A recent decision by the U.S. District Court for the Northern District of Texas (Unites States v. Davenport, No. 3:09-cv-02455-L) held that costs for modifying a commercially available software package did not qualify for the research and development (R&D) tax credit.
The case addressed members of the Davenport family, who own Burly Corporation. Burly Corporation manufactures metal roofing and standalone metal buildings through its subsidiary, Mueller Supply Company. In 2006, the Davenports filed refund claims for the 2002 and 2003 tax years for the "OneWorld" software project developed to manage, automate and integrate all aspects of Mueller's business, including the automation and integration of manufacturing, design, sales, accounting and shipping. The Mueller OneWorld system was created using the J.D. Edwards OneWorld commercially available enterprise resource planning (ERP) software application suite. Mueller also purchased a license from IBM for an ERP bridge (which consists of base code that is used as a starting point) and contracted with both IBM and other contractors to modify the software for its specific needs.
The IRS sought the return of $292,095 in erroneously issued refund checks to the Davenports for the 2003 tax year, while the Davenports claimed the IRS had wrongfully disallowed its R&D tax credits totaling $196,898 for the 2002 tax year.
The IRS argued that the Mueller OneWorld project involved "mere adaptation of existing commercially available software and quality control type testing" that was not eligible for the credit. The Davenports contended that the project involved a complex process of custom software design, customization, testing and implementation that met the R&D credit qualification tests. The court sided with the IRS in determining that the software project did not meet the requirements for qualified research.
Specifically, in granting the motion for summary judgment, the court ruled that the Mueller OneWorld project did not involve a process of experimentation. In its decision, the court cited the Tax Court decision in Union Carbide Corp. v. Commissioner (T.C. Memo 2009-50, discussed below). The court reasoned that to constitute qualified research, a valid process of experimentation must have a result that was uncertain at the beginning of the research. In reaching this finding, the court noted that "although the Project may have taken a considerable amount of man hours to complete, there is no indication that it involved anything other than a straight-forward configuration of JDE OneWorld and ERP Bridge as contemplated by IBM's proposals to Mueller."
While the IRS made many arguments regarding why the project did not qualify for the credit, the court focused only on the process of experimentation requirement and did not address the other IRS arguments.
The court also noted that the taxpayers relied "solely on conclusory arguments and neither offer any evidence nor cite to any evidence in the record in response to the government's motion." The IRS provided more than 500 pages of documentation to support its assertions.
The court's ruling highlights the need for companies claiming the R&D credit to pay special attention to documenting how the activities claimed as qualified research constitute a valid process of experimentation and to gathering evidence that substantiates the qualification of specific R&D projects.


