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Winnipeg Free Press - PRINT EDITION
They rocked and roared
We Day crowd moves to the beat of music, inspiration to create philanthropic pandemonium
By Nick Martin
Can 18,000 children be inspired to change the world by an 81-year-old Russian great-grandfather speaking through a translator, a man whose greatness came before any of them was born?
As they say at We Day, "Yes, we can."
Work for peace -- only in a peaceful world can great change be achieved, former Soviet president Mikhail Gorbachev implored 18,000 Manitoba children Tuesday morning at the MTS Centre.
And they responded, with standing ovation after standing ovation.
"We need to move to a world without nuclear weapons," said the Nobel Peace Prize laureate at the second We Day Manitoba. "You should also work for freedom, so people can decide for themselves."
The kids from 396 schools earned a trip to We Day because they've committed to changing the world through supporting global projects for social justice and projects in their own backyards.
"I like you guys -- I love you!" he said to thunderous applause.
And at times, you could feel the entire arena shuddering as dread ran rampant.
"During the Cold War, about 100,000 nuclear weapons were produced. Each one of those warheads had the power to destroy a city like Winnipeg," said Gorbachev.
Introducing Gorbachev, Winnipeg Free Press editor Paul Samyn said, "President Gorbachev's widespread reforms changed the course of humanity."
Full-time pep rally, frenetic and beyond loud, dazzling light show; We Day is four hours of motivational speakers both renowned and ordinary, peppered with plenty of top music performers such as Lights, Shawn Desman and Victoria Duffield.
Singer Lights talked about growing up in a missionary family in the Philippines, where water is plentiful, but pitifully little of it is fit to drink. We Day's clean water campaign's brilliance is how easy it is to achieve, she said.
University of Manitoba medical student Tito Daodu returns to her native Nigeria to provide pediatric oxygen services to children -- who face the terrifying reality that 200,000 kids under the age of five die of pneumonia every year.
Blind bullying victim Molly Burke had the entire arena near tears -- especially when her guide dog Gypsy joined her onstage.
The Kielburger brothers, Marc and Craig, took the kids back in time to worlds where contemporary life here was not taken for granted -- Mahatma Gandhi standing up to an empire, little children integrating a school in Little Rock, Ark., Nellie McClung leading the movement to win women the vote.
"Everyone, not just the women, owes Nellie McClung a huge show of gratitude," said Marc Kielburger.
And with social media, students have tools of which Gandhi and McClung never dreamed, he said: "Today, you have the means to change the world faster than at any point in human history."
Manitoba Children and Youth Opportunities Minister Kevin Chief gave a huge shoutout to young people in Shamattawa, who are making the only safe place in the community available to kids.
"These youth volunteer four nights a week to make sure the gym stays open," he said.
Chief Justice Murray Sinclair said he wants a world in which his granddaughter can grow up with respect.
Too many schools still teach that aboriginal children are inferior, said Sinclair, head of the Truth and Reconciliation Commission on residential schools.
"They were told they were savages and heathens," Sinclair said.
Craig Kielburger said there was considerable thought given to a campaign urging Canadian students to help build schools on First Nations, but, "The costs were prohibitive."
Instead, in discussions with people such as national chief and Free the Children patron Shawn Atleo, they opted to break down barriers and promote a national aboriginal awareness month in schools.
"It starts with an understanding, breaking down prejudices" and the lack of knowledge, Kielburger said.
Through former prime minister Paul Martin's work with aboriginal education, Free the Children is making aboriginal awareness curricula available to all schools.
Meanwhile, Free the Children provides the resources for Sacred Circle, in which young aboriginal leaders work with aboriginal communities to tackle issues they've identified together.
Premier Greg Selinger saluted We Day sponsors Hartley Richardson, Mark Chipman and Bob Silver. "I call them the three wise men, because it was their idea to bring We Day to Manitoba," said the premier.
Selinger gave an impassioned plea for all 18,000 kids to reach out to kids being bullied: the child sitting alone, the kid who dresses a little differently, the new student who can't speak English.
"Can we reach out to them? Yes, we can," Selinger said.
"Yes, we can," echoed 18,000 voices, rivalling the noise with which any Jets crowd has ever rocked the arena.
But how silent can 18,000 kids be? Beyond deep space silent, when Spencer West described climbing Mount Kilimanjaro, though he lost his legs when he was five.
A few minutes from the top, his friends were collapsing on the slopes of the African summit this summer, West told We Day. "Sometimes, it's the people you don't think need help, need it the most," he said.
Climbing hand over hand, West urged his friends to make it to the top.
And they did.
Republished from the Winnipeg Free Press print edition October 31, 2012 A1
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- Indiana boxes itself into legal corner
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Beloit Regional Hospice Board elects new members
The Board of Directors of Beloit Regional Hospice has elected Doris Mulder and Charldene Schneier to serve the organization as board members. Mulder is a vice president within Beloit Health System, and Schneier is an associate general counsel at Regal Beloit Corporation.
"We welcome Doris and Charldene," said Beloit Regional Hospice Executive Director Mary Anne Miller. "We value their perspectives, which complement the current qualities of our board members."
To sum up why she made the decision to become a board member, Mulder pointed out, "It is important that Beloit Health System and Beloit Regional Hospice continue to partner to improve end-of-life care in this community."
Schneier noted that for her, "It's a great opportunity to represent Regal Beloit. In addition, I have an elderly father with health concerns; being involved here will be interesting and helpful to me."
From the April 18-24, 2012, issue


text:
The managed services program is the facility that allows the company to manage the information technology services for other companies over internet. It makes the managed IT services provider the company that gives permanent outsourcing for the IT functions. These types of the services that are the managed work on inveterate model of the revenue; repairs and monitors things mainly on online basis opposed to physical labor at office of a client. Some features incorporated in managed services allow providers of such services to setup the remote desktop, security and network monitoring services. It's equipped with the technical help, the data backup, which is remote besides patch management solutions. Model of the business behind such services was made when conventional computer VAR model continued to give minimal profit margins. Also, there were some other factors than the lower profits, which led to development of MSS. They included the minimal profit margins on hardware and software reselling, high competition from the direct computer sellers besides unanticipated revenues from the knee-jerk way of break fixing the technical support.
Managed service gives a lot of facilities to the clients after software installation. They include following services:
• Simple spotting & fixing of the problems before experience of the downtime.
• Continuous network monitoring procedure that is done 24hours per day and 7 days week.
• Do frequent checks of system at the intervals of five to fifteen minutes.
• Requirement to invest at novel hardware can be done away with.
• The commercial cost remote management & monitoring solution
• The production of the regular reports for monitoring status and health of network.
Users of such type of the software are MSPs that put up for the sale managed services besides offering many different cost structures. Most common structure is monthly fee however there are some costing models like material & time model besides price per server, desktop or network device models. MSPs assure to monitor IT infrastructure of clients by using software besides resolving any kind of issues that might come up within infrastructure. It leaves client with complete peace of mind besides anticipated inveterate revenues of provider of such managed services. Software for the managed services give small and medium sized company an option for fulfillment of the IT needs without even engaging services of the on-site employees for more information you can visit http://www.calitso.com/.
Good number of managed IT services providers outsources such type of the technology from companies that are been referred as the managed Services providers. Since platforms are given by the sellers, providers of the managed services are the companies who use given platforms for offering solutions in information technology to the consumers in sector for small and medium sized companies. Larger companies use MSP to outsource IT requirements since these sellers give enterprises with the platforms for use with the organizational IT employees. It means that enterprise models will give more of value to their staff in IT department since they need to stay on-the-site at http://www.calitso.com/.


