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Upon the initial few minutes, Polo's new single 'On my End' is quite jarring and raw, but keep listening and intricate beats and instruments peek through and eventually it is like a jagged jigsaw puzzle coming together smoother over by satin vocals.
Alt-pop trio Polo from Leeds released their debut EP, 'Alice' last Spring and shortly after, completed their first UK tour, selling out small venues across the country.
Vocalist Kat describes the meaning of their new single out today, "I feel OME really captures my attitude to a lot of things a lot of the time. It's basically me putting my fingers in my ears to all the external nags and pressures to do things a certain way. It's a bit cheeky and to be taken with a pinch of sarcasm."
Fellow band members Luke and Dan also comment, "OME is one of the rare occasions where the first idea, was the only idea. We didn't even compromise at all. Every idea any one of us had was completely accepted by the rest, we threw everything at it and nothing got rejected, and we resulted in a song that is uniquely Polo, yet still shines as a personal achievement for all of us. The riff, chords, and percussion were written on the first ever session and remained unchanged throughout the process. We worked fast, didn't second guess ourselves and I think you can hear this in the track."
Last year Polo released their single 'Louder Than Words' which was showcased at key festivals such as The Great Escape, Live at Leeds, Hit the North, Evolution Emerging, Tramlines and The Great Exhibition of the North. As well as their largest and most successful tour to date, a string of Radio 6 plays and to top 2018 off, signed to Tipping Point Records (The Lake Poets, Eliza and The Bear), Generator's dedicated Artist Development programme & record label, Polo has certainly been propelled to stardom.
Polo's Live Dates
23 – Manchester – Jimmy's
24 – Hull – Polar Bear
26 – Liverpool – Sound
27 – Newcastle – Cluny 2
28 – Sheffield – Cafe Totem
1 – York – Fulford Arms
2 – Leeds – Headrow House


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OQP team members at our final quilt documentation day, April 27, Montavilla Sewing Center, Lake Oswego. L-R: Mary Brooner, Susie Berke, Janis Pearson, Claire Kellogg, Joan Beck, Eileen Fitzsimons, Renee Miller, Kathy Yee, Ann Pfrender, Lori Friedman, Melora Neminsky.
The final two quilt documentation days of the Oregon Quilt Project took place in the spring of this year.
Sisters, Oregon was the location of our second to last event, held on Friday and Saturday, March 8th and 9th. With advance planning by the Three Sisters Historical Society, East of the Cascades Quilters, and the Sisters Outdoor Quilt Show, we set up in the classroom of the Stitchin' Post quilt shop and began our recording process. As we have done throughout the Project's nine-year history we invited members of local guilds to work alongside us to learn how to document quilts so they can continue in their community after the event.
Using OQP documentation forms (available on-line), a few common tools and standard reference books, local volunteers took turns examining the quilts and recording their findings. We were able to operate two tables and with the half-hour-per quilt appointments added 47 quilts to the almost 1800 that the OQP team has recorded since 2010. The information and photographs have now been entered into the Quilt Index by patient volunteers Sheryl Miles and Janis Pearson, where it may be viewed by everyone with internet access. Although not deliberate, the March trip connected the beginning and end of the Oregon Quilt Project: the first documentation event took place in Sisters in April, 2010, under the leadership of Martha Sparks and Bill Volckening.
As we planned a documentation day in late April, the realization that the event would be our "swan song" brought eleven volunteers to the classroom of the Montavilla Sewing Center in downtown Lake Oswego. As in Sisters, we operated two tables and recorded 31 quilts, which are in the process of being added to the Quilt Index.
A day of mental focus and many hours of standing over exam tables is exhausting, but as always, it was exciting to see the quilts as they unfolded before us and we heard the stories of their creation. It was a poignant moment when we completed our paperwork and packed our tool box and bags, knowing that the documentation phase of the Oregon Quilt Project was finally complete. It was serendipity that the final quilt of record was "54-40 or Fight", a pattern that refers to the mid-19th century, when national debate was underway to determine placement of the northern boundary line of what became the State of Oregon on February 14, 1859.
While our documentations have ended, Project volunteers are working with our sponsor, the Willamette Heritage Center in Salem to open an exhibit on the discoveries of the Oregon Quilt Project. It is scheduled to open to the public on Friday, September 27 and run through December 23. See this page for a flyer/announcement of the exhibit, which we hope you, our readers, and quilt owners, will be able to attend.
Because the records of all recorded quilts are on the Quilt Index, we will not be producing a book on the Oregon Quilt Project. However, as part of the WHC exhibit, team members are creating a "storybook" about the Project and some of the quilts that will stay in their memories. Further details on this publication will be posted at a future date.
Stitches and Stories
Discoveries from the Oregon Quilt Project
An exhibit at the Willamette Heritage Center, Salem
September 27 – December 23, 2019
The Willamette Heritage Center is located at 1313 Mill Street, S.E.. An admission fee provides access to the rest of the WHC site, as well as the exhibition gallery.
Open hours are Monday – Saturday, 10 a.m. – 5 p.m. Hours may vary due to holiday events. Please confirm the gallery is open via their website before you come.

subset: commoncrawl


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Posted on March 25th, 2020
During these very difficult times in the midst of the Corona Virus. We need to do everything we can to boost our immune systems and deeply rest. One of my favorite ways to do this is to drink a nightly cup of India's Ayurvedic gem, "Golden Milk".
Every night before going to bed, I drink Ayurveda's "Golden Milk". It helps me calm down and relax so that I can get a good nights sleep. The golden color as well as it's potent immune boosting power comes from tumeric, the gem of Indian medicine and cooking. Tumeric has incredible digestive, anti-inflammatory as well as antioxidant properties. Coupled with ginger and the spices, it becomes a digestive powerhouse and immune booster, as well as a magical sleepytime elixir! I've been drinking it every night. I love the ritual of grating the fresh tumeric & ginger and grinding the spices. The pepper unpacks and increases the benefits of the tumeric. Cardamom helps cleanse the body during the night with it's detoxification properties. Cloves are a digestive stimulant as well as a great boost for the immune boost system helping release sinus & bronchial congestion. Cinnamon is also a digestive tonic. Nutmeg is an amazing sleep aid. The coconut cools all the heating spices down creating balance.
I find it the ritual of making Golden milk comforting and helps me start to relax. Then I light some candles, and put some essential oils in my diffuser (lavendar, clary sage, geranium, and bergamot are my favs these days) and curl up on my couch and sip my "Golden milk". It makes me deliciously relaxed and sleepy! Try it – You'll love it!
Golden Milk
Serves 1
1 mug of milk (almond or other nut milk, or coconut milk )
1 tsp fresh tumeric
¼ tsp fresh ginger
4 black peppercorns
2 cloves
2 pods green cardamon
Pinch cinnamon
Pinch nutmeg
½ tsp coconut mana
honey to taste
Heat up the milk and simmer. Grate the fresh tumeric & ginger and add to the milk. Grind the peppercorns, cloves and cardamon and add with coconut mana, cinnamon, nutmeg to the milk. Whisk until it becomes a gorgeous golden color. Strain & add honey. It's deeeeelicious!
Enjoy & stay well!
Posted on December 29th, 2019
This is it. It's 2020 and we're entering The Decade of Evolution.
We are invited to the Conscious Evolution of our species!
It really sunk in as I stood in the freezing cold outside NY City Hall with 1,000+ teens and a few adults, for the Dec 6. Climate Change protest. A 15 year old girl spoke passionately and eloquently about how the 2020's must shift everything. How when we look back from 2029, our energy sources, lifestyles, values, consumption patterns must all be carbon free in order to save our planet. With the latest IPCC reports, according to the Scientists, we only have about 10 years to make the changes necessary to reduce carbon in the atmosphere before we hit the tipping point. How are we going to do this? Entrenched patterns are so difficult to unravel and change. It takes so much mindfulness, intention, and compassion. Are we up to the task?
It is difficult to let go of what we know and even scary to be in the unknown. In the last few days of 2019, I've been reflecting on what is holding me back from making the transformations in my life that I envision. What do I need to let go of in order to invite full manifestation. I am feeling caught up in old patterns of personal difficulty and hopelessness. No matter how much I seem to work on these issues, some patterns just feel intractable! So I've been diving deeper into practices of mindfulness and compassionate loving attention with these issues. Tara Brach's podcasts and her, RAIN has been helping me alot- Recognize, Allow, Investigate, Nurture. I notice a softening and an opening. Transformation is happening. These practices of mindfulness and compassion give me hope. They are powerful tools to help us navigate the emotional process of transformation both personal and cultural in the Decade of Evolution.
It is exciting to be a part of this transformation of consciousness. Powerful to be moving into a way of inhabiting the world by living more consciously in tune with and connected with ourselves, each other, and our world. We as individuals making daily mindful choices for a healthier planet and life, as well as connecting with each other in community and evolving the emergent culture through working together. And sometimes we get lazy or tired, and sometimes we can't do it, but we do our best, and join with others on the path which helps us stay focused and buoyant. And little by little the shift is happening already! Conscious Evolution.
Posted on October 9th, 2019
Fall is here and I find myself deepening my self-care practices, embracing the yearly journey now turning inwards in our seasonal cycle. It can be difficult to release the Summer, with the fun travel & sun – the glorious fire element's expansion and outward expression. So to help me release in tune with the Season, I'm leaning into and consciously encouraging practices that summon a letting go. On retreat, as in this photo from our Nicaragua retreat, I offer restorative practices as the sun sets to help the students align with the daily cycle gracefully. Fall in our Seasonal cycle, is like the sunset. In the transition inwards that Fall offers, I often struggle with unraveling the constant outward and upward activity because it is so encouraged in our society and we are rewarded for our busyness and constant production. But all this constant activity all the time is exhausting and out of tune with the rhythms of the Earth. I find that when I move with the Season's invitation to go inwards and slow down, I feel more whole, more in touch with myself, more aligned with the Earth. I feel very nourished and rested. I'm inviting in more yin activities that welcome slowing down, honoring processing and reflection: sound healing, restorative yoga, Seasonal yoga practices, singing, pranayamana, meditating, and eating to balance vata dosha, the air element which gets agitated in Fall. Please join me this Fall for my offerings to help you slow down, go inwards, reflect and find balance in harmony with your inner self and connection with the Earth inside and outside. Self-care is Earth-care.
This Fall enjoy some of my sound healing offerings – sign up for a private sound healing sessions and join me for one of my Fall Rest & Renew: Restorative Yoga Sound Baths. Rest in deeply nourishing restorative yoga poses while basking in sonic bliss as Tibetan singing bowls, tuning forks, chimes, and drums lull you into an extraordinary meditative relaxed state.
Join me for a cozy Afternoon Tea YTT Info Session, on Sun Nov. 3 at my house for my Yoga Sukhavati 200 Hour Yoga Teacher Training. Don't miss the opportunity to save $150 with the Early Bird Special – Nov 25 – Level I – EVOLVE YTT – Costa Rica, March 12-22. I am thrilled to offer the first 100 Hour training on the glorious Osa Peninsula of Costa Rica, one of the most diverse places on the planet. It is a sublime setting to begin my Earth-focused 200 HR Teacher Training which fuses both the body and the earths' cycles, encompassing everything from anatomically based vinyasa yoga to ayurvedic wellness and women's practices to support the whole self in a lifelong journey of self awareness.
Ayurveda has given me so much insight and very tangible simple self-care practices, to help me find balance through the seasons and in my daily life. I am delighted to offer you the opportunity to explore these practices. Join me at Ayurveda Teacher Training Immersion, Nov 9-10 at Sacred Sounds. Invite health and well-being into your life!
joyous heart!