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Home » Blogs » Dairy Foods Blog » The importance of milk: You can easily take it for granted. But don't
The importance of milk: You can easily take it for granted. But don't
Milk doesn't always get its just due. It's worth focusing attention on the lifelong benefits derived from milk.
KEYWORDS dairy and childhood nutrition / the importance of milk in the dairy
By Michael Zacka, president and CEO, Tetra Pak North America
On most days, millions of Americans jump out of bed, take a quick shower and grab a bowl of cereal before they start their commute. They never think about the significance of this ordinary routine. Our quality of life comes from the little things we take for granted - from a bed to sleep in, running water for a shower or the milk to pour on cereal.
Milk doesn't always get its just due. We consume very few things as nutritionally dense and wholesome as milk, the first food we experience as babies. So it's worth focusing attention on the lifelong benefits derived from milk, and hoping it will soon be available to billions more children, adults and seniors worldwide in nothing short of a nutrition revolution.
So why do we take milk for granted?
Perhaps out of habit. It's the staple our great-grandmother had in her old-fashioned icebox and the staple we have in our modern one, in part thanks to its ready availability in America.
It is the first food we all consume ¬– whether it is from the breast or the bottle – for a reason. The latest dietary research by the USDA finds it ageless, and indicates it deserves a place at our tables throughout our lifetimes.
It is the only beverage that we consider a food, and is so nutritionally important and multi-purpose that it has its own classification on the USDA'S food pyramid.
It also represents powerful yet contradictory views, for we perceive it to be old fashioned and modern, local and global, a treat and a necessity and natural and innovative.
Milk was the original health food
Long before health foods and drinks were a trend, milk was an important and significant source of protein, vitamins and minerals. Humans' unique capacity to digest the milk of cows and other animals was developed some 7,500 years ago as a way to access a rich supply of Vitamin D in sunlight deprived regions of Central Europe, according to research in the Public Library of Science's Journal of Computational Biology.
While Vitamin D is the most well-known vitamin found in milk thanks to the way it's emblazoned on every label, it's far from the only one. Milk also supplies bodies, bones, and brains with protein, vitamins A and B12, calcium, potassium, phosphorus, riboflavin, niacin, zinc and magnesium, report University of Wisconsin nutritionists. They also note that the ratio of calcium to phosphorus is roughly the same one required by the body for bone creation and maintenance.
So, milk's cache of calcium is another remarkable asset for humans. Scientific studies have stressed this wonder food's value in bone creation from cradle to grave – particularly in adolescents as they go through their awkward and gangly growth spurts, according to a study published in the Journal of International Medical Research and available for review online at the National Institute of Health's PubMed. Research published in the Journal of Nutrition also encourages dieting adults to consume dairy products, as does a study on osteoporosis for seniors trying to head off bone loss in the Journal of the American College of Nutrition.
Expanding global access to milk benefits
According to projections in our 2011 Dairy Index, we will see surging demand for milk as emerging middle class families in China, India, Vietnam, Kenya and elsewhere have the ability to improve their family diets. Our forecasts anticipate world demand for liquid dairy products will grow by 30 percent between 2010 and 2020. Spurred by these developing markets, companies and industries all over the planet are going into hyper-drive to meet global need as dairy sales worldwide are forecast to approach $500 billion by 2015, according to a January 2012 report on the dairy products industry from Global Industry Analysts.
The way these emerging middle-class families will consume milk is different. Unlike the United States, where we're used to seeing refrigerated bottles and cartons, children who may have never seen a refrigerator in developing countries will drink milk in a shelf safe carton that was placed on store shelves right off the bed of a pick-up truck. And as rural dwellers move to the cities in India, Pakistan, Sri Lanka and Bangladesh, they will no longer be supplied by "milkmen" carrying loose unpasteurized milk from nearby farms in large metal cans on their bicycles and mopeds, but rather have access to safe, hygienic and nutritious milk in aseptic packaging.
Our vision at Tetra Pak is 'we commit to making food safe and available everywhere.' We pioneered the use of aseptic shelf-safe cartons, a remarkable technology that enables even sensitive products, such as milk, to be delivered over long distances without refrigeration or preservatives. This development helped, and will continue to help, expand the access and consumption of nutrition-dense milk both in developing and developed countries by enabling parents to give milk to their kids in a safe way anytime, anywhere.
A future of safe, mobile and easily stored milk worldwide is unquestionably a good thing from a global health perspective. Milk has always been a major component in American children's diets. But when millions more children around the world also have the benefit of this nutrient boost, we will know that observances such as World Milk Day and National Dairy Month have been effective.
It seems our busy lifestyles cause us to take much for granted. We should pause to remember that the glass of milk in your hand or your baby's fist is really something special.
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Tharp & Young on Ice Cream: An Encyclopedic Guide to Ice Cream Science and Technology
An at once an all-inclusive guide to the meaning of hundreds of technical terms and ideas needed for ice cream manufacturing, as well as a practical introduction to the ingredients, freezing methods, flavoring, and packaging of ice cream, sherbet, sorbet, gelato, frozen yogurts, novelties and many other kinds of frozen desserts.