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A little bit about me
I've been releasing music under the alias of Theves for a number of years. To date, I've composed 3.5 feature films and dozens of short films. I'm currently releasing my first LP "Different Places", one song at a time, all of which are available for free download through this link. Independent creators may use music from my soundtracks non-commercially under the Creative Commons BY-NC-SA 4.0 licence. Get in touch if you want to use a track and need the audio stems.
But wait, there's more!
In a fit of passion, curiosity and anger, I took a hiatus away from music to make a documentary about the Occupy Wall Street movement in 2011 and onward. *I can already detect your silent judgment*, which is exactly why you should watch this film if you're at all a political person. The film was well received by audiences, critics and festivals alike and serves as a valuable hindsight into protest movements of the modern age.
As I was developing my skills as a producer of music and film over the years, I kept a roof over my head as a freelance web developer. I was fortunate to have computers around from a very young age and naturally gravitated towards coding websites as soon as Geocities hit the scene. I keep programming projects that I'm particularly proud of in the coding section if you're into that sort of thing.
Music and computing are so closely related in many aspects but then poles apart in others. Nowadays, I'm developing technology that bridges the gap between the two.


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Dec 4, 2012
Brazil's Fibria is aiming to get its leverage below 3.0x by the end of next year, in order to set up a possible upgrade to investment-grade status, its CFO says, with additional liability management operations in the cards for 2013. "We continue to pay down debt. We are not going to issue additional debt. You will see more debt repayment, and we are monitoring the markets for liability management," Guilherme Cavalanti tells LatinFinance....
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Apr 16, 2015 | Hotel Unique, Sao Paulo, Brazil
2015 is an important year for Brazil. In the rear-view: the 2014 World Cup and Presidential...
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Will a strong dollar deter investors from LatAm bonds?
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The strong demand that we saw in the yen market was historic, which allowed us to extend the maturity in a way that we wouldn't have thought possible in the past.
Alejandro Díaz de León, Mexico's head of public credit
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text:
If I Have Ars Will I Have Itchy Rash
Here's a taste of what TheBody.com has to offer on this topic:
Ask the Experts
Itchy rash Vs Non Irch Rash in ARSOne question Dr. that I hope you can answer: Can you tell us why the Rash associated with ARA doesn't Itch or have pain???. I had a low risk exposure (Oral Sex - received and performed) and I have not been tested for HIV - If you can provide a...
Response from Keith Henry, M.D.
University of Minnesota, School of MedicineThe rash seen in primary HIV infection is presumably immunologic in origin. Different persons have different immune responses to viral infections. In some patients, the rash may itch (? IgE or mast cell triggered) and in others there may be no... Read more »
PEP itchy rash ? combivir/nelfinavirHello. I started combivir/ nelfinavir(1.25gm bd) 8 days ago for a needlestick from a HIV+ve... Read more »
Itchy rashes?Hi Mr Kull, I had an unprotected oral sex encounter with a guy months ago. Weeks later some... Read more »
DNCB (Dinitrochlorobenzene)...a week to a 2-inch square area. Then the area was bandaged and kept dry for 10 hours. After the first skin response (a red, itchy rash), the strength of the DNCB solution was lowered. Instructions for using DNCB originally provided by DNCB Now!... Read more »
An Overview of Selzentry (Maraviroc, Celsentri)mah-RAV-er-rock Brand Name: Selzentry Other Name(s): Celsentri, MVC, UK-427,857 Drug Class: Entry and Fusion Inhibitors Maraviroc, also known as Selzentry, is a type of medicine called an entry inhibitor. Entry inhibitors work... Read more »
An Overview of Fuzeon (Enfuvirtide, T-20)...in the mouth, constipation, cough, depression, diarrhea, eye infection, flu-like illness, fatigue, herpes simplex infection, itchy rash, loss of appetite, muscle pain and weakness, pain and tingling in the hands and feet, nausea, nervousness,... Read more »
RescriptorCommon Name: delavirdine Brand Name: Rescriptor Class: non-nucleoside analogs (also called non-nucleoside reverse transcriptase inhibitors, NNRTIs or non-nukes) Standard dose: Two 200 mg tablets or four 100 mg tablets three... Read more »
Connect With Others
rash symptom
Posted by Anonymous...Does anybody know if the only symptom of ARS could be a rash? I have an itchy rash on my stomach, back, legs, chest and arms but it is worse on my stomach. I have no other symptoms and have had the rash for nearly Read more »
Rash after 1 week, please someone help
Posted by preocupado1...possible HIV exposure, 5 days later I practiced oral sex on a girl and the masturbated her. 1 week after that she develped an itchy rash on neck a belly. Can someone please tell me the chances that I infected her (if im an infected) and the... Read more »
PLEASE help! Rash, no fever...worried SICK
Posted by molls1992, 4 Replies...just had an HIV test in April after being with him....NEGATIVE. But now after this last instance, I've developed a HORRIBLY itchy rash. Like...itch till I give myself bruises type of rash!!! Its mostly right around where the band of my bra sits,... Read more »
Posted by Anonymous, 3 RepliesIf there is ARS i have a question. When would the rashes apear. During Serconversion or rashes can appear months after?? Read more »
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text:
Preheat oven to 400°F.
Cut sausage lengthwise into quarters; cut each quarter into 5 pieces.
Remove biscuits from can; separate.
Using fingers, flatten each biscuit to about 4 inches.
Place small amount of honey, barbecue sauce or ketchup, if desired, in center of each biscuit; top each with 1 piece of sausage.
Bring up edges of biscuit and pinch together to seal over top of sausage.
Place on baking sheet.
Bake about 10 minutes or until lightly browned.


text:
I am looking to get myself an asks for christmas but the most common ones i see are either yugo or chinese wich is best? What other sks's are out there? How can you tell what they are? I only know a yugo sks is a yugo beacuse of the greanade launcher so wich is the best sks? What is your favorite?
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I have some sort of pre ban Chinese sks. All orginal. I love it. Fun to shoot and accurate.
Oh dern....
Revelation 19:11
And I saw heaven opened, and behold a white horse; and he that sat upon him was called Faithful and True, and in righteousness he doth judge and make war.
I'd say the Russian SKS's are the best but both the Yugo and Chinese are very good and either will be a great rifle to have. It mostly comes down to personal preference which to go for.
I had a Yugo that I made a few mods to. It was a great gun. It was battle accurate and functioned flawlessly (after my mods). I could dump 30 rounds down range pretty quick.
I sold it for a Nice profit during the panick.
I don't need No stinking signature.
I really like the looks of the Yugo SKS and have considered getting one. They are heavier than a regular SKS but also supposed to be more accurate.
I personally have owned a Norinco (Chinese) and thought it was a great shooter. However, the stock was very short as it was made for a smaller person. I ended up replacing it, but never had a problem with the weapon itself.
"For every problem there is always a solution that is simple, obvious, and wrong." - Mark Twain
Chrome lined barrel, very accurate, never fails, shoots any type ammo, and most of all FUN!.
My very fist time out at a range I shot a Norinco SKS. I hit a 10" target at 100 yards. I was sitting and the rifle was on a rest as it was kind of heavy for me. Those rifles are great shooters, indeed. It was this trip to the range that made me fall in love with shooting. I mostly shot an Uzi and the SKS. I'm short so the shorter stock was a pretty good fit for me.
I like the Norinco I have. There was a Yugo model that didn't have the goofy grenade launcher, M59 I think.
Dead Bears, the only good kind.


text:
for felony possession of marijuana with intent to distribute.