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A new study that zeroed in on a single city in Michigan found that where men are scarce, youth were more likely to commit assaults.
Researchers from the University of Michigan analyzed youth arrests and U.S. Census Bureau data for Flint, Mich., an industrial city whose fortunes have risen and fallen with General Motors Corp.
They found that "adult male scarcity" – a low ratio of adult men to women – was closely tied to the share of households that had meager incomes or were getting government assistance, as well as the share of single parents.
But a lack of men also seems to have added effects of its own, beyond its ties to poverty and family structure. Even after controlling for other kinds of disadvantages, male scarcity explained 36% of the difference in youth assault rates from census tract to census tract, researchers found.
"Male scarcity is actually a driver of conditions," said Daniel Kruger, research assistant professor at the University of Michigan and one of the authors of the study. "It's the most powerful predictor."
When education is factored in, it becomes an even more powerful predictor: Male scarcity, plus the percentage of people with less than a high school degree, explained 69% of the difference in youth assault rates, the study found.
Researchers tied the shortage of men to a slew of other troubles: Nationally, men have been more likely to lose jobs than women in recent decades, possibly pulling them away from Flint. Men are also at higher risk of dying from accidents, violence and some diseases, and those differences are bigger in poorer and less-educated communities. On top of that, high incarceration rates pull men away.
Based on the results, researchers suggested that strengthening relationships with fathers and adult male role models could help cut down on youth crime. Kruger said the study offered evidence that a scarcity of men, already known to shape marriage and relationships, also affected communities in other ways.
"When we look at population trends, not just in Flint but around the world, we see that the sex ratio really shapes a lot in our social world," he said.
The study, published online this month in the Journal of Community Psychology, was based on assault rates for people ages 10 to 24 between June 2006 and 2008, as well as data from the 2000 U.S. Census.
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Colorado Fans: Do Good and Order a Doc Holliday Salad
Our special fundraiser benefiting the National Multiple Sclerosis Society, Colorado-Wyoming Chapter (MS Society) is almost over! Until April 30, MAD Greens is donating $1 from every seasonal Doc Holliday salad sold to the MS Society at all of our Colorado restaurants. Check out some of the great media coverage we've received so far:
MS is an unpredictable, often disabling disease of the central nervous system that interrupts the flow of information within the brain, and between the brain and body. The MS Society mobilizes people and resources to drive research for a cure and to address the challenges of everyone affected by MS.
Pictured: A tasty salad for a great cause!
A tasty salad for a great cause. Now - April 30. Time's running out so we hope to see you soon, Colorado! To join the movement or learn more about creating a world free of MS, visit: www.cureMSco-wy.org.


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Presenting Signature residences at Centria by Kolte-Patil Developers in Pune's most promising location. Experience Ultra-luxury when you step into your signature 3 BHK Garden Home, design thoughtfully with East –West Facing. As part of the 30 acres Hills & Dales Project, that Home to Almost 500 happy families. It's proximity to schools, malls & hospitals ensure that you live the convenient life you always aspired.
· Most Promising Location
· Signature 3 RLK Garden Apartments.
· Each apartment design thoughtfully with East-West Facing.
· Close Proximity to School, Mall & Hospitals
· As part of the 30 acres Hills & Dales Project where 500 happy families already living.


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It's All In The Details.
At CDC Designs, we're inspired to create the perfect balance of interiors and lifestyle in every space we design. Each detail is selected to harmonize with every element, creating lasting designs that match our clients' objectives.
Our mascot, Duffy
From coast to coast and border to border, our team is ready to rock. We'd love to meet up and discuss your project. We look forward to hearing from you.Contact Us


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filter properties
Newly built luxury apartments for sale in Bodrum. In the heart of Bodrum Town with full sea & castle views. Ideal Bodrum investment 300m to the sea.
Ref: BOD2570APT
2 bedroom Apartment for Sale
Sea & Castle view Turkey real estate for sale in Bodrum Town. A modern 2 bedroom apartment with a prime location for an ideal investment in Turkey.
Ref: BOD1255APT
2 bedroom Apartment for Sale
Luxury sea, marina and castle view apartments for sale in Bodrum Town, Turkey. Ideally situated apartment with private pools for the ideal investment.
Ref: BOD1194APT
3 bedroom Apartment for Sale
Luxury Bodrum property for sale. Off-plan apartments close to the marina in Bodrum Town with 0% terms and a rental guarantee. Ideal Bodrum investment.
Ref: BOD3976APT
3 bedroom Apartment for Sale
Off-plan luxury apartments in Bodrum for sale. Walking distance to the marina in Bodrum town with a 2-year rental guarantee. Ideal Bodrum investment.
Ref: BOD3886APT
2 bedroom Apartment for Sale
Searching the Internet and looking at prices for modern apartment in Bodrum Town? - Bodrum Town is the life and soul of the Bodrum peninsula in southwestern Turkey, an area that offers everything from ancient history to a very vibrant nightlife and everything else in between, very popular for holidays and year-round living for many Turkish people and a large number of other nationalities who love the laid-back lifestyle and the wonderful climate.
A modern apartment in Bodrum Town for sale will not come cheap as property prices are on the whole premium and often with these high prices for modern apartment in Bodrum Town comes a stunning view of the Aegean Sea which can also tale in the marina and the ancient St. Peter's Castle along with a luxury lifestyle.
When looking to buy modern apartment in Bodrum Town for investment purposes these views if the sea will play an important factor, as they will help you to achieve a very high rental income as the area attracts a rather wealthy visitor who are always willing to pay that little extra to wake up to the perfect view, therefore, our recommendation is to search for a modern apartment in Bodrum Town for sale with a view of the sea and one that is located on the luxury market should your budget to buy modern apartment in Bodrum Town be high enough.
Contact Turkey Homes if you are looking at prices for modern apartment in Bodrum Town and we will direct you to the ideal property to suit your budget and lifestyle requirements and guide you through every aspect of the Bodrum property buying process and all the pros and cons about real estate in Turkey.
We appreciate your interest in Turkey Homes. One of our consultants will get back to you as soon as possible.
In the meantime if you require any assistance please call us on + 44 (0) 207 272 0002 or email us at firstname.lastname@example.com.


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Minor's Trust Attorney in DuPage County
Effective Legal Counsel to Care for Young Children in Western Cook County
Part of a good estate plan is to ensure that children under the age of 18 are cared for following an estate guarantor's passing. At AvoidProbate, our DuPage County minor's trust attorney can guide you through the legally complex and nuanced process of funding a specialized trust for your young family members. We offer flat-fee pricing to give you the counsel and guidance you need to move forward with your estate plan.
Call our firm today at (844) 798-7878 or contact us online. We offer free consultations.
Why Form a Trust?
It is a well-known fact that children under the age of 18 cannot legally own assets. They are not able to open a bank account or buy stocks in their name. This doesn't stop parents, grandparents, or other family members from giving money or assets to a child, usually through custodial (UTMA or UGMA) accounts. However, these accounts can be dangerous.
Any assets in a custodial account will automatically transfer to the child when they reach the age of majority (18 or 21). This means that if the child is in financial trouble or has substance abuse problems when that transfer occurs, having money to their name could hurt them in the long run. The solution is to work with one of our attorneys to prepare a 2503(c) minor's trust for each child.
A minor's trust can be structured to hold assets until a child is well into adulthood. An added benefit is that the trust doubles as a way to reduce the parent's or grandparent's taxable estate and can name them as beneficiaries of either life insurance or retirement plans. Overall, the minor's trust is vastly superior to custodial.
Client-Focused Service for Over a Decade
At AvoidProbate, we believe this valuable estate planning tool is greatly underused mainly because of the cost that most attorneys charge for them. Our firm offers minor's trust formation services at a low, flat fee so that they can be used in a variety of situations. You can count on us to look out for your best interests.
Call (844) 798-7878 now to begin working on your minor's trust.


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Syria – Opening of the 8th session of negotiations in Geneva (28 November 2017)
France welcomes the resumption today of the inter-Syrian peace negotiations under UN auspices in Geneva. We reaffirm our support for the mediation efforts of Special Envoy of the UN Secretary-General for Syria Staffan de Mistura.
After nearly seven years of war, the toll exacted by this conflict continues to rise. The cessation of hostilities and the unconditional, full and unrestricted delivery of humanitarian assistance to all populations in need are essential, both for the civilian populations and in terms of finding a political solution. The conference in Riyadh laid the groundwork for a unified, credible and pragmatic opposition. We also expect the regime's delegation to engage in good faith in these discussions in order to make progress toward a negotiated political solution, in accordance with the Geneva Communiqué and UNSCR 2254.
The Geneva process remains the only framework agreed by the international community aimed at finding a political solution in Syria. The international efforts to that end must be undertaken within this framework. In order to support the Geneva process, France is working to mobilize the permanent members of the Security Council in order to support Staffan de Mistura's mediation efforts and to produce the outlines of a political settlement. This is the reason for the meeting initiated by France between the five permanent members due to take place today in Geneva aimed at bringing their points of view closer together and effectively supporting the negotiations as


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Actor and Producer Ronald Guttman (Mad Men, The West Wing) joins his brother, Musical Director Michael Guttman and Symphony Napa Valley in narrating the gripping and rarely-performed orchestral poem of ancient origin, Genesis. Timeless works by Mendelssohn and Bloch complete the afternoon's spiritual journey.
Genesis, a symphonic poem by Dr. Baruch Berliner is inspired by the story of the world's creation that is described in the "Book of Books", the Bible. The work is particularly relevant in these days of tragic rifts between nations. In Genesis, ancient Biblical stories, intertwined with music that describes them, creates a universal bridge of peace and freedom, tolerance and acceptance.
Sunday, April 3, 2016 @ 3:00 PM
Lincoln Theater/Symphony Napa Valley
Purchase any price ticket through the Lincoln Theater Box Office at lincolntheater.com and simply add on the $15.00 VIP reception to your ticket price.
Reception to benefit Jewish Historical Society of Napa Valley
The VIP reception will take place in the upstairs mezzanine after the concert. There will be a 'meet and greet' with Maestro Guttman and Ron Guttman.