redpajama_set_name: RedPajamaCommonCrawl


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Digital Civilisation, Connected Cities and the 'New' Silk Roads
digitalChina Speculation Digital Civilisation, Connected Cities and the 'New' Silk…
Presentation text:[1]
Michael Keane, Professor Curtin University, Australia. Program Leader Digital China Lab
The Power of Brand: Brand Building and National Image Communication in the New Age Grand Canal Culture Belt, Yangzhou University, Yangzhou, China, 18 May 2019.
People are talking about civilizations a lot recently, especially the values of civility that have made cultural exchange possible, including the respect for diversity of views that foster technological progress and innovation. Some call this cosmopolitanism. An example relevant to this forum is the Silk Roads of the past.
The "New" Silk Roads, including the Belt and Road (BRI) and the Digital Silk Roads, are contemporary symbols of technological progress and innovation. Although I will focus on digital connectivity, and explain what "digital civilisation" entails, I will mention some examples of material civilisation, including food and cultural sites, many of which are evident in the city of Yangzhou, and more broadly in Jiangsu Province. These material forms are in turn rendered in digital formats for promotion (branding), consumption (via e-commerce) and sharing (communication).
In China today people sometimes talk about the "four great new inventions": mobile payments, high speed rail, e-commerce and bike-sharing. While these are not new, their usage has expanded in China so much that they are part of peoples' daily lives. It can be stated that they are all part of an "emerging digital civilisation", which also includes Internet of Things, Artificial Intelligence, Virtual Reality, and Blockchain.
Historically Yangzhou illustrates examples of material civilization and shared learning. In the Tang Dynasty the city was connected to maritime trade routes; it was a meeting place of diverse cultures from the Silk Roads: Arab, Persian, Chinese. Later Yangzhou had a Muslim community, and even an Italian community.
With the BRI (一带一路) discussion now engaging with the European Union (EU) and various partners in the Muslim world, the expectations are high for a renewal of pan-civilizational discourses. Yangzhou has a key role to play here. The cosmopolitan image of the city is set for regeneration, and technology will undoubtedly play a key role in rebranding.
Connected cities
Yangzhou is famous for its role in connecting cities via its intricate canal system. The Cities are now being connected in a new model of "digital civilisation". The Internet of Things, Artificial Intelligence (AI) and Machine Learning (ML) combine to make life better for people in crowded cities. Cities of the future will have "brains", which will engage in Deep Learning.
Algorithms will do a lot of the thinking. Self-driving cars may even solve problems of congestion; energy will be generated rather than lost; the environment will be protected. And criminal behaviour will be tracked efficiently via extensive social credit systems.
China's "digital civilization" is likely to consolidate as cities, connected across the BRI region, become ever more dependent upon China's technological platforms, expertise and algorithms, and as people take up Chinese apps, affordances and smart phones, particularly young consumers in these regions.
In this way Information and Communications Technologies (ICTs) "afford" people the possibility to connect to other people and cultures in the region in real time; for instance, the take up of WeChat among non-Chinese or Alipay financial technologies, the use of Tik Tok, and SHAREit in parts of Asia, including BRI nation-states. As people in Eurasia use these services, they are likely to be drawn closer into a Chinese cultural universe.
Digital civilization thus equates with a kind of digital cosmopolitanism, a "community of shared destiny". This is the ideal future of humanity—as long as machines, corporations or governments don't create new social divisions, which is the subject of much science fiction.
Digital civilisation: what is it?
To understand digital civilisation, it is worth looking at the various meanings of civilisation found both in the media and in the academy. The first is human civilisation (the species): this is the most general meaning of civilisation and includes all nations, all people.
Secondly, there is the idea of a universal civilisation: for instance, there are dominant civilisations, we might say Western civilisation, or Confucian civilisations, hence we have the "clash of civilisations" idea.
Third, civilisations are ethnographic constructs: i.e. varieties exist and all are good in their own way. This is the work of anthropologists.
Finally, there is an archaeological approach to the study of civilisations. Scholars can reconstruct the progress of civilisations and cultures from texts, relics and excavations; for instance, the caves at Dunhuang (Gansu), Kizil (Xinjiang), and Luoyang (Henan).
Digital civilisation is the latest addition. According to the strategist Parag Khanna, digital civilization is now expanding "along digital rivers and tributaries much as human civilization has grown along natural ones". The Internet is constantly remapping the digital world, enabling new communities to emerge, some of which are fast-changing and even dangerous to the ideal of social harmony.