Brandon Morris dismissed at Georgia
Updated: July 14, 2014, 5:34 PM ET
ATLANTA -- Georgia forward Brandon Morris has been kicked off the basketball team following his arrest on a marijuana charge.
The Athens-Clarke County Jail said Monday he was arrested early Sunday and later posted bond. He was charged with felony possession of marijuana with intent to distribute.
Morris was suspended for three games last season for a violation of team rules. Coach Mark Fox said on the team's website that Morris has a "very clear understanding of our expectation. Unfortunately, I've had to dismiss him from our team."
Morris, a junior, was set to return as one of four starters for a team that won 20 games last season before losing in the second round of the NIT to Louisiana Tech. He averaged 8.6 points and 3.4 rebounds.
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UGA's Brandon Morris Arrested & Dismissed From Team
Started by GatorUnvrsty, Jul 14 2014 11:28 PM
Posted 14 July 2014 - 11:28 PM
Posted 15 July 2014 - 10:02 AM
Any Dawg basketball experts on the site? Article says he'd have been a returning starter; but that doesn't really say how vital he was to Fox's team and rebuilding efforts.
Posted 15 July 2014 - 10:40 AM
1. Who cares about basketball.
2. Who cares about a little pot?
2. Who cares about a little pot?
Posted 19 July 2014 - 07:50 PM
We'd suck anyways with or without him
Trenton Thompson save us
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The Man with the Compound EyesKnopf Doubleday Publishing Group 2014; US$ 15.95
When a tsunami sends a massive island made entirely of trash crashing into the Taiwanese coast, two very different people?an outcast from a mythical island and a woman on the verge of suicide?are united in ways they never could have imagined. Here is the English-language debut of a new and exciting award-winning voice from Taiwan, who has written... more...
Heavenly Stems and Earthly Branches - TianGan DiZhiJessica Kingsley Publishers 2014; US$ 65.00
A detailed explanation of the building blocks of Chinese metaphysics, the 22 Heavenly Stems and Earthly Branches upon which Chinese medicine, astrology and FengShui are based. Poetic descriptions dating from the Song dynasty give the reader a deep understanding of the meaning of each and of their interrelationships. more...
Asian American Studies NowRutgers University Press 2010; US$ 38.95
� Asian American Studies Now �represents the changes occurring in Asian American communities and the world that require a reconsideration of how the interdisciplinary field of Asian American studies is defined and taught. The editors have selected essays for the significance of their contribution and their clarity, brevity, and accessibility to readers... more...
30 Great Myths about the RomanticsWiley 2015; US$ 74.95
"Wu is not a scholar who trades in faddish or modish opinion, and as its title implies, this is by its very nature an exercise in controversy and debate. The book represents a triumph of individual scholarship over what is claimed as often flawed, albeit consensual, critical opinion. Wu's fluid, readable prose is accessible to all, and... more...
Cloud Data Centers and Cost ModelingElsevier Science 2015; US$ 99.95
Cloud Data Centers and Cost Modeling establishes a framework for strategic decision-makers to facilitate the development of cloud data centers. Just as building a house requires a clear understanding of the blueprints, architecture, and costs of the project; building a cloud-based data center requires similar knowledge. The authors take a theoretical... more...
China and Christianity: Burdened Past, Hopeful FutureTaylor and Francis 2015; US$ 52.95
This collection offers fresh perspectives on Sino-Western cultural relations, with particular regard to the experience of Christianity in China. The contributors include authorities from China (including Hong Kong and Taiwan), Europe (including Russia and Eastern Europe), and North America. more...
Zinc CatalysisWiley 2015; US$ 120.95
Filling the gap in the market for comprehensive coverage of this hot topic, this timely book covers a wide range of organic transformations, e. g. reductions of unsaturated compounds, oxidation reactions, Friedel-Crafts reactions, hydroamination reactions, depolymerizations, transformations of carbon dioxide, oxidative coupling reactions, as well as... more...
Our Global FamiliesBaker Publishing Group 2015; US$ 22.99
Utilizing the latest research data, this book shows how we can better connect with our global Christian family to bring about positive change in the world. more...
The Double ScreenReaktion Books 1996; US$ 35.00
In the first exploration of Chinese paintings as both material products and pictorial representations, The Double Screen shows how the collaboration and tension between material form and image gives life to a painting. A Chinese painting is often reduced to the image it bears; its material form is dismissed; its intimate connection with social... more...
Handbook of Manufacturing and Supply Systems DesignTaylor and Francis 2001; US$ 129.95
Manufacturing Systems Management (MSM) is a functional domain that involves all of the activities for regulating and optimizing a manufacturing system as it progresses through its life cycle. These include the tasks of strategic analysis, design, implementation, operations and monitoring. Handbook of Manufacturing and Supply Systems Design: From Strategy... more...


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Swizz Beatz - Money In The Bank (Rmx) ft. Young Jeezy, Eve & Elephant Man
Artist: Swizz Beatz
Featuring: Jeezy, Eve, Elephant Man
Producer: Young World Music
Album: One Man Band Man
Avg Rating: 4.6 ( 11 votes )
Swizz Beatz isn't the only male whose pet peeve is women with no Money In The Bank; for the remix, three friends offer their opinions. Young Jeezy stays on point, rapping about how the money-hungry women are after him, Eve turns the tables and raps about her own bankroll and the Energy God, Elephant Man (who is arguably the highlight of this track) focuses on the money and doesn't say too much about the females. Hopefully, the remix will help Swizzy improve the disappointing sales of One Man Band Man.
Swizz BeatzJeezyEveElephant Man Club
Written by Burmy on 09/5/07
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Monster Music Group
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Country Brook Farms:
"We have always had a website stock-piled with tons of information for our customers, yet with little to do about us as a business. After meeting with GCN and hearing that they were a small business (like us) focusing solely on garden centers, our decision to utilize their skills was an easy one. Our old website was very basic and more of a resource center. With the help of the GCN team, our site has cleaned up and become much more attractive. We still have a great source of information on our new site for our customers, but it's more much organized and appealing. Now our website drives people to come in and smell the flowers that they can see form our site. Every Monday we get a call from our team asking for updates and opinions. The customer service is unbeatable. Technical service can actually help you without being on hold for hours. They are there for you, when you need them and that is one characteristic other web hosts can't offer."
Springhouse Gardens:
"This helps to keep our customers engaged by making it relevant to them and their needs throughout the changing year. All in all, we would highly recommend Garden Center News to you and your company, should you need a fresh and new perspective, that is ever changing and growing as your company does."
K&W Greenery (Phyliss – Owner) :
August 21, 2012 – Now, more than ever, small business owners are challenged to do more & more with fewer resources. That can mean attempting to overfill your already full plate with more tasks that you most likely don't have the skills to do efficiently or effectively.
That was certainly true with our website before Garden Center News.
We were stuck with a very minimal site, or increased production time that frankly, we just couldn't manage. Working with Garden Center News, has changed a constant headache to a "dream it, they will do it" scenario.
You know your business, your hot items, your rapidly changing customer's needs….Garden Center News knows how to bring it!


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Proactive Parenting with Sharon Silver on The Mompreneur Model Show
in Women
Take a healthy dose of mom, mix it with a parent educator, and sprinkle with a seekers spirit and you have Sharon Silver. Sharon has lived what every parent is living; a real life parenting real children. She knows the challenges that daily life brings families and shares tips, methods, and concepts that help parents' correct behavior in a calm, yet firm, way to produce listening, accountability, respect, responsibility and resolutions.
