1 polypeptide of 35/HA-160T exhibited decreased mobility relative to that of DKGX/35 as a result of the single amino acid mutation of A160T, whereas when the HA1 polypeptides of these two viruses were deglycosylated with PNGase F, they showed similar mobility (Figure 6). These results suggest that the potential N-linked glycosylation site at amino acid positions 158–160 in the HA protein of the 35/HA-160T virus is glycosylated.
Lysates of H5N1 viruses treated with or without PNGase F were incubated with chicken anti-H5N1 antiserum. Binding was visualized with 3,30-diaminobenzidine after incubation with peroxidase-conjugated secondary antibodies. The locations of marker proteins are indicated on the left.
The amino acid A160T mutation in the HA protein of the AIV BHGQH/3 altered its receptor-binding preference and abolished its transmission in guinea pigs
BHGQH/3 bears the amino acid A at the position 160 in its HA gene, as does DKGX/35 (Table 4). To test how the HA amino acid mutation A160T affects the receptor-binding and transmission properties of BHGQH/3, we introduced this mutation in the BHGQH/3 background by using reverse genetics. The rescued wild-type virus, r-BHGQH/3, bound to both α-2,3 and α-2,6 glycans (Figure 7A), whereas the mutant BHGQH/3-A160T bound to only α-2,3 glycans, as determined by dose-dependent direct binding assays (Figure 7B). These results were confirmed by use of hemagglutination assays (data not shown).
Receptor-binding preference of r-BHGQH/3 (A) and BHGQH/3-160T (B) were performed by dose-dependent direct binding assay as described in the text. (C) and (D) Transmisson of H5N1 duck viruses in guinea pigs. (C) r-BHGQH/3-inoculated group. (D) BHGQH/3-160T-inoculated group. The dashed blue lines in these panels indicate the lower limit of detection.
We also compared the transmissibility of BHGQH/3-A160T in guinea pigs to that of the rescued r-BHGQH/3 virus. As shown in Figure 7C, the r-BHGQH/3 virus was detected in guinea pig nasal washes obtained between days 2–8 p.i. The virus was also detected in contact animals between days 4–14 p.i. BHGQH/3-A160T, however, was found in the nasal washes of only the inoculated animals, not the contact guinea pigs (Figure 7D). These results indicate that the amino acid mutation A160T in the HA protein of BHGQH/3 also abolishes the its binding affinity for α-2,3 glycans and its transmissibility in guinea pigs.
Analysis of the receptor distribution in the respiratory system of guinea pigs
To understand the underlying mechanism of the receptor binding specificities and replication phenotypes in guinea pigs of the viruses used in our experiments, we examined the receptor specificity of the respiratory tract of guinea pigs. The alveolar surface was covered by MAAII-binding α-2,3 glycan (Figure 8A, red), as was the surface of tracheal cells (Figure 8B, red), although some cells also expressed SNA-binding α-2,6 glycan (Figure 8B, green). The nasal respiratory region contained a mixture of cells expressing α-2,3 glycan and α-2,6 glycan (Figure 8C). In the nasal olfactory region, the cell surfaces were mostly covered by α-2,3 glycan (Figure 8D). The lack of α-2,6 glycans on the alveolar surface may explain the inability of the 35/HA-226L/228S virus to replicate in the lungs of guinea pigs (Figure 4A).
(A) Alveoli. Red staining indicates MAAII-binding α-2,3 glycan. (B) Tracheal mucosa of guinea pig. Red staining indicates the presence of α-2,3 glycan and green staining indicates the presence of α-2,6 glycan. (C) Nasal mucosa (respiratory region) of guinea pig. (D) Nasal mucosa (olfactory region) of guinea pig. Green staining indicates the presence of α-2,6 glycan.
H5N1 AIVs have caused the deaths of more than half of the humans they have infected since 1997 and clearly represent a threat to public health (World Health Organization [WHO]; http:// www.who.int). Most human cases of infection resulted from direct exposure to H5N1 virus-infected poultry or poultry products. However, human-to-human transmission, albeit limited, has been detected ,. The effects of specific amino acid changes on the transmissibility of H5N1 highly pathogenic AIVs remain largely unexplored. Here, we evaluated the replication and transmission in guinea pigs of six H5N1 AIVs isolated in China between 2001 and 2005. We found that two of these viruses, DKGX/35 and BHGQH/03, not only replicated but also transmitted efficiently in guinea pigs. We demonstrated that the amino acid 701N in the PB2 protein is required for DKGX/35 transmission in guinea pigs, and that DKGX/35 and BHGQH/03 bind to both α-2,3 and α-2,6 glycans. The amino acid mutation A160T in the HA protein, which creates a new potential N-linked glycosylation site, abolishes the ability of viruses to bind α-2,6 glycans and to transmit in guinea pigs. Our results demonstrate that the PB2 and HA genes play important roles in the transmission of H5N1 influenza viruses in a mammalian host, and we are the first to report that the lack of glycosylation at amino acid positions 158–160 in HA is important for H5N1 AIVs to bind to the human receptor and to transmit in a mammalian host.