It is sometimes said that we live in a "global village", a term coined by the media theorist Marshall McLuhan. McLuhan was fascinated by the idea of the extended mind, which has come to be known as "external symbolic storage." We outsource our thinking to machines and these machines have extended memory. The human brain by contrast has very limited memory.
If we allow smart machines (and apps), to do the work we can create more space in our minds. The best example is perhaps Google Maps. The machine will not forget. This is not without problems: we can be the object of digital surveillance. In order to work, the software needs to know where we are and we have to give permission. Many people accept this "invasion of privacy" for the benefits that accrue. We can call these "affordances", a term that comes from ecological studies.
We are a hyper-connected species. Parag Khanna speaks of "connectivity as destiny". The human species has certainly evolved rapidly. Our ancestors searched for recurring patterns and symbols, and found these in nature. The ancient tradition in China is evidence of this, particularly the use of symbols to represent reality. The Chinese written language recombined visual patterns, for instance, representations of trees, people, animals, objects. This recombination produced a great civilisation. Now recombination proceeds at a rapid pace.
Ray Kurzweil, the Director of Engineering at Google, who has received 21 honorary doctorates, refers to the human neo-cortex (brain) as a large pattern recognizer. John Lent, author of the recent book, The Patterning Instinct, identifies pattern recognition as the basis of human culture, and civilisation. The Silicon Valley based virtual reality pioneer, Jaron Lanier, argues that digital globalization "repatterns" the world, shifting our collective organizing protocols toward a new kind of network efficiency. The question is not whether this shift is happening, he says, "but rather the degree to which everyone participates."
In China, it seems everyone is willing to participate.
Affordances and civility
One of the great benefits of mobile devices is that they "afford" greater decision-making certainty. For instance, if we are travelling overseas as tourists, to Yangzhou for instance, we might 打卡, that is, we can check in online that we have visited the most important sites, for instance, Daming Temple. In English we say that we have 'ticked that box'. If we are looking for a place to eat in Yangzhou, we can check beforehand the menu, prices, and customer rankings to reassure ourselves that we will not be disappointed.
Two thousand years ago in China a person of authority would consult the Yijing, which perhaps we can say was a pre-modern algorithm? Should I have Yangzhou Fried rice (扬州炒饭) today or dazhu gansi (大煮干丝)? The Yijing provided a contextual or situational guide to an informed decision: the immediate consequences and the future results; perhaps it will be a fine occasion with friends but it may lead to gaining weight!
The Yijing is not the best way to make decisions today. We don't have time for abstract commentaries. The restaurant app offers a lot more. We can book a table, order and pay for the meal using WeChat or Alipay; then we can share our experience with our friends all over the world, who will no doubt 'like' what they see. The same applies to cultural artefacts like the Dunhuang Caves in Gansu. Digital technology and visualisation technologies makes these "wonders of civilisation" accessible to anyone. You don't even need to go there.
This is how it should work. Clever machines should allow us to be more efficient, to make better use of available resources. But there is a problem that we are losing in danger of losing touch with real life. We see reality through the interface of devices that are monitored by third parties. In China users are locked into the WeChat ecosystem; are there any real alternatives? In the West there are multiple choices. You don't have to be on a digital site like WeChat to pay for your purchase. When people visit China from abroad, they find that credit cards and paper money are inconvenient.
Digital civilisation has its downsides. Once we were called homo sapiens; now according to the Korean-German philosopher Byung-Chul Han, we are homo digitalis. We use our thumbs more than our hands. We have online "profiles", which can be easily curated; many people even have "avatars". People use emojis instead of expressing our real thoughts in words; and 'like' a post on Facebook because they feel obliged to like it, because online friends have liked it.
Alternatively, we should not forget ancient wisdom. In the Buddhist tradition of insight meditation, which is part of an ancient Asian civilisation, we deliberately free ourselves of such stuff, that is, we get rid of distractions. We recognize the "patterns of mind" as illusions. We try to be more "mindful". But with our new digital lifestyles we use the accumulation of patterns to make our lives meaningful. We add more and more layers to our extended minds, our external symbolic storage. We multi-task, "liking" posts on Facebook while watching television, adding "friends" and connections.
In many respects this is an example of mimetic behaviour that has deep cultural roots. In primitive times before we had language, people (hominids) communicated with nods, grunts, facial gestures. Even when language developed people used rituals to convey respect, particularly in China. This is the fundamental basis of any "harmonious" civilisation.
The concept of civility is thus described by the Chinese word wen (文), which in the Warring States period came to refer to refined ethical behaviour. A similar connection between civility and refinement took place in Europe, almost two thousand years later, leading to the idea of cultural capital.