Join me on The Mompreneur Model Show as I share Sharon Silver, founder and instructor of Proactive Parenting ~ helping parents raise children, not just correcting behavior! Imagine transforming reactive parenting into confident and calm, yet firm responsive ways to teach children about behavior. Sharon and Proactive Parenting delivers ways to correct misbehavior that inspire a child to think about his or her behavior, which creates respectful and responsible children who are ready for the world outside your front door!
Our show is being sponsored by Order out of Chaos and Phisco Marketing. Meet them and all of our sponsors in our Mompreneur Marketplace.
Be sure to connect with us on Facebook, Twitter and Google+ and email firstname.lastname@example.com with questions and comments.
Word of Mom Radio - sharing the wisdom of women.
"Empire" Season Premier: Genocidal Media Assassination of the Black Mind
in Television
"Empire" has aired with it's cast of "homosexuals", Mobutus (race-traitors), mulattoes, whores, feminists, degenerates, Sambos, integrationists, europeans, and smallhats. The "homosexual" Lee Daniels (producer of Monster's Ball, Precious, and the Butler) is the producer. His self-hatred leaps off of the screen when watching this travesty to Black culture and sanity.
Daniels and his smallhat (white so-called jews) sponsors invoke just about every negative hat-type (formerly known as stereotypes) we can think of. The checklist of offenses to Black people include, but are not limited to:
white-sex promotion (homosexuality and/or pedophilia)
Miscegenation (Interracial Dating/Sex/Marriage)
Black Female Decadence and Whorish Misbehavior
Oversized Black Mammy
Black-on-Black Jealousy, Hatred, and Murder
The Black Man and Woman Cannot Get Along
Black Gansterism and Thuggism
Perpetual Adolescence Demonstrated by Adult Black Males
Sambo and Zip-Coon Characters Present and Accounted For
Mobutu (Race Traitor Misbehavior)
Black People Going Crazy Over Chicken
Sexual Decadence and Salaciousness
Hat-hop (hip-hop) Buffoonery
Anti-Black Male Sentiment from Black Women
Promotion of Mulattoism and white Standards of Beauty
etc . . . . .
In this program, we will explore the current agenda to wipe out the last remaining vestiges of Black family through the promotion of sophisticated media death. "Empire" is one of the most disturbing media production in the last 5 years.
Straight Black Pride
Successful Writers – Picture Books to Kids' Misbehavior
in Books
Dianne Ochiltree is a nationally recognized author of books for the very young. Authors Beat interviewed her recently at the 2013 Florida Writers Association conference. This highly successful and motivated author offers a fascinating insight into a very different kind of writing and the hard work involved with her creations. We then interviewed Dr. Alan M. Davick, asking him whether kids are really wilder now than ever before or is it something we are doing wrong. What is "mis-kidding" and how can we help children choose better behaviors. Every parent, teacher, grandparent and anyone having something to do with children will find this chat with Alan M. Davick, M.D., author of Managing Misbehavior in Kids, a very practical and useful experience. The question is: Are we really hurting our kids by "Permissive Idiocy?"
Abraham/Lot Connection
in Prayer
The famine ended, and Abram and his household, among them his nephew Lot, returned to their old place in Canaan, between Beth-El and Ay. Abram was now very rich. He had flocks, silver, and gold in abundance. Lot also had a great number of sheep and cattle. But whereas Abram's shepherds abided strictly by the rules given to them by their pious master concerning trespassing upon the property of others, Lot's shepherds were rough men who did not respect this spirit of justice. Thus there were constant arguments and strife between the herdsmen of Abram and Lot. Soon complaints reached Abram about the misbehavior of Lot's herdsmen and the strife between the shepherds. Abram therefore called Lot and said to him: "Let there be no strife, I pray thee, between me and thee, and between my herdsmen and thy herdsmen; for we are brethren. Is not the whole land before thee? Separate thyself, I pray thee, from me; if thou wilt take the left hand, then I will go to the right, or if thou take the right hand, then I will go to the left."
War on the Horizon with Irritated Genie
War on the Horizon (WOH) is an organization designed to prepare Afrikan people worldwide for survival in the 21st Century. As a Race of people, we are under the international threat of an active program of genocide. If we do not prepare to defend our interests and those of our future generations, we will face the same fate that the Native inhabitants (Red man and woman) of the Western Hemisphere faced in the 19th Century – genocide.
white-sex is a term coined by the PanAfrikan community to describe the psychology of european thought and action. It provides deep insight into european thinking and a primary social reference point from which to analyze european culture. More than anything, it provides the most useful paradigm ever created to understand european psychopathology throughout history to include white aggression in the world today. Traditionally, Black people have discussed white sexual misbehavior separately and distinctly from white aggression and/or racism. For the first time in modern psychology, and perhaps the history of the world, a paradigm exists that can be used to comprehend white social misbehavior as a collective, organized mentality that governs the way whites behave in:
What Is Your Misbehaving Child Trying To Tell You?
in Parents
Join Sara Bingham, WeeHands Founder and author of The Baby Signing Book, each week as she shares parenting tips, language development strategies, and the joys of connecting with your baby, toddler and/or preschool child.
This week, Sara will chat with Sharon Silver, the founder of www.proactiveparenting.net, an author, speaker, blogger, coach, radio and TV segment host with a passion for bringing awareness to the communication between parent and child so understanding, teaching and learning can be achieved as a parent corrects behavior.
Sharon believes that all misbehavior is a form of communication. The key is to discover what's motivating your child's behavior so you can help them truly learn. Parents need to communicate, and ask questions versus assume, in order to decode behavioral situations.
Learn more about how WeeHands and signing with your baby at www.weehands.com or share with us on Twitter here: https://twitter.com/weehands.
Clete Bulach Discusses About Creating a Culture for A High Performing School
in School
Dr. Cletus R. Bulach is a retired Ohio school superintendent and associate professor emeritus at the University of West Georgia. He is the author of numerous articles in educational journals and is co-author of the book Creating a Culture for A High Performing School: A Comprehensive Approach to School Reform, Dropout Prevention, and Bullying Behavior. His website is www.westga.edu/~cbulach. The reform and school culture described in the book create a learning environment where students' five basic needs are met. This leads to improved test scores and a 75% reduction in student discipline and misbehavior problems.
Your Kind Voice
in Inspiration
My guest Sarah Adams-Nowlin is the author of the hit essay Be Cool To The Pizza Dude. Sarah grew up in the Midwest and graduated with my BA and MA out of Wisconsin. She currently lives in Washington State, working at a community college. Sarah (used to work for a phone company while living in Minneapolis and finishing up my MA. But lost her job, along with a lot of others, due to corporate misbehavior. That's when she moved to Washington.) Sarah is married and the busy mom to a toddler who is currently in her princess phase. She say's I'd normally say, I enjoy hiking in the Cascades and Olympics, but have been a little busy with my daughter.
Hosted Dominick Domasky
Monday Madness with your host Queen Ebony and Queen Nee'Nee
in Radio
A dysfunctional family is a family in which conflict, misbehavior, and often child neglect or abuse on the part of individual parents occur continually and regularly, leading other members to accommodate such actions. Children sometimes grow up in such families with the understanding that such an arrangement is normal. Dysfunctional families are primarily a result of co-dependent adults,[1] and may also be affected by addictions, such as substance abuse (alcohol, drugs, etc.), or sometimes an untreated mental illness.[1] Dysfunctional parents may emulate or over-correct from their own dysfunctional parents. In some cases, a "child-like" parent will allow the dominant parent to abuse their children.