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Rachel McAdams opens up about welcoming first baby
Rachel McAdams in an interview confirmed that she welcomed a baby boy with boyfriend Jamie Linden in April
Actor Rachel McAdams has opened up about her pregnancy, which she had earlier kept as a secret. The 40-year-old, in an interview with The Sunday Times, confirmed that she welcomed a baby boy with boyfriend Jamie Linden in April.
'It's the greatest thing that's ever happened to me, hands down,' the Oscar nominee revealed, as reported by People magazine. '[People say] your life is not your own anymore, but I had 39 years of me, I was sick of me, I was so happy to put the focus on some other person.'
McAdams, who was 39 when she gave birth seven months ago, said she didn't want to have children before 'it was the right time,' and that she is happy she 'waited a long time' to embrace motherhood.
'The Notebook' star further said her new role as a mother has been one of the most enjoyable experiences of her life. 'I'm having more fun being a mum than I've ever had,' the 'Doctor Strange' actor explained. 'Everything about it is interesting and exciting and inspiring to me. Even the tough days ' there's something delightful about them.'
Catch up on all the latest entertainment news and gossip here. Also download the new mid-day Android and iOS apps to get latest updates
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Recent survey conducted by Graduate Management Admission Council (GMAC), the owner of the Graduate Management Admission Test ( GMAT) found that 96% employers thought hiring recent business school graduates created value for their companies.
The survey results revealed that demand for recent graduates of master-level business programs specially MBAs as well as for bachelor's degree-holders and experienced direct-from-industry hires. Over 75% employers expect to hire MBA graduates and 70% employers agreed that hiring MBA graduates and specialized business master's programmes is a priority in their company's hiring plans .It was also found that more than 50% graduates planning to increased average salaries for new MBA hires.
All rights reserved © 2020 Wisdom IT Services India Pvt. Ltd


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Students in grades 10, 11, and 12 will receive their schedules in homeroom on the first day of school, Wednesday, August 19. For students who do not know their homeroom, lists will be posted outside the office. Tuscola High staff is looking forward to the start of the school year. See you soon!
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DALLAS Jan. 31, 2007 New research suggests that different treatments may be needed for chronic asthma, depending on whether it results from allergies or lung infections.
Previous studies have shown that certain lung infections such as Mycoplasma pneumoniae can linger on and contribute to a person later experiencing symptoms of asthma.
Researchers have now identified a particular gene that influences how severe a M. pneumoniae infection may be, which in turn suggests that a different strategy might be needed for treating asthma resulting from this and similar lung infections rather than allergies.
"What this shows is that infectious asthma might have a different mechanism than allergic asthma. Most people think asthma is asthma, but it may be multifaceted," said Dr. Robert Hardy, an infectious disease specialist at UT Southwestern.
That's an important implication because the latest statistics show that asthma is on the rise. According to the U.S. Centers for Disease Control, more than 20 million Americans currently have asthma and another 10 million have been diagnosed with asthma at some point in their life. Roughly 6.5 million American children, or nearly 9 percent of the nation's pre-adult population, have asthma, figures released in December show.
Dr. Hardy, an assistant professor of internal medicine and pediatrics, has been using mice to study how certain pneumonia bacteria contribute to chronic asthma and, in this latest study, identified how a particular gene may contribute to more severe lung infection. The research appears in the January edition of Infection and Immunity.
Pneumonia is a lung infection typically characterized by breathing difficulties and spread by coughing and sneezing. Symptoms often include headache, fever, chills, coughs, chest pains, sore throat and nausea. Dr. Hardy's research involves pneumonia caused by the bacterium M. pneumo
Contact: Russell Rian
UT Southwestern Medical Center
Related medicine news :
1. Study, meta-analysis examine factors associated with death from heatstroke
2. Study says COPD testing is not measuring up
3. Study suggests loss of 2 types of neurons -- not just 1 -- triggers Parkinsons symptoms
4. Study reveals gaps in vaccine financing for underinsured children
5. Study suggests nonpharmaceutical interventions may be helpful in severe influenza outbreaks
6. Study shows radiofrequency ablation highly effective in treating kidney tumors
7. Study says normal but out-of-control enzyme may be culprit that signals some cells to become cancer
8. Study finds HIV protease inhibitor drugs may adversely affect the scaffolding of the cell nucleus
9. Study outlines how stroke, head injury can increase risk of Alzheimers disease
10. Study identifies new regulator of fat metabolism
11. Study shows Diachrome improves blood sugar control in people with type 2 diabetes


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Display Boxes in Chester
If you're in need of display boxes in Chester for retail, look no further than Cheshire Packaging. As an award winning print finishing company with more than two decades of experience behind us, we have a reputation for delivering exceptional promotional packaging to various sectors across the UK. From the initial enquiry through to the finished product, we work closely with our clients to provide them with display boxes which encourage sales whilst promoting brand and product memorability.
Bespoke Display Boxes with a "Wow" Factor
All of our display boxes are manufactured from the best possible materials and we use the latest high spec machines, including automatic die cutting machines, to produce unique features such as cut out shapes, embossments and unusual signs. If you already have a design in mind or your already have branded artwork, we can print these onto your chosen boxes.
Alternatively, if you need some help with the design, we also have our own team of in-house designers who will use their creative flair and marketing know-how, to produce innovative display boxes in Chester with a "wow" factor. Our main aim is to provide you with a high standard product which sets your brand apart from other competitors in your market.
To ensure that we have something for everyone, we offer a range of display boxes in Chester including, transit boxes such as promotional direct mail packaging, cartons, shelf ready packaging and window boxes – you can find out more about our products here.
Speak to us today for display boxes in Chester
With all of our packaging options, we offer total flexibility on design, colour and shape and we take great pride in operating to BSEN ISO 9001:2008 standards. To find out more about our display boxes in Chester, please get in touch, a member of our team will be more than happy to help and can answer any questions that you may have regarding cost, design processes and production processes.


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The RETRO-10 is specifically designed as a replacement for your traditional halogen lamps in most applications. It's as easy as removing the standard MR16 lamp and transformer and replacing it with the RETRO-10 lamp and LED driver. Featuring the Mono Lens, the RETRO-10 will look almost exactly the same as what you have just replaced.The RETRO-10 incorporates the latest Sharp COB (Chip on board) which is then matched with a specifically designed heat sink to disburse heat away ;from the LED. All this is then perfectly matched with a ;fully mains dimmable LED driver complete with flex and ;plug for easy installation.Variants: 20490 – 3000K,20491 – 5000K
Domus Retro-10 Led 10W Kit 60°


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In a circle, you in middle. The kids copy you running on the spot, jumping etc. When you say quick they get into the defensive position. However if you say get down they continue to do what they were beforehand they are only out if you see them.
5 upwards
Try to say 'quick' in the middle of a sentence.
Triple threat position.
Teaches the kids to listen to the coach carefully all the time.
Improves reaction time on defence
This basketball drill is licensed under a Creative Commons License. This drill is from Pål Degerstrøm at www.degerstrom.com/basketball.
Copyright 2020 BasketballCoach.com & Championship Productions, Inc.


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The Internet Chamber of Commerce presents Psychologists in Cave Creek, Arizona. Click on a company name for full contact, product and service details.
Ellen R Diamond Phd
7171 E Cave Creek Rd
Cave Creek
More information about Ellen R Diamond Phd
Veronique Pangia
7518 E Elbow Bend Rd
Cave Creek
More information about Veronique Pangia
Note: If you are aware of any errors or ommissions on this page, there is a link on each business record's page where it can be reported.


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You will realize that there is a tax that is time and again associated with the estate of a deceased. This will often include all properties, monies and even possessions. It is necessary to indicate that there is a distinction between estate and inheritance tax. It is necessary to point out that inheritance tax will every so often be based on the worth of a specific item or rather bequest. This is unlike inheritance estate tax that is based on the overall value of the estate. You will find it necessary to be familiarized with a number of matters regarding inheritance tax. They will every so often include the following.
You will learn that the executor of a will shall often be the ones to handle the payment of this tax. On the other hand, if there is no will, the administrator of this estate will be held responsible. To learn more about Inheritance Tax, visit Mr Probate. You will learn that this tax will be paid from the monies within the estate. Such will include money that has been raised for the sale of assets of the given estate. It is only after the tax has been paid that the remains will be distributed to the heirs. In most cases, you will note that this tax will be expected to have been settled within six months after the demise of the owner of this estate. This will ensure that there is no accumulation of interests. There is room for you to pay this tax over a span of a period of time. There will be room for instalments but you will be expected to pay on time. This will keep you from further interests.
It is necessary to mention that there are a number of gifts that are exempted from this inheritance tax. Such will time and again include wedding gifts as well as agricultural assets. It is necessary for these gifts to have been given out at least 7 years before the death of the owner of the estate. For more info on Inheritance Tax, click Mr Probate's frequently asked inheritance tax questions. You will note that the tax will often vary depending on when the gift was given and who gave it. You will learn that there are a couple of ways that will help you reduce this tax. Such will time and again include leaving your assets in the hands of a trust. You can also choose to leave the legacy to charity. You can also opt to leave your property with your spouse or even civil partners.
You will learn that it is possible for you to use life insurance to pay this tax. You will have to make sure that the policy is in a trust. Such a policy will only be paid after the owner dies. Learn more from https://en.wikipedia.org/wiki/Inheritance_tax.


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RSS-BJP likely to deliberate upon Ram Temple and Kumbh Mela in Lucknow on October 24
New Delhi, Oct 19: To discuss the strategy further on for the construction of Ram Temple in Ayodhya and asses preparations of Kumbh Mela in Allahabad, senior leaders of the Rashtriya Swayamsevak Sangh (RSS) will be meeting in Lucknow on October 24, 2018. This will be a big meeting of the RSS office bearer in which two joint general secretaries - Dattatreya Hosabale and Krishna Gopal - will be participating along with other RSS leaders.
The meeting is being called as a coordination meeting so it will also have representatives from the state government and the state BJP as well. Besides Hosabale and Krishna Gopal, Uttar Pradesh chief minister Yogi Adityanath and party president Mahendra Nath Pandey will also be participating in the meet. Some of the issues that the meeting is likely to discuss included devising new strategy for the 2019 Lok Sabha elections, assessment of the work done by the Uttar Pradesh government, Preparation of Kumbh Mela in Allahabad so far and also to discuss the strategy for the construction of Ram Temple after RSS Sarsanghchalak Mohan Bhagwat talked about exploring legislative rout for it.
Also Read | Right wing players intensify pressure on the govt for construction of Ram temple
As usual every secrecy is has been maintained about the meeting but sources said that around 500 senior workers from the RSS and its affiliate organisations will be participating in this meeting as the time ahead is very crucial for the BJP in view of the Lok Sabha elections and Ram Mandir issue being raised. The Ram Mandir issue is also has to be dealt with legally for which hearing will start on October 29, 2018 in the Supreme Court.
A big number of the RSS workers will be coming from outside Lucknow so arrangement has already made for them. RSS joint general secretary Krishna Gopal has been looking after both the issue - Ramjanmabhoomi and Kumbh Mela from the Sangh however for the political matter the entire team of the RSS deliberate upon under the guidance of general secretary Suresh (Bhaiya ji) Joshi.