PB2, together with the viral proteins PB1 and PA, makes up the viral RNA polymerase. PB2 is an important determinant for the host range and virulence of influenza viruses –,. Two amino acids in PB2, 627K and 701N, have been found in H5N1 influenza viruses isolated from humans . These amino acids are known to affect the replicative efficiency of H5N1 influenza A viruses in mice ,. Indeed, the amino acid 627K has been reported to favor the replication of H5N1 influenza virus in the upper respiratory tracts of mice and may be important for efficient person-to-person virus transmission . Steel et al.  found that the amino acid residues 701N and 627K of PB2 increased the transmission of both human and avian influenza viruses in guinea pigs. In our study, we found that the amino acid mutation of N to D at position 701 of PB2 completely abolished the transmission of DKGX/35 virus among guinea pigs. The D to N mutation at position 701 of PB2 dramatically increased the replication of DKGX/22 in guinea pigs, but it did not confer transmissibility. Similarly, the mutant viruses 35/HA-160T and BHGQH/3-HA160T were unable to transmit in guinea pigs, even though both viruses contain either 701N or 627K in the PB2 protein. These results indicate that 701N or 627K in the PB2 protein are prerequisites, but not sufficient, for H5N1 virus transmission in a mammalian host.
The receptor binding specificity of HA has been implicated in the transmissibility of influenza viruses ,. Despite the ability to infect and cause severe disease in humans, most H5N1 viruses do not bind the α-2,6 glycan receptor with high affinity ,,. This low receptor-binding affinity is likely a major factor preventing H5N1 viruses from efficiently transmitting from person to person and causing a pandemic . Although several reports have documented mutants of H5N1 influenza viruses that have modest affinity for α-2,6 glycans –, it is unknown whether these mutations affect the transmission of the H5N1 influenza viruses in mammals. Here, we found that two H5N1 viruses, DKGX/35 and BHGQH/3, exhibited binding affinity for both α-2,3 and α-2,6 glycans and were able to transmit among guinea pigs. We demonstrated that the HA amino acid 160A plays a key role in the affinity of these two viruses for α-2,6 glycans. The single HA amino acid mutation of A160T abolished not only the binding affinity of DKGX/35 and BHGQH/3 to α-2,6 glycans, but also the transmission of these viruses among guinea pigs. It should be noted that viral transmission is a polygenic trait and that mutation of HA alone does not confer transmissibility to a virus.
Previous studies have reported that N-linked glycans close to the HA receptor binding domain can affect receptor-binding preference through steric hindrance or other mechanisms ,. The potential N-linked glycosylation site at HA amino acid positions 158–160 is commonly detected in H5N1 influenza viruses, but the majority of the clade 2.2 H5N1 isolates that were first isolated from wild birds in western China  and then in Europe, Africa and the Middle East, notably lack this potential N-linked glycosylation site . Here, we demonstrated that the lack of this potential glycosylation site at HA amino acid positions 158–160 is critical for the H5N1 influenza viruses to bind α-2,6 glycans and to transmit in a mammalian host.
The HA amino acids at positions 226 and 228 play key roles in the receptor binding preference of influenza viruses ,. The HAs of human influenza viruses bear Leu at position 226 (226L) and Ser at position 228 (228S) and preferentially recognize α-2,6 glycans . Although H5N1 influenza viruses have caused over 400 cases of human infection, the HA residues 226L and 228S have not been detected in any isolates, based on the available sequence information. Some naturally occurring H5N1 influenza mutants and lab-created H5N1 influenza viruses that contain mutations in the receptor-binding site of the HA protein, including the HA Q226L and G228S double mutant in the A/Vietnam/1203/2004 virus background, exhibit increased binding affinity for α-2,6 glycans, yet retain binding affinity for α-2,3 glycans ,. Here, we found that the mutant 35/HA-226L/228S, with the double HA amino acid mutation of Q226L and G228S in the DKGX/35 background, completely lost its ability to bind to α-2,3 glycans and only bound to α-2,6 glycans. It is likely that the HA amino acid 160A, which does not encode a potential glycosylation site at 158–160, also contributed to the inability of 35/HA-226L/228S to bind α-2,3 glycans.
Although the mutant virus 35/HA-226L/228S acquired human influenza virus-like receptor binding preference, its transmission in guinea pigs was slightly impaired relative to that of the wild-type DKGX/35 virus. Our receptor specificity analysis revealed that both α-2,3 and α-2,6 glycans were present in the nasal mucosa and the trachea mucosal surface, yet only α-2,3 glycans were detected in the alveoli of guinea pigs. This type of receptor distribution may favor the replication and transmission of an avian virus that can bind both α-2,3 and α-2,6 glycans, although human influenza viruses, which bind to only α-2,6 glycans, transmit well in guinea pigs . Therefore, although the 35/HA-226L/228S virus cannot transmit efficiently in guinea pigs, it may be able to transmit easily in humans, which have a different receptor distribution than guinea pigs .