To Spank Or Not To Spank
in Christianity
You get a call from your school and your son/daughter was caught misbehaving. How do you deal with that situation? What's the difference between abuse and discipline? What does it mean to "Spare the rod...spoil the child" How can you discipline your child the way God wants you to? Before you grab the belt make sure you listen to this show!
2014 New Beginnings Cleanse #7 - Break a Rule – Be Popular – Guest Rick Lewis
in Self Help
2014 New Beginnings Cleanse #7 - Break a Rule – Be Popular – with Author - Rick Lewis
Welcome to the 2014 Revolution of Engagement Series with Skip Lackey!
Show #7 – Break-a-Rule - Be Popular - Over 7-straight days we will be exploring a way to start off your year by cleansing yourself of old unrecognized rules that cause us to have lead a mediocre life.
Rick Lewis - Founder and Author of the "7 Rules You Were Born to Break" and BreakaRule.com with be sharing his highly regarded platform and method for ANYONE to recognize the impact that following certain societal rules, because we are programmed to do so... and have NO idea of how these unwritten rules are at play in our lives.
Rick travels around North America sharing his message and is a highly sought after keynote speaker for conferences and meetings. What he has to share will shatter old paradigms that keep you bound and help you let go of old unwanted behaviors and habits that hold you back to creating the life you long to lead.
If you are interested in finding out more about Rick and "Break A Rule" or the Engagement Supplements to practice rule breaking with "actions of intelligent misbehavior" - go to: www.BreakaRule.com to find out if you are a rule breaker and how you can practice breaking rules that bind you and be free!
Darren Kavinoky Criminal Behavior Expert
in Legal
The Original air date of this show was on May 25th. 2013. The topic of the show is "My guest Darren Kavinoky's career as an attorney, inspirational speaker, television personality/ host, legal analyst. The focus will be on current trials that are happening including the Arias Trial Verdict and on Darren's history and how he has gotten to the successful point of where he is today". Darren Kavinoky is a criminal behavior expert, trial lawyer, TV analyst, author, and keynote speaker, we can truly say that he is an extremely accomplished man amongst all trades. Darren is known as an award-winning criminal defense attorney. Darren has authored two legal textbooks for major legal publishers in the field of criminal law. Darren is the creator and host of the television show Deadly Sins, which airs on Investigation Discovery. As the show's criminal behavior expert, Darren explains the root causes of the most extreme forms of human misbehavior. He also guest-hosts live courtroom coverage for In Session on truTV (formerly Court TV). Darren's legal expertise and off-center wit make him an especially engaging contributor on many other TV shows as well. He has worked as both a field correspondent and as in-studio talent for Entertainment Tonight and The Insider.
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Calum Macneil and Dirk Platvoet Could artificial structures such as fish passes facilitate the establishment and spread of the 'killer shrimp' Dikerogammarus villosus (Crustacea: Amphipoda) in river systems? Aquatic Conservation: Marine and Freshwater Ecosystems 23
Article first published online: 22 MAR 2013 | DOI: 10.1002/aqc.2337
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|Industry Recognition
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An Orange County native, William Weinberg limits his practice to criminal defense matters across Southern California and federal courts. He invites prospective... moreView all photos and/or videos
I have known Mr. Weinberg for over twenty years and he is one of the best criminal defense attorneys in the state of California. There is no other... more


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Sunday, 17 Feb 2008 10:53 AM
BEIJING -- China is concerned by U.S. plans to shoot down an ailing spy satellite and is considering what "preventative measures" to take, the Foreign Ministry said on Sunday.
"The Chinese government is paying close attention to how the situation develops and demands the U.S. side fulfill its international obligations and avoids causing damage to security in outer space and of other countries," spokesman Liu Jianchao said.
President George W. Bush has decided to have the Navy shoot the 5,000-pound (2,270 kg) satellite with a modified tactical missile after security advisers suggested its re-entry could lead to a loss of life, U.S. officials said on Thursday.
"Relevant departments in China are closely watching the situation and studying preventive measures," Liu said in a brief statement posted on the Foreign Ministry's Web site (www.fmprc.gov.cn).
On Saturday, Russia's Defense Ministry said the U.S. plan could be used as a cover to test a new space weapon.
It will be the first time the United States has conducted an anti-satellite operation since the 1980s. Russia also has not conducted anti-satellite activities in 20 years.
China launched a ground-based missile into an obsolete weather satellite in January 2007, drawing international criticism and worries inside the Pentagon that Beijing has the ability to target critical military assets in space.
© 2015 Newsmax. All rights reserved.
Friday, 16 Jan 2015 09:25 AM
The Swiss National Bank spoiled the party for the country's luxury watchmakers just as they were preparing for some of t . . .
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Israel is refusing to allow New Zealand's new foreign minister to present his credentials to President Reuven Rivlin sin . . .
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Malaysia Airlines will slash 6,000 jobs, trim its route network, and replace its CEO under plans announced Friday to sta . . .
This serial criminal, like at least half the Democrat Party, belongs in federal...
Another disgusting betrayal of an ally by this Administration -- driven solely...
I don't feel anyone should be denied a seat at the Lunch counter..
But I do...
Iran is intent on destabilizing the Middle East to export its Shiite Revolution...
First order of business, at 12:01 AM first day of the new President's term:...
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Weyerhaeuser continues to face pressure from Wall Street to sell more businesses. The company has already shed $7 billion worth of businesses in the past two years, but investors are still looking for the company to become a pared-down cash cow. Weyerhaeuser is expected to record a full-year loss for the first time since 1991.
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Photos from the first round of the 2015 Tampa Bay Buccaneers Cheerleader tryouts. CLICK HERE
Interested in being a Bucs Cheerleader? Get all of your audition information including requirements, audition and training camp overviews, registration forms and more. CLICK HERE
Check out photos from the Pro Bowl in AZ and relive Cassie's experience through social media! MORE


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Riviera Hotel & Casino Tickets and Upcoming Events
Mar 29Sunday March 29, 2015 / 9:30pm (Doors Open at 9:10pm)
Mar 30Monday March 30, 2015 / 9:30pm (Doors Open at 9:10pm)
About Riviera Hotel & Casino

Image: https://www.flickr.com/photos/53979184@N04/16767742542

All attendees must be age 18 or older with valid photo ID. No cameras, video cameras, or audio recording devices allowed inside the venue.
25 Riviera Hotel & Casino Tips
- Casual 1
I wore if you get chilly easy take a sweater as the a/c does give you a chill.star this tip starred
I recommend picking up your tickets a little early at the box office 1/2 hour before the show at the latest. When we arrived the girl didn't know how to find me in the system eventhough I had my ticket confirmation printed out with my confirmation number etc. She had to ask 3 other employees and several minutes later came back and was able to finally find me in the system and print out our tickets. Luckily there was nobody else in line when we went to pick up our tickets or else I feel we might have been late for the show if they had this many problems with everyone's tickets.star this tip starred
Get there a little early and watch the video they play before the show!star this tip starred
I wore a sundress and was cold. Girls bring a sweater!.star this tip starred
Bar is located outside the theater.star this tip starred
Parked at the Riviera lot.star this tip starred
Parking Near Riviera Hotel & Casino
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A method for detecting the presence or absence of a polymorphism at a particular position within a nucleic acid sequence in a sample, said method comprising i) annealing a pair of probes to said nucleic acid sequence, said probes being designed such that they anneal adjacent each other, and only completely anneal to one form of the sequence, in a reaction mixture which is substantially free of ATP; ii) ligating together any completely annealed pairs of probes in the presence of NAD and a nucleic acid ligase which uses NAD as a substrate/ iii) phosphorylating any AMP released by a ligation event to ATP, and iv) detecting ATP, in particular using a bioluminescent reaction, in the resultant reaction mixture. Kits for conducting this method are also described and claimed.