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My eldest son and my youngest son after our Mum&Baby Yoga class yesterday, I turned to see them playing and my heart squeezed
I've been teaching Mum&Baby Yoga since my first son was 4 months old – he's now 9 years old!! 🙉🙈
I've learned so much from every mum that has come to the classes over the years and made such wonderful friends.
So thank you 💖
Feeling very grateful 🙏🙏🙏


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If you are due to have a colonoscopy, you may have questions about the procedure as well as what type of anesthesia will be used.
A colonoscopy is a procedure in which a doctor examines your large intestine (also called your colon) to look for and remove growths that could be precancerous or cancerous.
During the procedure, a doctor explores the colon with a colonoscope, which is a thin, flexible tube with a video camera attached to it. This instrument helps doctors look for growths such as polyps or tumors as well as inspect for inflammation, bleeding and ulcers.
According to Ira G. Rock, MD, director of Out of Operating Room/Endoscopy Anesthesiology in the Department of Anesthesiology at Yale Medicine, there are two kinds of anesthesia used during colonoscopies. If you have conscious sedation, it is usually administered by the doctor who performs your colonoscopy. If you undergo general anesthesia (also known as full or deep sedation), you can expect to be monitored by a credentialed anesthesiologist and nurse anesthetist.


text: Was getting ready to change the oil yesteday. Hooked up the fake a lake and started the engine to warm the oil. About 5 minutes in i reached in to see if i could spin the oil filter by hand and the intake line on the external oil filter housing popped off! What a mess! Oil everywhere. Of course I jumped up with oil all over me and killed the engine after at least 2 qrts pumped out in the engine compartment. Thank God i didnt have the carpet in. I spent the next 2 hours cleaning up the mess. I was able to pop the line back in the housing and now i cant seem to get it to pop back out. I went ahead and changed the oil and filter and and started the engine and the line held fine. How did this happen and will it happen again possibly on the water? Anybody ever had experience with this?