At the same time, however, online behaviour is increasingly uncivil; much of the uncivil (trolling) behaviour is due to the politics of fear, that is fear of the other. This is the downside of digital civilisation.
Shared destiny: the view from Australia
Many people of Chinese descent identify with the concept of a great civilisation—some commentators call China a "civilizational state", in contrast to a nation-state model that has characterised the rules-based global order for the last fifty years. China's leaders call for a "community of shared destiny", which entails a flexible concept of national sovereignty. The BRI with its historical legacy of trade and cultural exchange, seems like the best place to test this idea of shared destiny in practice.
Because I come from Australia, the "land down under", I don't naturally identify with the greater BRI project, although the BRI extends to Australia when one considers that Australia is a composite of European (western) civilisation, indigenous Australians, and many cultures from the Silk Roads. 70,000 years ago, homo sapiens moved "out of Africa"; many of our ancestors moved south and east. 10,000 year ago, the land mass now known as Australia was physically connected to Asia.
With modern communications we are even closer; we are also in the same time zone, compared with Europe and the US. So, we are truly connected. In the latest statistics of the Australian population (2019), more than 30% of people were born overseas; and 262 different nationalities were registered. In 2019, Australia has 557,690 people born in China, and 489, 410 born in India.
Significant numbers of resident migrants were born in countries (and regions) prominently listed in the BRI: South Africa (100,480), Zimbabwe (41,570), Afghanistan (53,670), Sri Lanka (124,500 ), Pakistan (69,660), Singapore (59,120), Philippines (252,690), Vietnam (243,220), Thailand (71,250), Hong Kong SAR (97,590), Mongolia (54,040), Malaysia (152,900), Iraq (74,680), and Indonesia (78,970). These numbers do not include children born of these parents: it does not account for the Diaspora as such.
Australia has relatively few large cities but most people live in cities contiguous to either the Indian or Pacific Ocean i.e. this is the (maritime) Road in the BRI construct. More than any other nation in the world we approximate a model of Eurasia. Australia might be a "European outpost" according to Malaysia's Prime Minister Mahathir, but with respect to the population base Australia is increasingly "Asianized". With respect to civilisations and intellectual traditions, Europe has a dualistic worldview. China, and much of Asia, has a holistic or integrated model. The former model celebrates a global rules-based order whereas the Chinese "community of shared destiny" privileges a degree of cultural respect.
So, in our national debates Australians are asking some difficult questions: how do we balance contending civilizational forces? We already have a multi-cultural society model that works well. Could a "community of shared destiny" model apply to a liberal democratic nation like Australia? We hope that we can learn much from China's experience and build new relationships.
[1] Not all of this content will be possible in 15 minutes. Focus will be on first section.
Chinese culture and the power of digital platforms
New Book is out and its Open Access
Civilisation, China and Digital Technologies
Masking power in the age of contagion: the two faces of China in the wake of coronavirus
China's digital media industries and the challenge of overseas markets
When push comes to nudge: a Chinese digital civilisation in-the-making
Inside the world of the Chinese shoppers who are unnerving Australians
China's 'social+' approach to soft power
Cultures of Creativity and Innovation in Greater China
Creativity, Affordances, and Chinese Traditional Culture
online literature Creative Economy creative production Alibaba state-owned media enterprises creative Huawei online platforms Dalian Wanda techno-nationalism digital cultural heritage international co-production copyright soft power COVID-19 shanzhai copycat nation cloud computing One Belt One Road Internet trends in China China Chinese Media culture digital culture PRC 'going out' campaign Chinese Entertainment Confucius Institutes Tencent streaming Cultural products Field Digital China 'champions' Willing Collaborator modern China Digital Disruption rage comics cultural power : Internet + policy CCTV iqiyi Entrepreneurship SL sreaming services Tudou innovation data privacy social credit Internet of Things Cross-straits Online Collaboration going out Chinese cultural products and services Dapeng Youku Chinese culture media Baidu platforms cultural policy digitisation User-generated content Asia audio-visual market machine learning internet companies digital entrepreneurship positive energy Collaborative Innovation international collaboration informal labour amateur BAT online video BRI online consumption digital civilisation China online and digital platforms China's international image AI digital China networking 'digital power' Digital Silk Roads Taiwan digital future China's 'going out' policy Sohu South Korea Silk Roads Big data analytics innovation nation data mining Chinese Diaspora State Owned Enterprise grassroots innovation Network convergence chinese entertainm diaosi Singapore 2018 digital transformation algorithms professionalization