1. A method for detecting the presence of a polymorphism at a particular position within a target nucleic acid sequence in a sample, said method comprising
i) annealing a pair of probes to said nucleic acid sequence, said probes being designed such that they anneal adjacent each other, and only completely anneal to one form of the sequence, in a reaction mixture which is substantially free of ATP;
ii) ligating together each completely annealed pair of probes in the presence of NAD and a nucleic acid ligase which uses NAD as a substrate, whereby AMP is released;
iii) phosphorylating the released AMP to ATP; and
iv) detecting ATP in the resultant reaction mixture.
2. The method of
3. The method of
4. The method of
5. The method of
6. The method of
7. The method of
8. The method of
9. The method of
10. A kit for detecting a polymorphism within a target nucleic acid sequence, said kit comprising a nucleic acid ligase which uses NAD as a substrate, a phosphorylating agent, and one or more reagents able to produce a bioluminescent signal.
11. The kit of
12. The kit of
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The present invention relates to a method for detecting specific nucleic acid sequences and in particular in identifying sequences which contain polymorphisms or mutations, as well as to kits for carrying out the assay.
A wide variety of methods are becoming available to detect particular nucleic acid sequences, and, in particular to determine the precise sequence of nucleic acids. Many of these are based upon nucleic acid amplification reactions such as the polymerase chain reaction (PCR) and the ligase chain reaction (LCR).
These methods are used in a wide variety of methods, to detect or diagnose for example diseases caused by pathogens such as bacteria and viruses. There are also particularly useful in genetic analysis or diagnosis, where the presence of a polymorphism, in particular a single nucleotide polymorphism (SNP) can give rise to a disease state, or a predisposition towards a particular disease state.
With the completion of the sequencing of the human genome, and increasing knowledge of the relationship of particular polymorphisms to particular diseases or predisposition to disease, the numbers of analyses which are being carried out for diagnostic purposes is increasing.
The ligase chain reaction is a particularly useful reaction for the detection of polymorphisms. In this reaction, a pair of complementary probes, which, when the correct template is available, hybridise next to each other on the template, are added to the sample under test. They are subjected to conditions under which such hybridisation takes place, and then ligated using a ligase enzyme, in the presence of a nucleotide which is commonly adenosine tri-phosphate (ATP), but nicotinamide adenine dinucleotide (NAD) may also be used.
A 3-step ligation event occurs during this process, as illustrated for example in an article by J. M. Pascal et al., Nature, (2004) 432, pp 473-478, resulting in the release of a molecule of adenosine monophosphate (AMP) as a by-product of each ligation event.
The ligated probes and the original template then serve as additional templates for a further round of hybridisation and ligation. This is effected by first heating the mixture so as to denature the nucleic acid strands, for example, to temperatures of from 80-90� C., suitably at about 85� C., and thereafter, cooling to a temperature at which the probes anneal and ligation occurs, for example from 40-60� C., typically at about 50� C. As subsequent cycles are carried out, the amplification but only if the correct template is present in the sample.
By using four probes, one pair corresponding to each strand in the original template molecule, which is double stranded, exponential amplification can result. In this case, in a preliminary step, the double stranded template DNA is denatured.
Furthermore, in some cases, the probes are designed so that, although they anneal close together, they are not directly adjacent each other. In these cases, a polymerase, nucleotides and other reagents necessary to allow the upstream probe to be extended across the small gap (generally of 2-3 base pairs) between the probes, is also included in the reaction mixture so as to cause the upstream probe (which functions then as a primer in an polymerase extension reaction) to extend across the gap prior to ligation. Extension will only occur if the 3′ base of the upstream probe is matched to the corresponding base on the template, and so if it does not, as a result of a polymorphism or the like, then no subsequent ligation will occur.
Thereafter the product of the probe ligation can be detected by separating the products on the basis of size, for example on an electrophoretic gel, and staining the gel so as to detect them. Bands corresponding to a ligated probe will be larger than the individual probes, and so the presence of such a band will indicate that ligation has occurred and that the correct template was present in the sample.
This separation step and detection step is however slow and complex to perform. It can only be conducted in a laboratory environment and requires a substantial level of operator skill, and it requires a number of separate reaction stages.
In some instances, fluorescent dyes which associate with double stranded DNA and whose fluorescence changes when associated with double stranded DNA as compared to single stranded DNA may be added to the reaction mixture. If amplification has proceeded, then this will be detectable from the nature of the fluorescent signal from the dye. In this case however, it is necessary to measure the signal using a fluorimeter, which is a relatively complex apparatus. Fluorescent light of a particular wavelength must be supplied to the sample, and the emission signal, which is at a different wavelength measured.
According to the present invention there is provided a method for detecting the presence or absence of a polymorphism at a particular position within a nucleic acid sequence in a sample, said method comprising
i) annealing a pair of probes to said nucleic acid sequence, said probes being designed such that they anneal adjacent each other, and only completely anneal to one form of the sequence, in a reaction mixture which is substantially free of ATP;
ii) ligating together any completely annealed pairs of probes in the presence of NAD and a nucleic acid ligase which uses NAD as a substrate;
iii) phosphorylating any AMP released by a ligation event to ATP, and
iv) detecting ATP in the resultant reaction mixture.
Step (i) can be carried out in a method conventional in such reactions. Thus, the probes may bind directly adjacent to one another on the template strand, so that ligation occurs in step (ii) if the adjacent ends of the probes are perfectly matched to the template.
Alternatively the probes may bind so that there is a small gap therebetween, provided that there are no adenine bases in this gap region. In this case, the reaction mixture contains a polymerase, nucleotides other than adenine and any other reagents necessary to extend the upstream primer to close the gap and allow ligation only if the 3′ end of the probe matches the corresponding base on the template. If this base does not match, then probe extension across the gap will not occur and ligation in step (ii) will not result.
Using this method, a ligation reaction in (ii) will only occur if the sample contains a sequence to which both probes bind fully.
The presence of a nucleic acid sequence which gives rise to such a match can be determined by detecting ATP, derived from the AMP released during the ligation reaction, in step (iv).
If, as a result of a mutation or difference in the nucleic acid sequence which forms the target of the assay, in particular at the position in which the probes bind adjacent to one another, an end of one of the probes does not bind to the target, then the ligation event will not take place in step (ii). As a result no AMP is generated, and so phophorylation in step (iii) will not give rise to any ATP. The absence of ATP in the reaction mixture in step (iv) will indicate that the sample does not contain the target nucleic acid, and therefore the presence of a different form may be likely.
Nucleic acid ligases which are able to utilise NAD as well as or instead of ATP as a substrate are known. A particular example of such a ligase is E.coli ligase.
The AMP by-product of the ligation may be phosphorylated during step (iii) to ATP enzymatically either directly or by way of the production of adenosine diphosphate (ADP).
he one or more enzymes necessary to convert AMP produced to ATP (step (iii) above), may for example be selected from phosphoenolpyruvate synthase which produces ATP directly from AMP in the presence of phosphate and phosphoenolpyruvate, which are also added as a reagent to the reaction mixture. Alternatively, a combination of a nucleoside triphosphate-adenylate kinase and NTP will yield adenosine diphosphate (ADP), which may then be converted to ATP by inclusion or addition of an enzyme such as adenylate kinase.
Yet further examples of suitable enzymes include pyruvate phosphate dikinase such as that described by Eisaki et al, Biochim. et Biophys Acta 1431 (1999) 363-373.