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O acetylation at carbon positions 7 or 9 of the sialic acid residues in the polysialic acid capsule of Escherichia coli K1 is catalyzed by a phase-variable contingency locus, neuO, carried by the K1-specific prophage, CUS-3. Here we describe a novel method for analyzing polymeric sialic acid O acetylation that involves the release of surface sialic acids by endo-N-acetylneuraminidase digestion, followed by fluorescent labeling and detection of quinoxalinone derivatives by chromatography. The results indicated that NeuO is responsible for the majority of capsule modification that takes place in vivo. However, a minor neuO-independent O acetylation pathway was detected that is dependent on the bifunctional polypeptide encoded by neuD. This pathway involves O acetylation of monomeric sialic acid and is regulated by another bifunctional enzyme, NeuA, which includes N-terminal synthetase and C-terminal sialyl O-esterase domains. A homologue of the NeuA C-terminal domain (Pm1710) in Pasteurella multocida was also shown to be an esterase, suggesting that it functions in the catabolism of acetylated environmental sialic acids. Our combined results indicate a previously unexpected complexity in the synthesis and catabolism of microbial sialic and polysialic acids. These findings are key to understanding the biological functions of modified sialic acids in E. coli K1 and other species and may provide new targets for drug or vaccine development.
Escherichia coli K1 is a versatile human and animal facultative pathogen that causes a variety of extraintestinal diseases including sepsis, meningitis, cystitis, pyelonephritis, cellulitis, pneumonia, and postoperative infections. The ability of E. coli K1 to invade and traverse the mammalian epithelial cell barrier also may contribute to inflammatory bowel syndromes such as Crohn's disease. A primary virulence determinant in these diseases is the polysialic acid capsule or K1 antigen, a homopolymer of 2-keto-3-deoxy-5-acetamido-7,8,9-d-glycero-d-galacto-nonulosonic, or N-acetylneuraminic acid (Neu5Ac, the most common sialic acid), residues connected by α2,8-glycoketosidic linkages (36). The kps and neu genes needed for polysialic acid synthesis and export map to a 17-kb accretion domain inserted near pheV (7). Mutation of neu (biosynthetic) genes generally results in no capsular polysaccharide produced while kps mutations usually result in intracellular accumulation of unexported polysaccharides (46, 47). Polysialic acid is also found on the mammalian neural cell adhesion molecule and comprises the group B meningococcal, Pasteurella haemolytica A2, and Moraxella nonliquefaciens capsular polysaccharides (41). Mammalian polysialic acid regulates cell migration, axon pathfinding and targeting, and plasticity in the embryonic and adult nervous system (6). Molecular mimicry of this antigen by the bacterial capsules is thought to account for the relatively low immunogenicity of microbial polysialic acid, which has limited the attempts to produce safe and effective capsule-based vaccines (41). Known functions of the capsule include inhibition of phagocytosis and other innate immune responses to microbial infection, but despite our understanding of capsule function during extraintestinal disease, we know little about its role in colonization of the mammalian large intestine. Increased understanding of the colonization process may suggest new targets for therapeutic development.
Unlike the neural cell adhesion molecule or group B meningococcal polysialic acid, the E. coli K1 capsule may exist in an alternate form in which the individual Neu5Ac residues are variably modified with O-acetyl esters at carbon positions 7 or 9. The O-acetyltransferase gene, neuO, responsible for these modifications is carried on a K1-specific prophage designated CUS-3 (13). In addition to lysogeny, neuO expression is controlled by a translational switch involving slipped-strand DNA mispairing of heptanucleotide repeats located in the 5′ coding region. This switch is designated the polyΨ domain, where loss or gain of heptad repeats in any number other than a multiple of three results in frameshift mutation and synthesis of truncated (inactive) neuO gene products. The neuO contingency locus and its mobile phage delivery vehicle account for at least five capsule forms: (i) permanently acetylation "off" because the cell is not a CUS-3 lysogen, (ii) stochastic variation in the proportion of "on" and "off" forms caused by neuO frameshifting, (iii) variation in the degree of acetylation, which may depend on the length of the polyΨ domain, (iv) variation in the positions of acetyl esters on individual Neu5Ac residues of the polysialic acid chains (carbon positions 7 or 9), resulting from nonenzymatic transesterification and (v) variation in the positioning of sialyl O-acetyl esters along the chains resulting from incomplete acetylation. Variation in neuO and its metabolic products thus has the capacity to alter capsule antigenicity and physiochemical properties of the K1 cell surface, with one locus accounting for potentially thousands of different capsular phenotypes (48).
In addition to neuO, the K1 neuD gene product annotates as an acyltransferase (4), and the group B Streptococcus (GBS) NeuD orthologue has been shown to be a monomeric sialic acid O-acetyltransferase responsible for modification of the streptococcal capsular polysaccharide (23, 24). Complementation of a GBS neuD mutant with K1 neuD+ restores sialyl O acetylation, indicating that K1 NeuD is also a monomeric O-acetyltransferase (23). In GBS, where O-acetyl esters are found at carbon positions 7, 8, or 9, the acetylated monomers are activated and transferred as terminal nonreducing residues of the capsule main chain. Therefore, neuD in E. coli K1 suggests there may be two acetylation pathways in this species, one involving neuO for modification of polysialic acid and the other, involving neuD, for acetylation of monomeric sialic acid. Furthermore, neuA encodes a bifunctional enzyme including N-terminal cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac synthetase) and C-terminal esterase in both the K1 and GBS systems (26, 52), suggesting a mechanism for converting acetylated monomeric sialic acid to the de-O-acetylated forms. In this communication we describe separate pathways for the O acetylation of polymeric and monomeric sialic acids and provide the first demonstration of a new class of esterase with activity against O-acetylated sialic acids.
Bacterial strains, plasmids, and growth conditions.The bacterial strains and plasmids used in this study are shown in Table 1. Plasmid pWV200b used for overproduction of E. coli K1 NeuA was derived from pWA1 (53, 54) as a template using PCR primers containing EcoRI and HindIII sites. Double-digested PCR product was cloned into similarly digested pKK223-3, placing K1 neuA + under control of the tac promoter and the ribosome binding site of pKK223-3. A truncated version of K1 neuA containing the N-terminal 254 amino acid residues (neuA′) was derived by PCR from pWS313 by mutagenesis methods described earlier (42) with the reverse primer Del 254R (5′-CGGCTCGAGCGTTATATCACTTACACTATCGAATTCCGG) to yield pWS319. This plasmid retains the synthetase domain but lacks the C-terminal esterase domain defined previously by Liu et al. (26). LB (Lennox formulation) was purchased from Fisher Scientific and used as the rich medium in all experiments. Minimal M63 salts medium (35) containing 0.4% glycerol as a carbon source was used as the defined medium where indicated. Ampicillin or chloramphenicol was used at 100 or 20 μg/ml, respectively, for plasmid maintenance. Cultures were grown at 37°C with vigorous aeration in a water bath equipped with a rotary shaker. Unless indicated otherwise, all cultures were supplemented with 20 μg/ml of Neu5Ac and grown to early stationary phase prior to cell harvesting by low-speed centrifugation.
Chemical reagents.Purified bovine submaxillary gland mucin (BSM) was kindly provided by Tony Corfield (Bristol, United Kingdom). Neu5Ac was purchased from ICN (Aurora, OH). N-acetyl-9-O-acetylneuraminic acid (Neu5,9Ac2) was a gift from Roland Schauer (Kiel, Germany), while samples of N-acetyl-7-O-acetylneuraminic acid (Neu5,7Ac2), N-acetyl-9-O-actylneuraminic acid (Neu5,9Ac2), N-acetyl-7,8 or 9-O-acetylneuraminic acid [Neu5,7(8),9Ac3], and N-glycolyl-9-acetylneuraminic acid (Neu5Gc9Ac), purified from BSM as previously described (43), were provided by Tom Warner (San Carlos, CA). N-glycolylneurminic acid (Neu5Gc), 3-deoxy-d-manno-octulosonic acid (KDO), para-nitrophenyl-acetate (pNP-Ac), and 1,2-diamino-4,5-methylenedioxybenzene (DMB) were purchased from Sigma (St. Louis, MO). All other chemicals were purchased from Sigma or Fisher Scientific and were of the highest technical grade available.
Enzymes.Recombinant, histidine-tagged endo-N-acetylneuraminidase (endo-N), from phage PK1E, was purified from a culture of IPTG (isopropyl-β-d-thiogalactoside)-induced E. coli BL21(DE3) essentially as described previously (30). CMP-Neu5Ac synthetase was purified as previously described (53, 54) from a culture of induced cells expressing pWV200b and had a specific activity of 610.3 units/mg protein, where 1 unit activates 1 μmol of Neu5Ac in 1 min at 37°C. Other enzymes were used as induced soluble extracts from cells (BL21 or DH5α) grown in overexpression broth (Zymo Research, Orange, CA) with 1 mM IPTG. Esterase activity was assayed as described by Yu et al. (52), with the absorbance of para-nitrophenol, resulting from hydrolysis of pNP-Ac, determined at 405 nm after 6 min of incubation at room temperature using a Beckman DU-640 spectrophotometer. Complete hydrolysis of pNP-Ac was accomplished by the addition of NaOH to a final concentration of 0.1 M. Data are expressed as activity relative to complete base hydrolysis, normalized for protein concentration as indicated.
Isolation and characterization of neuO form variants.Strains EV717 (neuO "off") and EV718 (neuO "on") were isolated from EV291 essentially as described previously (13). Briefly, EV291 was grown overnight in LB and plated for single colonies on agar medium containing 10% (vol/vol) horse-46 anti-polysialic acid antiserum. Colonies were examined for surrounding halos representing precipitation responses to polysialic acid, where acetylated capsular polysaccharide gives no or only a weak halo response. The lengths of the polyΨ domains in the two variants were determined by PCR analysis using flanking forward and flanking reverse primers (13) and were found to contain 20 and 21 heptad repeats in EV717 and EV718, respectively.
DMB analyses and TLC of α-keto compounds.For the analysis of capsular polysialic acid by thin-layer chromatography (TLC), cells from 10- to 30-ml cultures were harvested by centrifugation. The pellets were resuspended in 1/10 volume of 10 mM phosphate buffer (pH 7.0) and repelleted by centrifugation. Polysialic acid in the supernatants was either applied directly to silica gel thin-layer plates or treated with endo-N (50 μg) at 37°C for 2 h to release sialic acids prior to the application. Plates were developed with n-propanol:water (7:3, vol/vol) solvent, and sialic acids were visualized by orcinol spray as previously described (39). For DMB analysis, oligosialic acids released by endo-N digestion of whole cells were hydrolyzed completely to monomers by incubation at 80°C with an equal volume of 4 M acetic acid for 3 h. Any precipitate was removed by centrifugation in a microcentrifuge at 16,100 × g for 10 min at 4°C. Samples were concentrated at least 10-fold in a Savant SpeedVac system and recentrifuged, and 15-μl samples were subjected to DMB-labeling exactly as described in the original procedure (18). After being labeled in the dark for 2.5 h at 50°C, samples were centrifuged through 0.22-μm-pore-size nylon filters, and 6-μl aliquots were immediately analyzed fluorescently (373 nm excitation and 448 nm excitation) using a reverse phase (RP) high-performance liquid chromatography (HPLC) system equipped with Dionex AS50 Autosampler, GP50 Gradient Pump, RF2000 Fluorescence Detector, and Chromeleon version 6.50 software for data management. The solvent, acetonitrile-methanol-water (8:7:84, vol/vol/vol), was slightly modified from the 9:7:84 mixture originally described (18). One major modification was the use of a 4.6-mm by 10-cm TSKgel Super-ODS column (2 μm, 110 Å; Tosoh Bioscience, Montgomeryville, PA), which resulted in elimination of the reagent peak or DMB-breakdown product that elutes just after Neu5,9Ac2 (18). After the completion of any given set of experiments, the column was washed with 50% methanol before the next use. For the analysis of intracellular sialic acids, soluble cell extracts were prepared by sonic disruption and centrifugation to remove debris and then incubated with an equal volume of 4 M acetic acid for 30 min at 80°C before precipitated material was removed by centrifugation and RP-HPLC analysis as described above. Data were expressed as relative fluorescence versus time as previously described (23, 24), where elution of Neu5Ac quinoxalinone derivatives (25) varied between 9.5 and 13.6 min. However, on a given day's experiments the relative elution of all peaks was consistent. Where quantitative comparisons were made, the supporting data are posted on the Laboratory of Sialobiology website (www.cvm.uiuc.edu/path/sialobiology ). Confirmation of O acetylation was obtained by mild-base hydrolysis prior to DMB labeling (24).
Two molecular forms of CMP-sialic acid synthetase.Activation of sialic acid for transfer to nascent polysialic acid by NeuS (polymerase) is catalyzed by CMP-Neu5Ac synthetase (NeuA). Liu et al. (26) showed that the first 229 amino acid residues of NeuA comprise the synthetase, whereas residues 228 to 419 function as an acetylhydrolase (acylesterase) with predicted tertiary structural similarity to the α1-subunit of bovine brain platelet-activating factor acetylhydrolase (PAF-AH) isoform I. Similarly, the GBS NeuA orthologue C terminus was shown to be an acetyl esterase, and the authors (52) speculated that this activity might regulate accumulation of acetylated Neu5Ac derivatives synthesized by NeuD (23, 24). These observations suggest two molecular forms of NeuA: the long or bifunctional form, and a short form composed of just the synthetase domain. This hypothesis predicts that organisms with neuD might acetylate monomeric sialic acids and use a long form of NeuA to regulate intracellular O-acetylated monomeric sialic acid concentrations, while organisms that synthesize or activate sialic acids but that lack neuD might express only the NeuA short form that lacks esterase activity. We designated the putative sialyl O-acetyl esterase domain of the synthetase NeuA-star (NeuA*).
To investigate the correlation between the NeuA form and sialic acid acetylation, we carried out a BLAST (3) search for K1 NeuA and NeuD orthologues. As shown in Table 2, there was general concordance between acetylation/NeuD and NeuA*. Notable exceptions were the neuA gene products of Pseudomonas aeruginosa and Legionella pneumophila, which are known to acetylate the sialic acid-like molecules pseudaminic and legionaminic acid, respectively, and Neisseria meningitidis groups C, Y, and W-135, which are known to acetylate sialic acid but express the NeuA short form. Campylobacter jejuni was unusual because it contains both short and long synthetase forms. L. pneumophila expresses the NeuA short form while the P. aeruginosa long-form C-terminal domain was more similar to nucleotidyl transferase than esterase. When taken together, the results shown in Table 2 suggest that there may be two acetylation pathways in E. coli K1, one for polysialic acid catalyzed by the neuO-encoded O-acetyltransferase and another for acetylation of monomeric sialic acids (Fig. 1).
Detection of O-acetylated cell surface sialic acids.Note that the salient features of proposed sialic and polysialic acid synthesis in Fig. 1 show two O acetylation pathways interconnected at the substrate level by specific (NeuA*) or nonspecific O-acetyl esterases and the possibility that NeuO could act on the putative polysialic acid products of the NeuD-catalyzed acetylation pathway. Testing these hypotheses required a simple method of monitoring capsule and intracellular O-acetylated sialic acids. Unfortunately, published methods of polysialic acid isolation and characterization are laborious and require high polysaccharide concentrations, while chemical methods provide no information about the site(s) of O acetylation (28). To facilitate analytical investigation of acetylated polymeric or monomeric sialic acids from multiple strains or relatively small amounts of material, we took advantage of the specificity of K1-lytic phage endo-N. Figure 2 (lane 2) shows that digestion of polysialic acid from strain EV36 with a high concentration of endo-N results in a mixture of monomeric and oligomeric sialic acids. Note that the plate was overloaded so that minor O-acetylated sialic acid could be detected if present. A similar digestion pattern was observed when commercially available polysialic acid (colominic acid), also unacetylated (13), was treated with endo-N (Fig. 2, lane 4). When polysialic acid from EV36 harboring pSX785 (neuO +) was treated with endo-N, a sialic acid species migrating faster than Neu5Ac was detected (Fig. 2, lane 3). The relative mobility of this species was consistent with it being Neu5,7Ac2 or Neu5,9Ac2 (32). Sensitivity of this faster-migrating species to mild-base hydrolysis confirmed its identification as acetylated sialic acid (not shown). The colorimetric TLC method shown in Fig. 2 demonstrates that O acetylation neither prevents polysialic acid hydrolysis by endo-N nor affects the composition of the limit digestion products. This conclusion suggested a simple method for rapid and specific analysis of O-acetylated polysialic acids.
Figure 3 shows the details of our analytical method, indicating release of oligosialic acids by endo-N treatment and subsequent removal of intact cells by centrifugation. The released material is converted to a mixture of free Neu5Ac and O-acetylated monosaccharides (if present) by hydrolysis in dilute acetic acid under conditions that do not destroy sialyl O-acetyl esters. Labeling with DMB converts the sialic acids in the hydrolysate to their quinoxalinone derivatives that have characteristic retention times during RP-HPLC (Fig. 4A). Although all of our analyses were carried out with pmol amounts of sialic acids, DMB labeling is reported to detect as little as 50 to 100 fmol (18). Base treatment of BSM-derived sialic acids resulted in loss of peaks representing Neu5,7Ac2, Neu5Ac9Gc, Neu5,9Ac2, and Neu5,7 (8),9Ac3 and the expected increased ratio of Neu5Ac to Neu5Gc, which are both base resistant (Fig. 4B). Relative retention times of acetylated sialic acid derivatives were consistent with the originally described method (18), company literature (ProZyme Signal Product Code GKK-407), and tandem electron spray mass spectrometry (24).
Polysialic acid O acetylation is primarily dependent on NeuO.To detect acetylated polysialic acid independently of any CUS-3 open reading frames other than neuO, strain EV36 transformed with vector or vector expressing neuO + (pSX785) was subjected to DMB analysis. As shown in Fig. 5A, over 98% of the sialic acid from EV36 was unacetylated, indicating that if NeuD or any other O-acetyltransferase is active in this strain, it can account for only about 2% of the total capsular sialic acid residues. In contrast, polysialic acid derived from EV36 expressing pSX785 produced free Neu5Ac as well as detectable Neu5,7Ac2 and Neu5,9Ac2, which are the two known acetylated forms present in E. coli K1 strains lysogenized by CUS-3 (13, 18). Note the absence of triacetylated [Neu5,7 (8)9Ac3] derivatives, which would elute after Neu5,9Ac2 (Fig. 4A), indicating that NeuO most likely acetylates either the carbon-7 or carbon-9 position followed by nonenzymatic transesterification between positions. The results of DMB capsule analysis indicate that most of the acetylated Neu5Ac in polysialic acid is synthesized by the NeuO pathway (Fig. 1). Nonetheless, about 2% of the sialic acids appeared to be O acetylated by a NeuO-independent pathway that is likely to depend on NeuD (23).
To provide further evidence for the quantitative importance of the NeuO-catalyzed pathway, we carried out DMB analysis of strain EV291, a derivative of the prototypic K1 clinical isolate RS218 known to carry the CUS-3 prophage and undergo capsule form variation (13). Figure 5C shows the expected sialic acid profile of the isogenic neuO deletion mutant EV708. This profile is nearly identical to that of EV36 shown in Fig. 5A. Similarly, an "off" derivative (EV717) of EV291 produced mostly unacetylated polysialic acid (Fig. 5D). In contrast, an "on" form variant of EV291 (EV718) yielded a profile resembling that of EV36 harboring pSX785 (Fig. 5B), except that about 35% instead of 16% of the total sialic acid was O acetylated (Fig. 5E). Variation in the degree of acetylation is consistent with previously reported strain differences (19, 21, 28, 31). Note especially that in the "off" profile a small but detectable amount of material, consistent with Neu5,7Ac2 and Neu5,9Ac2, as well as a peak representing Neu5,8Ac2, was observed (Fig. 5D). These small amounts of NeuO-independent acetylated sialic acids may have resulted from leakage of intracellular monomers produced by a second O acetylation pathway in E. coli K1 (Fig. 1). However, this hypothesis is unlikely because, as shown below, neuA + strains do not accumulate O-acetylated sialic acids. Therefore, they most likely arose from activation and incorporation of a small proportion of O-acetylated monomeric sialic acid that escaped NeuA* recycling (Fig. 1). While some of the O-acetylated forms in strain EV717 may have arisen from the stochastic proportion of "on" forms expected to result during outgrowth of the predominant "off" form (13), this amount can be no more than 1 or 2% (compare O acetylation in Fig. 5D with that of Fig. 5C). These considerations suggest that there is a minor or secondary pathway for the O acetylation of monomeric sialic acid and that about 2% of these forms are ultimately incorporated into polysialic acid independently of NeuO.