In summary, here, we demonstrated that the PB2 and HA proteins are important for the transmission of H5N1 influenza viruses in a mammalian host. We confirmed that the PB2 amino acid 701N is important for the transmission of H5N1 influenza virus in guinea pigs, and found, for the first time, that the T to A mutation at position 160, which results in the lack of an oligosaccharide side chain at 158–160 of HA, is critical for the H5N1 influenza viruses tested to bind to human-like receptors and to transmit among a mammalian host. The absence of a potential N-linked glycosylation site at HA amino acid positions 158–160 may serve as an important molecular marker for assessing the pandemic potential of H5N1 field isolates. Moreover, it is worrisome that clade 2.2 viruses bearing PB2 and HA mutations that permit transmission among mammalian hosts continue to circulate in wild birds and poultry across a wide geographic area. Clearly, there is a critical need for continued surveillance of poultry and regularly updated control measures.
Materials and Methods
Studies with highly pathogenic H5N1 avian influenza viruses were conducted in a biosecurity level 3+ laboratory approved by the Chinese Ministry of Agriculture. All animal studies were approved by the Review Board of Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences.
Viruses and cells
Human embryonic kidney cells (293T) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and incubated at 37°C and 5% CO2. The six H5N1 AIVs used in this study (Table 1) were isolated from ducks in southern China and wild birds at Qinghai Lake in northwest China as described previously ,. The construction of two PB2 mutant viruses, 22/PB2-701N and 35/PB2-701D, was reported previously . An H1N1 virus isolated from humans in 2005, A/Baicheng/1/05(BC/05), was kindly provided by the Jilin Disease Control and Prevention Centers in China. The viruses were propagated in 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs and were stored at −70°C.
Guinea pig studies
Hartley strain female guinea pigs weighing 300–350 g and serologically negative for influenza virus were used in these studies. Ketamine (20 mg/kg) and xylazine (1 mg/kg) were used to anesthetize animals by intramuscular injection.
To investigate the replication of H5N1 AIVs, groups of four animals were anesthetized and inoculated intranasally (i.n.) with 106 EID50 of test virus in a 300 µl volume (150 µl per nostril). Two animals from each group were euthanized on day 3 post inoculation (p.i) and nasal washes, tracheas, lungs, brains, kidneys, spleens, and colons were collected for virus titration in eggs. The remaining two animals were observed for two weeks for signs of disease and death.
For the contact transmission studies, groups of three animals were inoculated i.n. with 106 EID50 of test virus and housed in a cage placed inside an isolator. Three naïve animals were introduced into the same cage 24 h later. Nasal washes were collected at 2 day intervals, beginning on day 2 p.i. (1 day post contact) and titrated in eggs. To prevent inadvertent physical transmission of virus by the investigators, the contact guinea pigs were always handled first, and gloves, implements, and napkins on the work surface were changed between animals. The ambient conditions for these studies were set as 20–22°C and 30%–40% relative humidity. The airflow in the isolator was horizontal with a speed of 0.1 m/s.
Receptor-binding analysis using hemagglutination assays
Hemagglutination assays using resialyated chicken red blood cells (cRBCs) were performed as described previously , with minor modifications. cRBCs were enzymatically desialyated with Vibrio cholerae neuraminidase (VCNA; Roche, www.roche.com), followed by resialylation using either α2–6-(N)-sialyltransferase or α2–3-(N)-sialyltransferase (Calbiochem, www.calbiochem.com) and CMP-sialic acid (Sigma, www.sigmaaldrich.com).