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Eugenia analamerensis är en myrtenväxtart som beskrevs av Joseph Marie Henry Alfred Perrier de la Bâthie. Eugenia analamerensis ingår i släktet Eugenia och familjen myrtenväxter. Inga underarter finns listade i Catalogue of Life.

Källor

Myrtenväxter
analamerensis

redpajama_set_name: RedPajamaWikipedia


Like most married couples, Allie and Tony have had their hard times. But when they are eventually forced to move from their house because of Tony's mistakes, things suddenly begin to change. Mark, their son, becomes convinced that their new next door neighbour, a grumpy old man called Jeremy, is none other than Santa Claus. But it's when he starts writing his letters to 'Santa' asking for his family to be happy and detailing his rather miserable school life, that events take a rather complicated turn . . .
Allie could pinpoint the exact moment her life with Tony changed. It was heralded by a ring on the doorbell at five o'clock on a mild Thursday afternoon in May. Tony was work and Mark and Owen, their two sons were playing football out the back garden. Owen stood in the goalmouth, sucking his thumb as Mark kicked the ball at him. Owen made no attempt to save the ball much to Mark's delight.
Alllie laughed and then the door bell rang.
A well dressed man in a grey suit and shiny shoes stood on her doorstep. His hair was cut military short so it was impossible to say what colour it was. His face was pale and lean, his eyes were blue. An ordinary face and instantly forgettable. Allie wondered idily what he was selling.
Looking at his shiny shoe, positioned so that she couldn't slam the door on him, Allie's legs grew suddenly weak. It was at that moment that she knew that everything she'd tried to keep a lid on was going to blow up in her face. All the stuff she'd tried to ignore was coming to her doorstep at last.
"Tony does," the man said, smiling at her which made things worse.
Allie swallowed and didn't respond. She knew if she spoke her voice would shake. She was afraid too that she might say the wrong thing.
It took a moment before Allie digested the threat. She swallowed hard. "What do you mean?" her voice trembled.
Owen could be heard howling from out the back garden.
She knew he jumped but she could sense him leaving.
"What does he owe you?" Her voice was stronger, firmer. She had to know, to find out how bad things were. To sort this mess out and keep her family safe.
"Has he not told you?" The man raised an eyebrow. To the casual observer, he'd look as if he found something amusing.
"You tell me how much he owes or you leave right now," Allie said.
The man's eyes narrowed and suddenly he didn't look so ordinary. "Oooh," he said, "I won't be going anywhere until I get my money." He smiled.
Allie recoiled as the smile only served to emphasise the coldness in his eyes. "How much does he owe you?" Despite her best efforts, her voice trembled.
Allie watched him walk away, his hands casually tucked into the pockets of his suit. He hopped into a car and was gone.
She closed the door, her whole body shaking. Then she made two phone calls, speaking calmly into the receiver, before slumping to the floor of the hall where her two kids found her ten minutes later.


Prominent Properties Sotheby's International Realty, one of New Jersey's leading luxury real estate firms, sponsors the 2019 Ridgewood Guild International Film Festival. A Spring staple for the village of Ridgewood, the festival features documentaries, animation, shorts, music videos, experimental videos, web, new media, and digital shots. This will be the 9th annual festival, with past festivals boasting special appearances by Danny Aiello, Tony-award winning actor Christian Borle, three-time Emmy Winner Martha Byrne, Oscar-nominated Chris Mann, and many others.
The Ridgewood Guild International Film Festival will feature films submitted by students from Bergenfield, Ridgewood, Allendale, Waldwick, Mahwah, Middletown, Hudson County, New York and elsewhere, with several of the films dealing with prevalent social issues faced by students. The festival will issue prizes for several film categories, and will also bestow a $500 scholarship to the director of the Best Film by a student.
The festival will feature its premiere movie, ROMA, on April 10th at the Ridgewood Public Library. April 25th will mark the closing night of the festival with a Red Carpet event at Bow Tie Cinema for the film premiere of Hollywould, starring actor Eric Roberts. After the premiere of the film Hollywould, the festival will host a Hollywood-style after-party at Felina's Restaurant in Ridgewood. The after-party guest list will include actor Eric Roberts, as well as the cast and crew of Hollywould, and other local filmmakers.
For more information about the festival, including movie schedules, special appearances, additional sponsors, and festival updates, click here.
To purchase tickets for the festival, click here.