A second O acetylation pathway for monomeric sialic acid.Lewis et al. (24) reported that a mutation in the GBS neuA orthologue resulted in accumulation of Neu5,7Ac2, Neu5,8Ac2, and Neu5,9Ac2, suggesting that NeuA normally out-competes NeuD or other O-acetyltransferases for Neu5Ac substrate. Alternatively, loss of the NeuA* esterase activity in this mutant might have accounted for the increase in acetylated forms (52). If a similar pathway is operable in E. coli K1, loss of NeuA in a mutant also lacking sialic acid aldolase (NanA) should result in accumulation of acetylated sialic acids (Fig. 1). Elimination of NanA would be necessary to detect this phenotype because in a neuA mutant the increased pool of Neu5Ac binds NanR repressor, leading to induction of the nanATEKyhcH catabolic operon and destruction of intracellular sialic acids (22, 29, 30).
When a whole-cell extract (sonicate) of the nanA neuA double mutant EV715 was subjected to DMB analysis, four peaks other than free Neu5Ac were observed (Fig. 6A). On the basis of BSM-derived standards (Fig. 4A) and analogy to the results of tandem electrospray mass spectrometry (24), the three peaks eluting with retention times greater than the Neu5Ac peak represent Neu5,7Ac2, Neu5,8Ac2, and Neu5,9Ac2, respectively. Confirmation that these peaks were derived from O-acetylated sialic acids was shown by sensitivity to mild-base treatment carried out prior to DMB-labeling (Fig. 6B). Note the increase in the ratio of peak c (Neu5Ac) to peak a in Fig. 6B, indicating conversion of O-acetylated sialic acids to Neu5Ac. Peak a, with the earliest retention time, eluted even before Neu5Gc (Fig. 4A), suggesting that it might be the 8-carbon α-keto sugar acid KDO (34). Spiking a sample of the EV715 extract with Neu5Gc prior to DMB analysis resulted in the addition of one new peak to the profile (Fig. 6C), showing that peak a was not Neu5Gc. Chromatography of derivatized KDO, Neu5Gc, or KDO plus Neu5Gc unambiguously confirmed that peak a was derived from KDO (not shown). We assume that KDO in the intracellular extracts results from partial release of free KDO during acid treatment from lipopolysaccharide present as contaminating membrane material. However, some kps genes (kpsF and kpsU) have been shown to function in the KDO biosynthetic pathway, warranting future studies of KDO metabolism in E. coli K1 strains. Because free sialic acid is not detectable by colorimetric or amperometric methods in wild-type E. coli K1 (29), we concluded that loss of NeuA allows accumulation of sufficient free Neu5Ac in a nanA mutant background to produce a detectable pool of acetylated forms, which by analogy to GBS may result from the action of NeuD (Fig. 1). Note that the combined amount of diacetylated forms is at least twice that of the intracellular Neu5Ac concentration (Fig. 6A). Although the concentration of all sialic acid derivatives was reduced in the nanA + neuA mutant, EV716, acetylated forms were still detectable (Fig. 6D). We assume that the known relative resistance of acetylated sialic acids to sialate aldolase encoded by nanA accounts for this observation (32).
Although LB contains a low concentration of free Neu5Ac (45), all of the acetylated forms synthesized by EV715 and EV716 (Fig. 6) were dependent on the mutant background. This conclusion was substantiated by analysis of an extract from EV78, an E. coli K-12 derivative of strain MC4100 that lacks the kps/neu accretion domain. Note the accumulation of Neu5Ac by EV78 but the absence of diacetylated forms (Fig. 7A). As expected, when EV78 was grown in nonsupplemented minimal medium, no free Neu5Ac peak was observed (Fig. 7B). However, when the medium was supplemented with exogenous Neu5Ac, accumulation of intracellular sialic acid was detected (Fig. 7C). Note that the failure of supplemented EV78 to produce diacetylated sialic acids means that E. coli lacking the kps/neu genes does not express Neu5Ac O-acetyltransferase. Furthermore, the failure to accumulate diacetylated forms by the nanA neuB neuS triple mutant, EV239 (compare Fig. 7D and E) suggests efficient de-O acetylation by NeuA*. Although as expected EV36 (wild type) does not accumulate detectable free sialic acid, the peak eluting with the longest retention time represents DMB-labeled pyruvate, another α-keto acid that most likely results from sialic acid accumulation and subsequent NanA cleavage during polysialic acid biosynthesis (Fig. 7F). Finally, to determine if activation of sialic acids protects acetylated forms from NeuA*, we analyzed an extract from strain EV136 that accumulates CMP-Neu5Ac due to a NeuS polymerase defect (29, 38, 51). Although the free Neu5Ac peak resulting from acid hydrolysis of CMP-Neu5Ac in this extract was apparent, acetylated forms were not detected, indicating that if acetylated sialic acids are activated by the synthetase, NeuA* deacetylates them prior to DMB labeling (Fig. 7G). We conclude from the results shown in Fig. 5 to 7 that the monomeric O-acetyltransferase, probably NeuD (23), produces diacetylated sialic acids that are normally deacetylated by NeuA* or nonspecific esterase (Fig. 1). As shown in Fig. 5, only a small percentage of the acetylated monomers are ever incorporated into capsular polysialic acid (Fig. 5). This is a major distinction with the NeuD catalyzed pathway in GBS, where 50 to 60% of O-acetylated monomeric sialic acids are added to the streptococcal polysaccharide (23, 24).
O-acetyl esterase (NeuA*) activity of NeuA.To determine if NeuA has sialyl O-acetyl esterase activity, recombinant synthetase was added to an extract of EV715. As shown in Fig. 8A, all three diacetylated peaks were sensitive to the NeuA* activity of the purified enzyme. Heating enzyme at 90°C for 5 min prior to addition to the EV715 extract eliminated esterase activity, indicating the absence of nonspecific de-O acetylation (Fig. 8B). Using half the amount of enzyme as the experiment shown in Fig. 8A resulted in incomplete deacetylation (Fig. 8C). That contamination of the enzyme preparation by nonspecific esterase is an unlikely explanation for these results was apparent from the extended (5 h) incubation of the EV715 extract at 37°C. Though this treatment resulted in some loss of Neu5,7Ac2, it had only a minor effect on Neu5,8(9)Ac2 (Fig. 8D), while all three acetylated forms were eliminated after incubation with purified NeuA for 30 min (Fig. 8A).
NeuA* has been shown to hydrolyze pNP-Ac between pH 7.5 and 9.0 (26, 52). As shown in Table 3, the normalized recombinant NeuA activity against this model substrate was as great as the hydrolysis produced by alkali treatment, indicating that NeuA* rapidly and completely deacetylated pNP-Ac. Similarly, an extract containing overproduced K1 NeuA, but not one containing the overproduced NeuA short form from group B meningococci, expressed NeuA* activity that was at least nine times higher than background (Table 3). This background activity was likely due to nonspecific esterase because the same amount of background was observed in an extract prepared from EV715, which lacks NeuA because of mutation (Table 3). In contrast, an extract containing overproduced Pm1710 from Pasteurella multocida (37) had elevated esterase activity while an extract containing the overproduced short form of P. multocida NeuA (Pm0187) did not (Table 3). When taken together, the results shown in Fig. 8 and Table 3 indicate that NeuA*, in addition to its expected activity against pNP-Ac (26, 52), is a sialyl O-acetyl esterase. This conclusion is supported by the identity of conserved blocks I, II, III, and V found in GDSL esterases of the SGNH-hydrolase family (2) whose members include conserved catalytic SGNH residues at the indicated positions of each polypeptide shown in Fig. 9, strongly suggesting that Pm1710 also is a sialyl O-acetyl esterase (37). Further confirmation that NeuA* is an acetyl esterase was obtained by showing that an extract containing overproduced, truncated K1 NeuA composed of just the first 254 residues (synthetase domain) lacked elevated activity against pNP-Ac (Table 3).
Contribution of NeuD to O acetylation of polysialic acid.To determine if the small amount of NeuO-independent O-acetylated sialic acids represents monomers that escaped NeuA* recycling and were subsequently incorporated into polysialic acid, we used exogenous Neu5Ac to rescue the nanA neuD double mutant RS2887 (12). In addition to its activity as a monomeric sialic acid O-acetyltransferase (23), NeuD is required for sialic acid synthesis (4, 12). The synthetic defect of a neuD mutant can be rescued by adding Neu5Ac to the growth medium as long as the cell lacks NanA to prevent the lyase from destroying transported monosaccharide. When polysialic acid from sialic acid-rescued RS2887 was analyzed by DMB labeling, there were no detectable O-acetylated forms (not shown), indirectly suggesting that NeuD is necessary for the minor O acetylation detected in wild-type strains lacking neuO (Fig. 5). We conclude that NeuD is a functional monomeric O-acetyltransferase in E. coli K1 but that NeuA* recycles most monomers to Neu5Ac before incorporation into polymer (Fig. 1). The exact function of this minor pathway is unknown, although we assume that NeuD is responsible for the O acetylation of monomeric sialic acids in species such as GBS, E. coli O104, and others (23). However, our results unambiguously demonstrate the quantitative importance of the NeuO-catalyzed pathway, suggesting that NeuD, at least in E. coli K1, plays only a minor role in capsule modification.
Identification and function of NeuA*.Our results indicate that sialyl O-acetyl esterase activity is catalyzed by the long form of NeuA, a bifunctional CMP-Neu5Ac synthetase in E. coli K1 and other bacteria that decorates their surfaces with sialic acids (Table 2). On the basis of three-dimensional molecular modeling and biochemical activity, Liu et al. (26) concluded that the K1 NeuA C terminus (residues 229 to 419) was a PAF-AH. These authors speculated that PAF cleavage may function in E. coli K1 invasion of the blood-brain barrier after proteolytic release of PAF-AH from the N-terminal synthetase domain (26). We think this suggestion is unlikely for at least six reasons. First, NeuA is a cytoplasmic enzyme, and even if it were proteolytically processed in vivo, how it gains access to PAF would still remain unclear. Even if PAF were somehow accessible as a substrate, it is unclear how hydrolysis would affect endothelial invasion. E. coli K1 invasion of the blood-brain barrier has been studied extensively in tissue culture, and the model seems unconvincingly complicated since mutants with in vitro invasion defects still cause meningitis in the rodent disease model and have only modest invasion defects in vivo (20). Second, our demonstration that O-acetylated sialic acids are NeuA* substrates provides the first direct evidence for this activity, suggesting that PAF may not be a physiological substrate. Third, the failure to accumulate O-acetylated sialic acids in a neuA + strain points to a function of NeuA* in regulating the intracellular monomeric O-acetylated sialic acid concentration. Fourth, the obligate animal commensal and facultative pathogen P. multocida expresses CMP-sialic acid synthetase encoded by the short form of neuA (pm0187). P. multocida also expresses a homologue of neuA* (pm1710), which maps as the first gene of a functional nan operon for catabolism of environmental sialic acids (37). The concentration of O-acetylated sialic acids in nonhuman mammalian serum is more than twice that of Neu5Ac (18), suggesting that the pm1710 gene product may function to remove O-acetyl esters after the cell transports acetylated sialic acids from the host. O acetylation is known to inhibit sialate aldolase (NanA), further suggesting that the NeuA* physiological substrates, independent of species, are O-acetylated sialic acids. Fifth, the active residues in blocks I to III and V of NeuA* are more similar to esterases such as Pm1710 and TesA than to PAF-AH (Fig. 9). Finally, other neuroinvasive strains like N. meningitidis and Haemophilus influenzae express the short form of neuA, indicating that NeuA* is not essential for cell invasion. Evidence for the in vivo function of NeuA* in vivo may be obtained by constructing an E. coli K1 mutant that lacks star (O-acetyl esterase) activity while retaining synthetase.
Human serum contains over 30 times less O-acetylated sialic acid than Neu5Ac (18), which may explain why an obligate human commensal like H. influenzae lacks a pm1710 homologue despite the functional expression of other nan catabolic genes (44). Note that H. influenzae neuA encodes the short form of the synthetase (Table 2), suggesting that it does not modify its surface with O-acetylated sialic acids. Because most E. coli strains do not synthesize or activate sialic acids, and therefore lack neuA, the NeuA* physiological substrates in E. coli K1 may be the monomeric O-acetylated sialic acids produced during sialic acid synthesis, which requires NeuC (epimerase), NeuB (synthase), and NeuD (Fig. 1), instead of environmentally derived sialic acids. The niche of the human and animal large intestine occupied by E. coli K1 and other E. coli is a complex and poorly understood microbial environment that includes a diverse set of organisms expressing sialidases, sialyl O-acetyl esterases, and other catabolic enzymes directed against mucins and mucin-derived monosaccharides (8-10), suggesting that the E. coli nan system may primarily scavenge Neu5Ac instead of O-acetylated forms. However, over half of all mucin sialic acid residues are O acetylated (8-10), suggesting that E. coli K1 NeuA* may confer a selective advantage over other E. coli lacking the esterase. For example, in a study of healthy pregnant females, E. coli K1 was the most prevalent aerobic species in 38% of the participants (31), indicating that it may express specialized colonization factors such as efficient deacetylation of environmental sialyl O-acetyl monomers. The temperature dependence of capsule synthesis and nicotinamide auxotrophy of many K1 strains also may indicate ongoing adaptation to an animal environment (48), in which case NeuA* could play an important role in colonization and persistence.
Contribution of the monomeric O acetylation pathway to capsule modification.Although NeuA*-sensitive O-acetylated sialic acids accumulate in a neuA mutant, most of the acetylated sialic acids in the capsule are derived from the NeuO-catalyzed pathway. However, 2 to 4% of residues in polysialic acid are O acetylated by the NeuO-independent pathway, indicating that all E. coli K1 stains, regardless of CUS-3 status, contain a small amount of modified residues. The apparent synthesis and reconversion of most O-acetylated monomeric sialic acids to Neu5Ac would seem to be a wasteful or nonessential process (Fig. 1). However, E. coli serotype O104 (Table 2) synthesizes O-acetylated monomeric sialic acid as a structural component of lipopolysaccharide (15). NeuA* offers a mechanism to regulate the degree of acetylation by controlling the concentration of O-acetylated sialic acids available for surface modification. In contrast, no O acetylation of sialic acid was reported for the E. coli O145 serotype (14), suggesting that there may be differences in the relative activity of NeuA* or activity of NeuD in some strains. In GBS the relative concentration of O-acetylated monomeric sialic acids is low unless the neuA orthologue is inactivated (24). Although Lewis et al. (24) ascribed this phenotype to a competition between the synthetase and O-acetyltransferase encoded by neuD (23), our results support NeuA* as the molecular explanation for the accumulation of O-acetyl forms in an E. coli K1 neuA mutant. Indeed, it is unclear how the levels of GBS polysaccharide O acetylation reported could occur in the presence of NeuA*, unless the activity of this enzyme in GBS is less than in E. coli K1. In vitro comparisons of NeuA* proteins from E. coli K1 and GBS against the model esterase substrate pNP-Ac indicate that the E. coli esterase is relatively more active (52).
Similarly, the groups C, Y, and W-135 meningococcus neuA homologues lack the genetic information to code for NeuA*, and the degree of O acetylation in these strains is 90%, 47%, and 66%, respectively (21), which is much higher than in E. coli K1 strains lacking neuO. Meningococcal strains also lack NeuD (Table 2), supporting the negative correlation between the degree of acetylation and NeuA*. In contrast to these NeuA*-negative meningococcal strains, the degree of capsule acetylation in E. coli K1 CUS-3+ strains may approach 100% (28) despite the simultaneous occurrence of sialyl O-acetyl esterase. Our current results show that capsule O acetylation is largely dependent on neuO. That NeuO acetylates polymeric instead of monomeric sialic acid (19) presumably accounts for the lack of an effect of NeuA* on the degree of polysialic acid O acetylation, since it is as yet unclear whether NeuA* recognizes O-acetylated polysialic acid. However, the degree of E. coli K1 polysialic acid acetylation is strain specific (13, 19, 28). Sialic acids from stains lacking neuO either due to mutation or naturally because they are not CUS-3 lysogens contain few O-acetyl esters. In contrast, CUS-3 lysogens expressing neuO in the "on" form vary over a wide range in the degree of acetylation (28), which may not be solely dependent on the stochastic proportion of cells in the "off" form (13). How or even whether the degree of acetylation is affected during host colonization relative to that observed in cells grown in vitro, as in the current study, provides fertile new ground for investigating the biological functions of variable capsule modification in the context of the host-microbe interaction.
Function of the NeuD-catalyzed pathway.Our results indicate that the NeuD-catalyzed addition of O-acetyl esters to Neu5Ac is a continuous process in E. coli K1 but that NeuA* limits accumulation of O-acetylated forms or their incorporation into capsule. However, while neuD + (pRS361) complemented a ΔneuD mutant (RS2887) for sialic acid synthesis, the plasmid did not result in detectable O-acetylated sialic acids when expressed in strain EV78 grown in sialic acid-supplemented medium (not shown). Thus, while NeuD is the most likely candidate for the E. coli K1 sialyl O-acetyltransferase, our results do not provide direct evidence for this conclusion despite the recent observation that K1 NeuD appears to acetylate Neu5Ac in GBS (23). Note that Lewis et al. (23) did not identify acetyl-coenzyme A as the presumed two-carbon donor nor demonstrate biochemically that either K1 or GBS NeuD functions as an acetylase in vitro. Both of these biochemical conditions have been demonstrated for NeuO (13). It may be that NeuD requires interaction with other kps or neu gene products in order to be an active O-acetyltransferase, and a direct interaction between NeuD and NeuB has been demonstrated in vivo (11). However, in unpublished experiments, plasmids expressing neuBAC or neuDBAC in EV78 did not produce detectable intracellular Neu5Ac, suggesting that stoichiometry or interactions with still other gene products not present in the K-12 background may be critical. Evidence that O-acetylated monomers are not necessarily protected from NeuA* upon activation by the synthetase domain was evident from the phenotype of EV136 shown in Fig. 7G. This mutant has a defect in neuS and accumulates high intracellular concentrations of CMP-Neu5Ac (29, 38) but no detectable O-acetylated forms, suggesting that K1 NeuA either does not activate acetylated monomers or, if so, then they, too, must be NeuA* substrates. However, because GBS NeuA clearly activates O-acetylated Neu5Ac, the low incorporation of NeuO-independent acetylated sialic acids in polysialic acid presumably reflects the efficiency of NeuA* and, perhaps, the efficiency of the polymerization process (39). It will be interesting to determine whether NeuA* is active against O-acetylated polysialic acid. Our current results suggest that the small but constant amount of NeuO-independent acetylation of polysialic acid may be a target for potential vaccination against all K1 strains.
Finally, the NeuD-catalyzed O acetylation pathway may be irrelevant to the biology of E. coli K1. This possibility follows from the bifunctional nature of neuD, where in addition to its acetylase activity, NeuD is required for sialic acid synthesis (12). Dissection of these two functions has been accomplished for GBS NeuD by site-directed mutagenesis, resulting in loss of the acetylase activity but retention of the sialic acid biosynthetic phenotype (23). Therefore, it may be that only the biosynthetic function of NeuD is important in E. coli K1. The situation where only one of two biochemical functions is important to polysialic acid biosynthesis might be analogous to KpsF, which is enzymatically active in KDO-precursor biosynthesis (27) but also includes a cystathionine-β-synthase or Bateman domain (5) that is now understood to bind adenosine ligands and function in a variety of cellular regulatory phenomena (33). E. coli kpsF mutants are defective in export of polysialic acid that accumulates intracellularly (7). Thus, the enzymatic functions of NeuD and KpsF may be of little or no relevance to polysialic acid synthesis or its in vivo functions. In either case our results indicate that the metabolism of microbial monomeric and polymeric sialic acids is far more complex than previously thought. The absence of at least some of these metabolic processes in mammals suggests new targets for potential drug or vaccine development (47-49).
This research was supported by NIH grant R01 AI042015 (E.R.V.). Y.-C. Lee was supported in part by the LG Yeonam Foundation, South Korea, during his sabbatical with the Laboratory of Sialobiology.
We thank Kerry Helms for expert photographic assistance. We are grateful to Mark and Theresa Kuhlenschmidt for technical training in the use of the Dionex HPLC components used for analysis of DMB-labeled α-keto compounds, and Richard Silver for stimulating discussions. We gratefully acknowledge the kind gifts of BSM or O-acetylated standards from T. Corfield, T. Warner, and R. Schauer and to U. Rutishauser for pEndo-N.
- Received 4 April 2006.
- Accepted 15 June 2006.
- Copyright © 2006 American Society for Microbiology