Dose-dependent direct binding to different glycans of H5N1 avian influenza viruses
Analysis of the receptor specificity of influenza virus was performed by using a direct solid-phase assay . Briefly, a streptavidin-coated, high-binding capacity 96-well plate (Pierce, www.piercenet.com) was rinsed with PBS and 50µl of a 2.4 mM solution of biotinylated glycans in PBS was added to each well and incubated overnight at 4°C. Two α-2,6 glycans (6′SLN Neu5Aca2-6Galb1-4GlcNAcb-SpNH-LC-LC-Biotin, 6′S-Di-LN Neu5Aca2-6[Galb1-4GlcNAcb1-3]2b-SpNH-LC-LC-Biotin) and two α-2,3 glycans (3′SLN: Neu5Aca2-3Galb1-4GlcNAcb-SpNH-LC-LC-Biotin, 3′S-Di-LN Neu5Aca2-3[Galb1-4GlcNAcb1-3]2b-SpNH-LC-LC-Biotin) were tested upon being kindly provided by the Consortium for Functional Glycomics (Scripps Research Institute, Department of Molecular Biology, La Jolla, CA). The plate was subsequently washed with cold PBS to remove any excess glycans. Virus was inactivated by adding 0.1% (v/v) β-propiolactone for 3 days at 4°C. Virus binding to the glycan-coated wells was performed by adding serially diluted virus in PBS containing 1% bovine serum albumin (BSA) to each well followed by an overnight incubation at 4°C. After being rinsed with PBS containing 0.05% Tween-20 to excess virus, the wells were incubated with chicken antisera against A/goose/Guangdong/01/1996 (H5N1) virus for 5 h at 4°C. The wells were then extensive washed and subsequently incubated with HRP-linked goat-anti-chicken antibody (Sigma-Aldrich, www.sigmaaldrich.com) for 2 h at 4°C. The wells were washed again with PBS containing 0.05% Tween-20, and incubated with O-phenylenediamine in substrate solution containing 0.01% H2O2 for 10 min at room temperature. The reaction was stopped by adding 50 µl of 1M H2SO4 and the absorbance was determined at 492 nm.
Site-directed mutagenesis and virus generation
The HA mutants of DKGX/35 and BHGQH/3 viruses were generated using reverse genetics as previously described . A site-directed mutagenesis kit (Invitrogen, www.invitrogen.com) was used to create specific mutations in the HA gene by using the primers shown in Table 4. The plasmids used for transfection were prepared by using the QIAfilter™ Plasmid Midi kit (QIAGEN, www.qiagen.com). All the constructs were completely sequenced to ensure the absence of unwanted mutations.
Deglycosylation using PNGase F
Deglycosylation was achieved by using PNGase F (New England Biolabs, www.neb.com). Virus was concentrated by ultrafiltration with Microcon YM-100 tubes (Millipore, www.millipore.com) and purified by using column chromatography with Sepharose 4FF (GE Healthcare, www.gehealthcare.com). The virus concentrates were denatured according to manufacturer's instructions, and then deglycosylated by incubation at 37°C for 16 h with the PNGase F enzyme in the buffer provided by the manufacturer and NP40 at a final concentration of 1% (provided with the enzyme). Deglycosylated samples were analyzed by SDS-PAGE and western blot.
Western blot analysis
Virus samples were analyzed by SDS-PAGE and Western immunoblotting as described previously . Chicken antisera induced by the pCAGG-HA DNA vaccine  was used as the primary antibody, and IRDye™700DX-conjugated goat anti-chicken immunoglobulin G (IgG) (Rockland, www.rockland-inc.com) was used as the secondary antibody.
Detection of α-2,3 glycans and α-2,6 glycans in guinea pig respiratory tissues
Paraffin-embedded, surgically removed, normal guinea pig upper-to-lower respiratory tract tissues were cut into 5 µm thick sections with a microtome. The sections were mounted on 3-aminopropyltrethoxy-silane (APS)-coated slides (Matsunami Glass Ind., Ltd., www.matsunami-glass.co.jp), deparaffinized in xylene, and rehydrated with alcohol. To detect sialyloligosaccharides reactive with α-2,3 glycan- or α-2,3 glycan-specific lectins, the sections were incubated with 250 µl of FITC-labeled Sambucus nigra (SNA) lectin (Vector Laboratories, www.vectorlabs.com) or biotinylated Maackia amurensis (MAA) lectin (Vector Laboratories, www.vectorlabs.com) overnight at 4°C. After three washes with Tris-buffered saline (TBS, pH 7.6), the sections were incubated with Alexa Fluor 594-conjugated streptavidin, (Molecular Probes, Inc., www.invitrogen.com) for 2 h at room temperature. They were then counterstained with 4′,6-diamino-2-phenylindole, dihydrochloride (DAPI; Dojindo Molecular Technologies, Inc., www.dojindo.com). After three washes with TBS, the sections were then mounted on cover glasses and observed with a fluorescence microscope (ECLIPSE TE300 with a fluorescence equipment mercury set, Nikon Co., www.nikonusa.com). Photographs were taken with a digital microscope camera (Olympus DP70, Olympus Optical Co., Ltd., www.olympus.com).
We thank Gloria Kelly and Susan Watson for editing the manuscript, the Consortium for Functional Glycomics (Scripps Research Institute, Department of Molecular Biology, La Jolla, CA) for providing the glycans.
Conceived and designed the experiments: Z. Bu, H. Chen. Performed the experiments: Y. Gao, Y. Zhang, K. Shinya, G. Deng, Y. Jiang, Z. Li, Y. Guan, G. Tian, Y. Li, J. Shi, L. Liu, X. Zeng. Analyzed the data: Y. Gao, Y. Zhang, K. Shinya, Z. Bu, X. Xia, Y. Kawaoka, H. Chen. Wrote the paper: Y. Gao, Z. Bu, Y. Kawaoka, H. Chen.