By using NAD instead of ATP, which is conventionally utilised in LCR, the possibility arises that a signalling system based upon ATP detection may be used.
Various systems of this type are known. However they can generally be carried out rapidly, and give for example clear visible signals in situ. As a result, the ligation reaction can be determined in a homogenous manner, without the need for analysis of the sizes of the ligation products.
In particular, the ATP is detected using a bioluminescent system. Examples of the application of such detection systems are described for example in WO 96/02665.
Particularly suitable bioluminescent reagents, which react to the presence of ATP, include luciferin and luciferase, accompanied if necessary by a source of magnesium ions such as magnesium acetate. In the presence of ATP, these reagents produce a luminescent signal, which can be readily monitored for example using conventional luminometer devices. These are generally much simpler to operate and use than say a fluorimeter.
In generating a signal, these reagents regenerate an AMP molecule, which in the presence of the enzymes and/or reagents of (iii), will be reconverted back to ATP. Thus the signal builds up exponentially and so will be readily and rapidly detected. An example of such a system is described by Sakakibara et al., Analytical Biochemistry, 268, 94-101 (1999). This exponential rise in signal may mean that detection can be carried out directly, in circumstances where amplification reactions may previously have been required.
Bioluminescent signalling systems are more sensitive than fluorescent systems.
Furthermore, detection of such a reaction can be carried out simply and easily, using simple a light detector such as a camera. The use of complex equipment, such as fluorimeters and the like, necessary to detect signals in the widely used fluorescent signalling systems is thus avoided.
Suitably, the method described above forms a part of a ligase chain reaction (LCR). This amplification reaction may be conducted in the usual way, for example by cycling the reaction mixture through denaturation, and annealing/ligation temperatures. Thus, effectively steps (i) and (ii) above are repeated until the desired level of amplification has been achieved.
Suitably the reaction is conducted with two pairs of probes, one corresponding to each strand of a double stranded template nucleic acid sequence, so that amplification can proceed exponentially. In this case, the double stranded template nucleic acid is denatured prior to step (i).
Where this is the case, at least step (iv) and suitably steps (iii) and (iv) above may be carried out at the end of the reaction. This provides a good end-point detection method.
If desired however, the phosphorylating and bioluminescent reagents are present or added throughout the amplification reaction so that the progress of the reaction can be monitored. Generally speaking, the thermostability of reagents such as luciferase is not sufficient to allow it to be present throughout an amplification reaction that involves significant thermal cycling, and thus, it is suitably added at the end of each cycle, and measurements taken before the next cycle commences. However, the selection of thermostable luciferase enzymes such as those described for example in EP-A-524448, WO95/25798, WO 99/14336, WO 00/24878, WO01/31028 and WO2003/0068801 may facilitate some monitoring reaction of step (iv) to be carried out continuously through the LCR.
In particular, this may be further facilitated if the reaction conditions or reagents used in the LCR are such as to reduce or minimise the extent of the thermal cycling in terms of the range of temperatures which are required in order to achieve the desired result.
For example, it is possible to lower the denaturation temperature required by addition of reagents such as Betain (N,N,N-trimethylglycine) or increasing the volume of the solvents such as dimethylsulphoxide (DMSO) or dimethylformamide (DMF).
Addition of enzymes which destabilise duplexes such as topoisomerase or DNA gyrase may also be effective in reducing the denaturation temperatures to those which may be tolerated by luciferase enzymes. The amounts of these enzymes should be sufficient to ensure that there is an equilibrium reaction going on so that annealing and ligation is possible, if the temperature is reduced below that at which the destabilising enzymes is fully active.
In yet a further alternative, denaturation may be effected in a substantially isothermal manner, for example by applying electrochemical potentials to the reaction mixture in order to bring about a denaturation step.
Additionally, a luciferin recycling protein, which regenerates luciferin from oxyluciferin, such as those described in U.S. Pat. No. 5,814,504 and WO 03/042693 may be added to the reaction mixture, to avoid the need to continuously add luciferin to the reaction mixture.
Such information may be used then in the quantification of the target nucleic acid sequence in the sample, using algorithms etc. which are known in the art.
The reaction as described above could be carried out in a variety of conventional equipment. These include for example a Pyrosequencer (available from Pyrosequencing AB, Sweden), which is already provided with appropriate signal detection means. Alternatively, the reaction may be carried out using block heating devices as described for example in EP-A-0810030 and supplied by The Perkin-Elmer Corporation, rapid hot air thermal cyclers such as the RapidCycler� and LightCycler� from Idaho Technologys Inc. or other types of thermal cycler such as those described in WO98/24548.
In order to ensure that no, or at least a very low level of ATP is present in the sample before the assay, it may be preferable to carry out a pre-treatment step in which ATP is removed. This may be done for example if the sample containing the nucleic acid sequence is treated with apyrase to remove ATP, although since this may lead to some AMP production, it may be preferable to apply a deaminase, which destroys the adenosine residue. However, the bead wash steps should ensure effective separation of nucleic acids from the adenylates due to the increased binding of DNA thereto, and so the process can be carried out in the absence of any such step.
The method can be used in any applications in which LCR is currently feasible, both in detection of sequences such as microorganisms, virus or other pathogens, or in the diagnosis of disease. Suitably therefore the sample is a genetic sample.
In a further aspect the invention provides a kit for carrying out a method as described above, said kit comprising a nucleic acid ligase which uses NAD as a substrate, a phosphorylating agent, and one or more reagents able to produce a bioluminescent signal.
Suitable phosphorylating agents include enzymes such as those described above including a combination of phosphoenolpyruvate synthase, phosphate and phosphoenolpyruvate; a combination of a nucleoside triphosphate-adenylate kinase, NTP and adenylate kinase; or pyruvate phosphate dikinase.
Suitable reagents able to produce a bioluminescent reaction systems comprise luciferase and/or luciferin. Optionally, the kit may also comprise magnesium salts required to activate the luciferase/luciferin system, but which may also be used in the LCR.
The kit may further comprise a pair of probes suitable for annealing to a target nucleic acid, and preferably two pairs of probes, each pair being capable of annealing to corresponding regions on opposite strands of a target double stranded nucleic acid.
The kit may further comprise other reagents suitable for conducting LCR, for example NAD, a buffer (such as 250 mM EPPS [N-2-hydroxyethyl)piperazine-N-(3-propanesulphonic acid), or potassium salts.
Additionally, it may also include apyrase enzyme or more suitably a deaminase enzyme to carry out the pre-treatment step outlined above.
The invention will now be particularly described by way of example with reference to the accompanying diagrammatic drawing, which illustrates schematically, a method according to the invention.
In this reaction scheme, steps 1 to 3 represent a conventional cycle in an LCR reaction, with the exception only that NAD is used as the substrate for the ligase enzyme (NH2-LysLig). In the first stage, the NAD becomes bound to the enzyme and disassociated to AMP (which is not available for phorphorylation at this stage). Pyrophosphate (PPi) is released by this process. In the presence of template DNA (1) having an upstream probe (2) and a downstream probe (3) hybridised thereto, the enzyme transfers its AMP to the 5′ end of the downstream probe (3), which then ligates to the free hydroxy group at the 3′ end of the upstream probe (2), forming a ligate (4) and releasing free AMP (Step 3).
The addition or presence of phosphorylating enzymes at this stage will result in conversion of the free AMP to ATP, which can then be detected in a conventional luciferase/luciferin bioluminescent reaction, illustrated at Step 4.
The system of the reaction therefore provides a reliable and easy to operate assay, in particular for the detection of polymorphisms, such as SNPs, in particular as set out in Example 1.