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In the fast-paced world of advertising, it's not uncommon for a company to use inspiration from an already existing piece. Usually this could include nods to layout, font, and color, maybe, but a pretty sizeable duplication of an artist's work isn't a normal thing, especially for Disney.
French Artist Hachim Bahous is claiming that Disney has, without the artist's consent, used his previous work for a Sony music release as the basis for their Solo: A Star Wars Story posters.
Bahous posted this side-by-side comparison on Facebook, and it's quickly gaining attention:
The post (originally in French but translated by the social media website) reads:
Left the official posters of the next Star Wars (Disney), right the compiles for which I made the creation in 2015 for Sony Music France / Legacy Recordings France following the brief of the project leader Romain 'Rpiz' Pizon.
I am flattered that the quality of my work is recognized, but it is still pure and simple forgery, I have not been asked for my permission, I wish to be credited and paid for this work I have done for Sony!
A thought for everyone who worked hard on this very beautiful project: Dj Reverend P (Patrice Larrar), Richard Lecocq, Aurélien Orel, David Hachour, Yazid Manou and Yazid Manou So Romain 'Rpiz' Pizon.
A simple search on Amazon results in the 2016 Legacy of Electronic Funk compilation, released by Sony Music Canada Inc. and labeled as an "import". The other albums listed in the above image are easily found too.
Obviously the art has been around since at least October of 2016, then, and more than likely was turned in by the artist to the company way before packing happened.
This isn't the first time this type of artwork has been used, though, it's worth noting. One of the original Ian Fleming James Bond novels Man with the Golden Gun had a 1965 hardcover jacket with a pretty similar style.
That's not to say someone before then hadn't used the general layout too, but in this case, it sure looks like Disney more than 'borrowed' from Bahous's art.
We're diving into this in more detail and tracking down the company responsible for creating the posters for Disney — we'll let you know when we get some updates on this situation.