- 1. Vong S, Ly S, Van Kerkhove MD, Achenbach J, Holl D, et al. (2009) Risk factors associated with subclinical human infection with avian influenza A (H5N1) virus–Cambodia, 2006. J Infect Dis 199: 1744–1752.
- 2. Chen H, Deng G, Li Z, Tian G, Li Y, et al. (2004) The evolution of H5N1 influenza viruses in ducks in southern China. Proc Natl Acad Sci U S A 101: 10452–10457.
- 3. Li Z, Jiang Y, Jiao P, Wang A, Zhao F, et al. (2006) The NS1 gene contributes to the virulence of H5N1 avian influenza viruses. J Virol 80: 11115–11123.
- 4. Claas EC, Osterhaus AD, van Beek R, De Jong JC, Rimmelzwaan GF, et al. (1998) Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351: 472–477.
- 5. Subbarao K, Klimov A, Katz J, Regnery H, Lim W, et al. (1998) Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Science 279: 393–396.
- 6. Webster RG, Peiris M, Chen H, Guan Y (2006) H5N1 outbreaks and enzootic influenza. Emerg Infect Dis 12: 3–8.
- 7. Chen H, Li Y, Li Z, Shi J, Shinya K, et al. (2006) Properties and dissemination of H5N1 viruses isolated during an influenza outbreak in migratory waterfowl in western China. J Virol 80: 5976–5983.
- 8. Maines TR, Chen LM, Matsuoka Y, Chen H, Rowe T, et al. (2006) Lack of transmission of H5N1 avian-human reassortant influenza viruses in a ferret model. Proc Natl Acad Sci U S A 103: 12121–12126.
- 9. Tumpey TM, Maines TR, Van Hoeven N, Glaser L, Solorzano A, et al. (2007) A two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission. Science 315: 655–659.
- 10. Lowen AC, Mubareka S, Steel J, Palese P (2007) Influenza virus transmission is dependent on relative humidity and temperature. PLoS Pathog 3: e151.
- 11. Lowen AC, Steel J, Mubareka S, Palese P (2008) High temperature (30 degrees C) blocks aerosol but not contact transmission of influenza virus. J Virol 82: 5650–5652.
- 12. Hatta M, Gao P, Halfmann P, Kawaoka Y (2001) Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science 293: 1840–1842.
- 13. Li Z, Chen H, Jiao P, Deng G, Tian G, et al. (2005) Molecular basis of replication of duck H5N1 influenza viruses in a mammalian mouse model. J Virol 79: 12058–12064.
- 14. Gabriel G, Dauber B, Wolff T, Planz O, Klenk HD, et al. (2005) The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host. Proc Natl Acad Sci U S A 102: 18590–18595.
- 15. Hatta M, Hatta Y, Kim JH, Watanabe S, Shinya K, et al. (2007) Growth of H5N1 influenza A viruses in the upper respiratory tracts of mice. PLoS Pathog 3: e133.
- 16. Steel J, Lowen AC, Mubareka S, Palese P (2009) Transmission of influenza virus in a mammalian host is increased by PB2 amino acids 627K or 627E/701N. PLoS Pathog 5: e1000252.
- 17. Van Hoeven N, Pappas C, Belser JA, Maines TR, Zeng H, et al. (2009) Human HA and polymerase subunit PB2 proteins confer transmission of an avian influenza virus through the air. Proc Natl Acad Sci U S A 106: 3366–3371.
- 18. Beare AS, Webster RG (1991) Replication of avian influenza viruses in humans. Arch Virol 119: 37–42.
- 19. Hinshaw VS, Webster RG, Naeve CW, Murphy BR (1983) Altered tissue tropism of human-avian reassortant influenza viruses. Virology 128: 260–263.
- 20. Shinya K, Ebina M, Yamada S, Ono M, Kasai N, et al. (2006) Avian flu: influenza virus receptors in the human airway. Nature 440: 435–436.
- 21. Nicholls JM, Chan MC, Chan WY, Wong HK, Cheung CY, et al. (2007) Tropism of avian influenza A (H5N1) in the upper and lower respiratory tract. Nat Med 13: 147–149.
- 22. Matrosovich M, Tuzikov A, Bovin N, Gambaryan A, Klimov A, et al. (2000) Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals. J Virol 74: 8502–8512.
- 23. Parrish CR, Kawaoka Y (2005) The origins of new pandemic viruses: the acquisition of new host ranges by canine parvovirus and influenza A viruses. Annu Rev Microbiol 59: 553–586.
- 24. Wan H, Sorrell EM, Song H, Hossain MJ, Ramirez-Nieto G, et al. (2008) Replication and transmission of H9N2 influenza viruses in ferrets: evaluation of pandemic potential. PLoS ONE 3: e2923.