1. Is Rental Car Coverage Primary Or Secondary?
2. How Do I Make Sure Coverage Applies To My Rental?
3. What Coverage Is Included?
Credit card coverage mostly applies to what's called a collision damage waiver or loss damage waiver, typically the most expensive coverage offered at the rental counter. This waiver isn't actually "insurance," although many people refer to it as such. The rental car company has insurance on the car; the waiver just means the rental company (and its insurance company) won't come after you financially if the car is damaged or stolen. Cards also usually include some coverage for towing expenses and administrative fees. Many cards also cover loss of use, which means compensating the rental company for time the car is out of service while damage is repaired.
Typically not covered are liability and injury concerns, such as damage to property other than the rental car, people you hurt and related lawsuits. But many people have those coverages elsewhere. Rental car companies must provide a minimum level of liability insurance, and your personal liability coverage often applies. Also usually not covered is loss or theft of personal belongings inside the car, but that is often already covered by homeowner's or renter's insurance. Credit card coverage also doesn't handle ambulance or medical bills following a crash, but your personal health and auto insurance policies probably do, according to the insurance institute.
5. What Vehicle Rentals Are Excluded?
Credit card coverage usually excludes certain types of vehicles, such as expensive or exotic cars as well as motorcycles, trucks and large vans. Mastercard, for example, covers vehicles meant for nine passengers or fewer. Credit card coverages typically address rentals from traditional car-rental companies, not car-sharing services like ZipCar and peer-to-peer car rental services. If you use those services often, confirm with your card issuer that it doesn't provide coverage.
6. How Long Of A Rental Will Be Covered?
The credit card coverage period is usually limited. For example, Visa generally covers rentals for 15 consecutive days in your country or 31 days outside it. But if your coverage is primary, 31 days for all rentals is common.
7. What If I Don't Have Personal Auto Insurance?
Generally, you get an even greater benefit from your card: secondary coverage becomes primary for collision. However, if you rent cars often, you should consider a non-owner liability policy, says the Insurance Information Institute.
8. How Does Coverage Vary For International Rentals?
Rentals in some countries may be excluded. For example, standard Visa and Mastercard coverage excludes rentals in Israel, Jamaica and Ireland. American Express won't cover in Italy, Australia and New Zealand. Aside from excluded countries, the good news is that if your personal auto policy doesn't cover you outside the United States, your secondary credit card coverage could become primary coverage automatically.
9. What Is The Procedure For Filing A Claim?
Ask your card issuer what documents you must submit. Examples include a copy of the accident report from the rental company, the rental agreement, a repair estimate, photographs and a police report, if there is one. If filing claims sounds like a hassle, you could instead buy coverage from the rental car company, which might result in less paperwork. If you have several credit cards that offer rental car coverage, find out which has the superior benefit. That way, you'll know which card to plunk down the next time you're at the rental counter. This article was written by NerdWallet and was originally published by The Associated Press.
Written by Gregory Karp for NerdWallet. Gregory Karp is a writer at NerdWallet. Email: gkarp@nerdwallet.com. Twitter: @spendingsmart.
The article Wreck Your Rental Car? Your Credit Card Could Help originally appeared on NerdWallet .


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Ворголка, Варгов (рос.) — річка в Україні, в межах Конотопського району Сумської області. Права притока Клевені (басейн Дніпра).

Опис
Довжина 18 км, похил річки — 1,7 м/км.  Площа басейну 132 км².

Розташування
Бере початок на південному заході від Сутисків. Спочатку тече на південний захід понад Покровським, а потім переважно на південний схід і біля Литвиновичів впадає у річку Клевень, ліву притоку Сейму.

Населенні пункти вздовж берегової смуги: Калашинівка, Зазірки, Воргол.

Річку перетинає автомобільна дорога .

Примітки

Джерела
 «Каталог річок України». — К. : Видавництво АН УРСР, 1957. — С. 97. — (№ 1611).
 Словник гідронімів України — К.: Наукова думка, 1979. — С. 121

Малі річки України
Річки Сумської області
Річки басейну Дніпра
Клевень
Статті про річки Сумської області без зображення в картці
Річки Конотопського району