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West End Condo - 106 1399 Barclay Street, Vancouver, BC - JUST SOLD!
West End - Condo - JUST SOLD!
100% owner occupied townhouse-style suite at The Westbriar. Faces Barclay Heritage Square (a city-owned park w/greenspace & beautifully restored heritage homes) Adults 19+. Rain screened heritage style bldg. Enter this garden level 1 BR home off a beautifully landscaped secured inner courtyard (handy for pet owners). Breezy east/west air flow. Features incl: laminate floors (great for pets), bathroom upgrades, wood-burning FP for chilly winter nights, pass thru from kitchen (easy to add a bartop), insuite WD & generous outdoor patio for year round BBQ's. There's a friendly community spirit here & pride of ownership is evident.1 storage,1 parking. Easy access to Park, Farmer's Market, Greenhorn café, & Robson shops. 2 cats or 1 dog allowed.
|1 Bedroom
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Check out other great Vancouver Condos for Sale.
Stephen Burke
Sutton Group - West Coast Realty


text: Thank you for selecting the Kittery Outlets as your next shopping destination. Groups of 15 or more will receive a complimentary VIP savings booklet upon arrival. Please complete this form to confirm your reservation. Upon submission, you will receive confirmation from the Kittery Outlets management office. If you need further assistance or more information please call us at 888-KITTERY.


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Where to stay in Bali: a guide for all types of travellers
Choosing where to stay in Bali is not the easiest thing to do. The information available on the internet often leaves us more confused about which is the best place or the best beach. That said, Bali is one of my favorite destinations of life. Do you know these places that we're already going crazy to go back? And I promised I would, to better explore all the wonders of the island. Relax on the beaches, take a yoga course in Ubud, get to know the volcanoes, dive …
The first thing you need to keep in mind is that in Bali, less is more. As in the rest of the country, the traffic there is chaotic and the absence of an efficient public transport system makes the locomotion between the beaches a headache. Small distances can take hours to be covered. Knowing this, it's best not to take a hectic travel pace. Even because this does not match the climate of the island. Choose few places and stay in them for days.
Which beach to choose in Bali?
The most famous and well-named beaches are the southernmost islands on the Bukit peninsula and nearby Kuta: Uluwatu, Padang Padang, Nusa Dua, Jimbaran, Sanur and Seminyak, which are also close to Denpasar airport, Bali's main airport. See now our suggestions on where to stay in Bali.
Ubud, the cultural capital of Bali
Ubud is the cultural capital of Bali. Surrounded by temples, mountains and rice paddies, the city also houses artists, art galleries, festivals, yoga schools and meditation centers. The tourist infrastructure is great. It is the perfect place for anyone who wants to take a break on a trip, relax, taste the typical gastronomy and learn about Balinese culture.
Even if you are going for the beaches, reserve at least three days for Ubud.
Kuta, Legian and Seminiak: the hottest beaches
Kuta is controversial. The capital of the island of Bali receives, every year, thousands of tourists looking for beaches, resorts, bustling nightlife and places to shop. But it's not uncommon the testimonials of people who say that staying there is a terrible choice. The criticisms are many: dirty beaches, environmental degradation caused by predatory practices of tourism and crowd. It passes away from the calm and mild climate for which the island is famous. Some say he hated Bali because he stayed there. On the other hand, it is there that concentrates the largest tourist structure of the island. It is certainly easier to get around and hire services and facilities staying there. It's your choice.
One way to stay close to Kuta facilities, but at a safe distance from the hustle and bustle, is staying at the nearby beaches, Legian and Seminyak. Legian is the closest to Kuta and still suffers from the crowds of tourists, but there the thing is more bearable. The beaches are cleaner and attract many surfers. Also not to be missed in the structure: restaurants by the beach do not lack there.
If Kuta has bad fame, Seminyak is the coolest region. It is full of boutique shops and trendy restaurants, among them the Ku De Ta, a world-famous meeting point on the beachfront. Speaking of it, the beach is quieter, but it does not come to impress with its beauty.
It is a region full of four or five star resorts and usually has higher prices than other parts of the island, but as in Bali nothing is too expensive, it is possible to find good accommodations for very little. Stay here if you want to see beautiful people, comfort, music and sophistication. Avoid if you are looking for a closer experience with the local culture.
Jimbaran: resorts in Bali
Next to Denpasar airport, Jimbaran lies between Kuta and the Bukit peninsula. It was formerly a small fishing village, but today it was invaded by five star resorts, both which is known as the Beverly Hills of Bali. It also has some middle-term and economic hotels, although it is not the stronghold of the region. The beaches are good for bathing and white sand. Jimbaran is perhaps the best place in Bali to eat the traditional seafood barbecue. The place is good for shopping, but the nightlife is almost dead.
Uluwatu and Padang Padang: awesome beaches
Uluwatu is the surfers paradise, these two beaches on the Bukit peninsula are beautiful, clean, crystal clear and are 5km apart. They do not usually attract crowds, but they can get fuller in high season. The region is less inhabited and therefore leaves little to be desired in the infrastructure. You will not get as many dining, nightlife and transportation options as nearby Kuta, but the scenery and nature make up for being more secluded.
Padang Padang is chosen by many surfers and backpackers, as prices are inviting. This was also our choice during our stay in Bali. We stayed in Uluwatu, a few meters from the famous temple, and we loved it. On a next trip, however, I would not repeat the lodging. You would choose a hotel closer to Padang Padang. I found the beach there better and had a minimal structure (type ATM and these basic things).
Find lodging in Uluwatu and Padang Padang
Nusa Dua: for those who enjoy luxury
Luxury resorts is what is not missing there. The place even has a private beach, for the exclusive use of the guests of these hotels. The sea is clean, blue and calm, almost without waves, which leaves the surfers away, but attracts a lot of people who just want a swim. It is the perfect choice for those who want rest and stewardship. It is located on the easternmost side of the Bukit peninsula.
Sanur: a more familiar beach
Sanur is a quiet and relaxed getaway but with an excellent structure of hotels and restaurants. It's more expensive than Kuta, but cheaper than Seminyak. In general, it is frequented by more mature people, families or those who do not have the patience for the spirit of hype that can stand out in other parts of the island.
Where to stay in Bali: other options
Did not like any of our suggestions? Do you have another place in mind? Do not worry! Booking has almost 3,000 properties listed in Bali. You can refine the search according to your preferences – hotels that look for the guest at the airport, for families, that accept pets and everything else. Check the map:


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How to Trade In a Car
Don't know how to trade in a car? Don't panic. These simple tips can help you get the best value on your vehicle.
Trading In a Car?
Trading in a car can be confusing, especially if you don't know how much your car is worth. You also might feel intimidated by the process, so take some time to understand what's involved.
Trade-In Value
When you trade in your vehicle, its value is deducted from the price of the new car you're buying. Find out how much your car is worth before starting negotiations, and do online research to see how much comparable cars are selling for to find a target trade-in price range.
Tips For Trading In a Car
Get quotes ahead of time from several dealers, and get trade-in appraisals from at least two different ones for better bargaining power. Keep your trade-in and purchase negotiations separate to get the best possible deals.
A Stress-Free Experience
Trading in your old car and buying a new one should be a fun, positive experience. At Biggers Mazda in Elgin, we like to educate our clients with vehicle buying tips and advice. Contact us today to get started on your trade-in. We're located near Streamwood and Bartlett.