- 25. Bouvier NM, Lowen AC, Palese P (2008) Oseltamivir-resistant influenza A viruses are transmitted efficiently among guinea pigs by direct contact but not by aerosol. J Virol 82: 10052–10058.
- 26. Belser JA, Blixt O, Chen LM, Pappas C, Maines TR, et al. (2008) Contemporary North American influenza H7 viruses possess human receptor specificity: Implications for virus transmissibility. Proc Natl Acad Sci U S A 105: 7558–7563.
- 27. Yen HL, Lipatov AS, Ilyushina NA, Govorkova EA, Franks J, et al. (2007) Inefficient transmission of H5N1 influenza viruses in a ferret contact model. J Virol 81: 6890–6898.
- 28. Mubareka S, Lowen AC, Steel J, Coates AL, Garcia-Sastre A, et al. (2009) Transmission of influenza virus via aerosols and fomites in the guinea pig model. J Infect Dis 199: 858–865.
- 29. Lowen AC, Steel J, Mubareka S, Carnero E, Garcia-Sastre A, et al. (2009) Blocking interhost transmission of influenza virus by vaccination in the guinea pig model. J Virol 83: 2803–2818.
- 30. Lowen AC, Mubareka S, Tumpey TM, Garcia-Sastre A, Palese P (2006) The guinea pig as a transmission model for human influenza viruses. Proc Natl Acad Sci U S A 103: 9988–9992.
- 31. Maines TR, Lu XH, Erb SM, Edwards L, Guarner J, et al. (2005) Avian influenza (H5N1) viruses isolated from humans in Asia in 2004 exhibit increased virulence in mammals. J Virol 79: 11788–11800.
- 32. Maines TR, Jayaraman A, Belser JA, Wadford DA, Pappas C, et al. (2009) Transmission and pathogenesis of swine-origin 2009 A(H1N1) influenza viruses in ferrets and mice. Science 325: 484–487.
- 33. Chandrasekaran A, Srinivasan A, Raman R, Viswanathan K, Raguram S, et al. (2008) Glycan topology determines human adaptation of avian H5N1 virus hemagglutinin. Nat Biotechnol 26: 107–113.
- 34. Stevens J, Blixt O, Chen LM, Donis RO, Paulson JC, et al. (2008) Recent avian H5N1 viruses exhibit increased propensity for acquiring human receptor specificity. J Mol Biol 381: 1382–1394.
- 35. Auewarakul P, Suptawiwat O, Kongchanagul A, Sangma C, Suzuki Y, et al. (2007) An Avian Influenza H5N1 Virus That Binds to a Human-Type Receptor. J Virol 81: 9950–9955.
- 36. Yamada S, Suzuki Y, Suzuki T, Le MQ, Nidom CA, et al. (2006) Haemagglutinin mutations responsible for the binding of H5N1 influenza A viruses to human-type receptors. Nature 444: 378–382.
- 37. Stevens J, Blixt O, Tumpey TM, Taubenberger JK, Paulson JC, et al. (2006) Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science 312: 404–410.
- 38. Ilyushina NA, Govorkova EA, Gray TE, Bovin NV, Webster RG (2008) Human-like receptor specificity does not affect the neuraminidase-inhibitor susceptibility of H5N1 influenza viruses. PLoS Pathog 4: e1000043.
- 39. Gambaryan A, Tuzikov A, Pazynina G, Bovin N, Balish A, et al. (2006) Evolution of the receptor binding phenotype of influenza A (H5) viruses. Virology 344: 432–438.
- 40. Beigel JH, Farrar J, Han AM, Hayden FG, Hyer R, et al. (2005) Avian influenza A (H5N1) infection in humans. N Engl J Med 353: 1374–1385.
- 41. Ungchusak K, Auewarakul P, Dowell SF, Kitphati R, Auwanit W, et al. (2005) Probable person-to-person transmission of avian influenza A (H5N1). N Engl J Med 352: 333–340.
- 42. Subbarao EK, London W, Murphy BR (1993) A single amino acid in the PB2 gene of influenza A virus is a determinant of host range. J Virol 67: 1761–1764.
- 43. de Jong MD, Simmons CP, Thanh TT, Hien VM, Smith GJ, et al. (2006) Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat Med 12: 1203–1207.
- 44. Suzuki Y (2005) Sialobiology of influenza: molecular mechanism of host range variation of influenza viruses. Biol Pharm Bull 28: 399–408.
- 45. Stevens J, Blixt O, Glaser L, Taubenberger JK, Palese P, et al. (2006) Glycan microarray analysis of the hemagglutinins from modern and pandemic influenza viruses reveals different receptor specificities. J Mol Biol 355: 1143–1155.