This can be carried out on an instrument such as the Thermo Labsystem KingFisher machine.
A blood sample (200 μl) is added to 300 μl of 3.8M guanidium hydrochloride. This is then mixed for 1 minute at room temperature to lyse cellular material and release DNA. Promega MagneSil KF paramagnetic particles (20 μl) are added (these may be stored in a well containing the first wash solution (see below) and transferred in from that well) and mixed for 2 minutes. Extracted DNA binds to the silica particles. The particles with bound DNA are then magnetically transferred into the first wash well which contains a 50% mixture of ethanol and TE buffer at pH 7.4 (TE buffer is 10 mM Tris.HCl with 1 mM EDTA). The beads are released and mixed with the first wash solution for 1 minute. This is repeated with two more identical wash solutions. After the 3rd wash the beads are transferred into a TE buffer at pH 8.3 at a temperature of >50� C. with mixing for 12 minutes when the DNA is eluted from the silica matrix. The beads are then removed and disposed of in one of the wash wells and the prepared DNA used for the next step.
This can be performed in a block thermal cycler such as the Perkin Elmer 9600.
The DNA is mixed with an equal volume of LCR Assay Mix to give a final reaction volume of 50 μl with the solution containing 20 mM Tris.HCl, pH 7.6, buffer containing 100 mM KCl, 10 mM MgCl2, 1 mM EDTA, 10 mM dithiothreitol, 10 mM NAD+, 4 μg/mL salmon sperm DNA, 15 units T.aquaticus ligase with the two diagnostic oligonucleotides at a concentration of 5 μM each. The reaction is carried out with a thermal profile of 94� C. for 1 min and 65� C. for 4 minutes, repeated for 40 cycles. Bioluminescent detection of AMP formed from the NAD is then used to indicate whether or not LCR occurred. The diagnostic oligonucleotides are designed to have a melting temperature about 2� C. above 65� C.
The assays can be performed, at room temperature, in 100 μl volumes in 96 well microtitre plates using a Labsystems Luminoskan Ascent plate luminometer from Thermo Labsystems.
The added bioluminescent assay mix contains 20 units/ml adenylate kinase, 5 mM acetyl phosphate, 2 units/ml acetate kinase, 3000 ng/ml firefly luciferase and 1 mM D-luciferin in 90 mM Tris-HCl, 10 mM MgCl2, 1 mM EDTA and 10 mM dithiothreitol, to give a final pH of 7.9. Light emission is high where the diagnostic oligonucleotides end-match the complementary bases in the DNA target eg where positive detection of a single nucleotide polymorphism (SNP) is indicated.
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Business Blog
Huge cold storage facility to be built in north Whatcom County
Posted by Dave Gallagher on March 26, 2015
A major cold storage warehouse is coming this fall to serve the local fishing and agriculture industries.
Preferred Freezer Services announced that it plans to build a 331,568-square-foot facility in Lynden, on Main Street west of Guide Meridian. Thats almost half the size of Bellis Fair mall in Bellingham. The company also has secured property nearby for possible expansion.
The facility itself will cost around $32 million to build and could employ up to 30 people on a seasonal basis, according to a company news release. Preferred Freezer expects the warehouse to be open for business in September.
Whatcom Business Alliance announces winners at awards dinner
Posted by Dave Gallagher on March 26, 2015
Whatcom Business Alliance honored several businesses and business owners at the 29th annual Whatcom Business person of the Year Awards dinner, held on Wednesday, March 25 at the Silver Reef Hotel Casino Spa. Here are the winners:
Catching up on Whatcom County restaurant, retail tidbits
Posted by Dave Gallagher on March 25, 2015
Local retail remodels and movement tends to kick into high gear in the spring, and this year doesn't seem to be an exception. Here's a few things I've seen:
Bellingham's Western Solar moving into a bigger space
Posted by Dave Gallagher on March 25, 2015
A local provider of solar energy systems is moving into a bigger space and making plans to expand its staff.
Bellingham's Tech Help acquires Ovation Technical Services
Posted by Dave Gallagher on March 24, 2015
Tech Help, a Bellingham information technology company and a division of Big Fresh, has acquired Ovation Technical Services.
Whatcom's unemployment rate remains unchanged
Posted by Dave Gallagher on March 24, 2015
Whatcom's unemployment rate was 7.2 percent in February, matching the revised January rate.
WWU to open office in downtown Bellingham
Posted by Dave Gallagher on March 23, 2015
Western Washington University will soon expand its presence in the downtown district geared toward better connecting with the local community.


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A weeklong wilderness escape from the electrical lights that illuminate most of our daily lives is enough to reset our internal circadian clocks to synchronize with sunrise and sunset, according to new research from the University of Colorado Boulder.
The spring semester grade numbers are in for the University of Colorado athletic program, and the most recent news parallels that of the last four years as the 300 student-athletes enrolled in the 2013 spring semester had a collective term grade point average of 2.892.
Perched on a mountain ridge at 10,000 feet, University of Colorado Boulder senior Taylor Stratton watches pikas scurrying among boulders and rocky debris. Lower down the wind-swept ridge Brian Shreve collects soil and plant samples in one of the 20 marked-off plots that dot the slope.
This summer the two students have been working in the alpine environment of the Colorado Rocky Mountains 26 miles west of Boulder. They are part of the 20 other undergraduates and 30 or so researchers and graduate students who are conducting research at the CU-Boulder Mountain Research Station, an interdisciplinary facility of the Institute of Arctic and Alpine Research.
The University of Colorado Boulder today announced that Ryan Chreist has been named assistant vice chancellor for alumni relations. Chreist, who most recently served as the director of recruitment, operations and system integration for the CU-Boulder Office of Admissions, starts this week.
Pieter Johnson, assistant professor of ecology and evolutionary biology at CU-Boulder, is having a pretty good year. He and a co-researcher have won an award recognizing outstanding contributions to ecology, and he has been named an Early Career Fellow by the Ecological Society of America.
Things have changed at CU. What Rick George steps into in 2013, both locally and nationally, bears little resemblance to what he left when he waved so-long to college athletics in 1998. Since then, he's remained in sports but in different positions at different levels – a factor that played well to the CU search committee.
"I've been in sports all my life and never done anything else . . . I don't want to do anything else," George told athletic department staffers about an hour before his introductory press conference in the Byron White Club Room at Folsom Field.
Read the whole story at CUBuffs.com >>
University of Colorado Boulder Chancellor Philip P. DiStefano today named Rick George as director of intercollegiate athletics at CU-Boulder, effective Aug. 12, 2013.
George currently serves as president of business operations for the Texas Rangers major league baseball team and has spent more than three decades as a sports executive in the collegiate and professional ranks, including a stint as assistant athletic director for football operations for CU from 1987-91.
In the months before elections, members of the University of Colorado community occasionally receive e-mails from political candidates, including members of the Board of Regents. Because some of you have inquired whether these e-mail communications are appropriate, I asked the University's legal counsel to advise me on this issue.
Amid the staccato clatter of sewing machines, the wardrobe artists snipped, stitched, embellished and ironed garments fit for a king. And a soldier, and a gaggle of nobles, murderers and a gardener.
In the Colorado Shakespeare Festival costume shop housed in CU-Boulder's Theatre and Dance Department, are stacks of bins and drawers of fabric, thread, lace and the sorts of sartorial bits and bobs that go into making theatre costumes.
Aspiring artists at CU-Boulder can learn some of what it takes to become a successful artist in the real world by rubbing shoulders with working professional artists through the Visiting Artist Program.
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