- 46. Klenk HD, Wagner R, Heuer D, Wolff T (2002) Importance of hemagglutinin glycosylation for the biological functions of influenza virus. Virus Res 82: 73–75.
- 47. Vines A, Wells K, Matrosovich M, Castrucci MR, Ito T, et al. (1998) The role of influenza A virus hemagglutinin residues 226 and 228 in receptor specificity and host range restriction. J Virol 72: 7626–7631.
- 48. Connor RJ, Kawaoka Y, Webster RG, Paulson JC (1994) Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates. Virology 205: 17–23.
- 49. Nobusawa E, Ishihara H, Morishita T, Sato K, Nakajima K (2000) Change in receptor-binding specificity of recent human influenza A viruses (H3N2): a single amino acid change in hemagglutinin altered its recognition of sialyloligosaccharides. Virology 278: 587–596.
- 50. Paulson JC, Rogers GN (1987) Resialylated erythrocytes for assessment of the specificity of sialyloligosaccharide binding proteins. Methods Enzymol 138: 162–168.
- 51. Ge J, Deng G, Wen Z, Tian G, Wang Y, et al. (2007) Newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous H5N1 avian influenza viruses. J Virol 81: 150–158.
- 52. Jiang Y, Yu K, Zhang H, Zhang P, Li C, et al. (2007) Enhanced protective efficacy of H5 subtype avian influenza DNA vaccine with codon optimized HA gene in a pCAGGS plasmid vector. Antiviral Res 75: 234–241.


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Development/Infrastructure Works on the largest irrigation project in Afghanistan's history, the Qushtepa canal is in progress. This is the result of two months of intensive digging.
Development/Infrastructure A 6-minute 3-part video (merged together) showing some of the achievements of Kabul Municipality past month
Development/Infrastructure Russia is Ready to Invest in Afghanistan Energy Production - Bakhtar News Agencybakhtarnews.af
Development/Infrastructure Kandahar Public Works Department officials: The Arghandab district's largest bridge, one-third of which was destroyed in previous wars, has been rebuilt and commissioned at a cost of more than four million afghanis.
Development/Infrastructure 1,000 Houses Provided with Electricity in Past 3 Months in Ghazni - Bakhtar News Agencybakhtarnews.af
Development/Infrastructure Construction work on a bridge over the Aqcha River in Balkh has begun, with the cost of 8 million Afghani. The bridge will be 30 meters long and 5 meters wide and will be constructed in six months, which will connect Charbolak and Chimtal districts of the province.
Development/Infrastructure Qatar’s Al-Gharrafah Foundation to Complete Its Unfinished Projects In Afghanistanbakhtarnews.af


text:
Fundraising for Tibet World and Donation
It’s important to know where your donations are going. We are proud that all of our donations go to cover the cost of our educational and cultural programs. These donations allow us to keep cost low to participants so those that are in most need are able to use Tibet World services. All of our programs are greatly subsidized and we are only able to do that with the help of YOUR donations.
rs/300 or about $5- helps purchase supplies like markers and pens for five classrooms for a week
rs/2500 or about $40- helps purchase larger one time purchases, like seat cushions for students to use in our five classrooms.
rs/3,500 or about $55.00- helps cover the cost of one cultural show to encourage the growth of performers and share important traditions with others.
rs/4,500 or about $75 helps for the rent of a classroom. These classrooms are used to teach language courses to those in Dharamsala including Tibetan exiles.

domain: economics


More than an accessory, a jewel!
Want to cover Le Petit Cordon UK in your magazine, blog or in the press? We've put together the essentials to make your job easier. Need an image format, a product photo, an interview? Just get in touch! We'll be delighted to help!
The story of Le Petit Cordon
Who hasn't struggled in summer between pairs of sunglasses and prescription glasses? Where to put them? The solution is to wear a cord or chain to keep them around your neck.
Matthieu Hamel wants to bring the famous glasses cord up to date. Obsolete ? Not at all," says the 32-year-old entrepreneur...
Le premier site de cordons à lunettes est breton - Article broadcast on February 13, 2019: https://www.francebleu.fr/emissions/breizh-positive/armorique/le-premier-site-de-cordons-a-lunettes-est-breton
Is the eyewear cord becoming trendy? - Article published May 24, 2018:


text:
Do cats have sensitive ears?
How often should a cat have a bowel movement?
Is a cat that is ten years old considered to be a senior cat, or of old age?
What are the natural instincts of a cat?
What do you call male cats and female cats?
What is the drug methergine prescribed for?
Why does my cat scratch her ear?
Neomycin and Polymyxin B Sulfates is prescribed for cats with eye infections, ulcers, scratches or allergies. It is usually in the form of drops that are administered every 2 to 4 hours.
polymyxin is a drug used for cats to improve health and keep cats healthy and fit. Vetenarians often use this when checking up on cats to help cats live longer.
Polymyxin is used to treat bacterial infections in cats. It can treat a variety of ailments from ulcers to scratches, and allergies. Polymyxin is seen in many cat products for the treatment of eye infections.
Usually Polymixin is combined with neomycicin is prescribed for eye infections in cats, but can also be prescribed for scabs and open wounds that need a little bit of extra help healing.
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text:
CNBC says mistresses will want diamonds this year, company accidentally purchases a parcel of synthetics, and other stories on the industry’s radar this week.
– Are there diamonds in Antarctica? Russia to mine diamonds in Sudan.
– Indian industry leaders call for separate “certification” for synthetic diamonds. Dalumi says it accidentally purchased a parcel of synthetics. H. Goldie’s Mark Boston wonders: “What now?”
– Goldman Sachs bullish on diamonds. Anglo American doesn’t plan to sell diamond assets.
– Indian companies worry about profitability. Indian bank cautious about lending to diamond companies.
– Media Watch: The New York Times on the “murky trail of diamonds.” CNBC says mistresses are going to want diamonds. A publication called Death and Taxes doesn’t like diamonds.
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Ahmedabad: The Gujarat government on Friday announced 10 percent reservation for all non-reserved categories on the basis of their annual income.
Chief Minister Anandiben Patel told the media that the reservation will be given to all those whose annual income is Rs.6 lakh or less.
The announcement comes amid ongoing protests for reservations in jobs by the members of Patel community.
Kareena Kapoor Khan looks in fab shape after weight loss! See photos
Mumbai food: 9 dishes to try during 9 days of Navratri
Photos: Hrithik Roshan, Nidhhi Agerwal and Adah Sharma walk the ramp
Photos: Shah Rukh Khan spotted with new set of wheels
Photos: Kareena Kapoor Khan, Kangana Ranaut at the Mumbai airport


text:
Pictures from Italy (Hardcover)
Usually Ships in 1-5 Days
* Although seldom overtly political, 'Pictures from Italy' often touches on the corruption and cruelty of Italian history, the grinding poverty and a sense of continuing oppression lurking just below the surface.
About the Author
Charles Dickens (1812-1870), one of the greatest British novelists of all time was also a journalist, an actor and a curious observer of the streets. In his novels, essays and journalism he brought to life the momentous nineteenth century, captive to commerce and greed, science and progress, conquest and colonialism. Human oddities, frailty, greed, sorrow and sentiment, the poor, meek and the laboring moved him. Acts of time and history, mortality and the compassion that may yet redeem us absorbed his attention and got written into his extraordinary fiction.
Livia Signorini is based in Italy and splits her time between Rome and Milan. She studied art at Temple University and Universita la Sapienza of Rome, and has worked as an artist and picture researcher since 1988. Her work has been exhibited in different galleries in both Rome and Berlin, and includes mostly collages or the reinvention of found objects.


text: Serum beta 2-microglobulin concentrations were determined in 21 untreated hyperthyroid patients (12 with Graves' disease, and nine with toxic nodular adenoma) and in 20 healthy controls. All subjects had normal serum creatinine concentrations and urine analysis. Both total and free thyroid hormones were significantly higher in the hyperthyroid groups than in controls. Beta 2-microglobulin concentrations were significantly increased in both groups of hyperthyroid patients compared with controls. No difference was found in the thyroid hormone and beta 2-microglobulin concentrations between both sets of patients. The beta 2-microglobulin and thyroid hormone concentrations were not correlated. These data show that hyperthyroidism is another cause of increased beta 2-microglobulin production along with viral infections, immunologically mediated diseases, and malignant neoplasms. The increased serum beta 2-microglobulin concentration in thyroid hyperfunction is probably related to metabolic rate, even if autoimmunity might contribute to its overproduction.


text:
New Animal Food Nutrition laboratory at Massey University complete
After a delay earlier this year following the demise of Mainzeal Construction, the new Animal Food Nutrition Laboratory at Massey University in Palmerston North is now complete and fully operational. The existing laboratory was housed within a number of small interconnecting rooms in Ag/Hort A dating from the 1970s. It was no longer adequate to accommodate the increased work load, new equipment or the technological demands of a modern testing and analytical laboratory.
The new laboratory is a vibrant architectural addition to the Ag/Hort complex. The open laboratory spaces have been designed to be as flexible as possible to allow for future changes in technology, installation of new equipment and the introduction of new assays.
The laboratory is set out with the analytical and testing areas positioned around the perimeter with shared work spaces such as the balance room, wash-up, centrifuge and storage areas in the middle of the building. Consideration has also been given to vision from the write-up area through to the laboratory and within the laboratory itself to improve observation of the staff safety and laboratory protocols. Mobile benching will allow for access to the rear of analytical equipment for servicing and maintenance without the need for dismantling or disconnection of equipment.

domain: chemistry