text:
Brent Goudarzi
Goudarzi & Young, L.L.P. - Gilmer
P.O. Box 910, Gilmer, TX 75644 United States
Brent Goudarzi is the founding partner of Goudarzi & Young, L.L.P. in Gilmer, Upshur County, Texas.
Mr. Goudarzi received his B.A. (1993) and J.D. (1996) from Texas Tech University. He is a past President of the East Texas Trial Lawyers Association (2011-2017) and currently serves on its board of directors in the capacity of President Emeritus. Mr. Goudarzi has been honored as a Texas Super Lawyer in 2012 (Super Lawyers recognition is a Thomson Reuters service), a National Trial Lawyers Top 100 Trial Lawyer (2013, 2014, 2015, 2016, 2017), a Trial Lawyers Board of Regents Litigator Award winner in 2014 and 2015 (ranked top 1% of lawyers), and a National Association of Distinguished Counsel member (2015) and a member of the American Academy of Trial Attorneys – Premier 100 (2015).
Mr. Goudarzi is also a member of the American Association for Justice, the Million Dollar and Multi-Million Dollar Advocates Forum (since 2013), the Honorable T. John Ward American Inn of Court and the Upshur County Bar Association.
Mr. Goudarzi is honored to have been invited, and proud to serve, on the Association of Plaintiff Interstate Trucking Lawyers of America ("APITLA") National Advisory Board—a team committed to achieving a substantial reduction in the thousands of traffic deaths and injuries that occur each year on America's highways that are caused by unsafe trucking companies who choose to operate in violation of the Federal Motor Carrier Safety Regulations.
Mr. Goudarzi's practice is devoted to the representation of victims in catastrophic injury and wrongful death cases, including commercial trucking and auto negligence, products liability, workplace injury and premises liability. His law firm has represented over 17,000 people. Mr. Goudarzi is admitted to practice in all State Courts of Texas and the United States District Courts for the Eastern and Northern Districts of Texas.
Mr. Goudarzi has tried cases all over the state of Texas and has secured record jury verdicts in Upshur County, Gregg County, Harrison County, Dallas County, Hopkins County, Marion County, Harris County, Camp County, Titus County, and Smith County. Additionally, he has achieved historic and record settlements throughout the state. In 2015, Mr. Goudarzi negotiated the 5th, 13th and 17th largest settlements in the United States and the 3rd, 4th, 6th, 11th and 16th largest settlements in Texas. Most recently, in July 2019, Mr. Goudarzi secured the largest single plaintiff settlement in American history—$140 million.
Mr. Goudarzi's successes have not gone unnoticed; even by his adversaries. Lawyers who have been opposite Mr. Goudarzi in lawsuits and witnessed first-hand his tenacity and advocacy skills often refer Mr. Goudarzi clients, including their own family members.
Texas Tech University School of Law – J.D. – 1996
Texas Tech University – B.A. – 1993
U.S. District Court, Eastern and Northern Districts of Texas
Memberships & Awards
East Texas Trial Lawyers Association, Past President (2011-2017)
East Texas Trial Lawyers Association, Board of Directors
Honorable T. John Ward American Inn of Court
Million Dollar Advocates Forum, 2013 – Present
Multi-Million Dollar Advocates Forum, 2013 – Present
Texas Super Lawyer (Super Lawyers recognition is a Thomson Reuters service), 2012
National Trial Lawyers, Top 100 Trial Lawyer, 2013 – Present
Trial Laywers Board of Regents Litigator Awards, Ranked Top 1% of Lawyers, 2014 – 2015
National Association of Distinguished Counsel, 2015
American Academy of Trial Attorneys – Premier 100, 2015
Provides pro bono services all over Northeast Texas
Active sponsor & coach of little league baseball, soccer & football


text:
Florida Race Gives GOP Chance to Flip House Seat Lost in 2018
Miami-Dade mayor challenging Democrat Debbie Mucarsel-Powell
Incumbent called too liberal for heavily Cuban-American district
August 19, 2020 1:51 PM	 By Samantha Handler
House Republicans see a chance to win back a seat in Florida where Miami-Dade County Mayor Carlos Gimenez has mounted an aggressive challenge to freshman Democratic Rep. Debbie Mucarsel-Powell.
Gimenez, who won his GOP primary Tuesday, has attacked the incumbent by tying her to Washington liberals and labeling her a socialist in a district with a large number of Cuban Americans.
The 26th District, which stretches from southwest Miami-Dade County to Key West, is one of the only House districts held by a Democrat that political analysts have recently shifted toward Republicans. Mucarsel-Powell won by 2 percentage points in 2018, ousting Carlos Curbelo (R) two years after Hillary Clinton carried the district by 16 points.
Photographer: Patrick Semansky/AP Photo/Bloomberg
Rep. Debbie Mucarsel-Powell (D-Fla.) faces a challenging race against the GOP mayor of Miami-Dade County Carlos Gimenez.
The Cook Political Report now rates the race as a Toss-up, after previously giving Mucarsel-Powell the edge. Florida Democratic and GOP strategists say the race now looks like a dead heat, but that Gimenez's ties to President Donald Trump and his fundraising deficit are significant disadvantages.
"Both parties have an equal interest in it," said Susan MacManus, a professor emeritus at the University of South Florida. "Democrats are hanging on for dear life, and Republicans would love to flip it."
Cuban-Americans in the district have a historically higher turnout rate than other demographic groups, and Republicans are capitalizing on their concern about socialism, MacManus said.
Too Liberal
Gimenez campaign consultant Cam Savage said Mucarsel-Powell is too liberal and partisan for the district. He contrasted that with Gimenez's role as county mayor that has him working with the county commission "where everyone is technically nonpartisan."
Republicans have attacked Mucarsel-Powell after her husband's employer, the Fiesta Restaurant Group, received a $15 million loan from the Paycheck Protection Program intended to help small businesses deal with the economic fallout of the pandemic. Fiesta employed more than 10,000 workers in 2019. Mucarsel-Powell said she opposes big businesses receiving such aid but that it was Republicans who inserted language in the law that allowed larger companies to benefit from the program.
Republicans have also circulated a video showing Mucarsel-Powell saying no one thought she was going to be able to "take on the Republican Cuban establishment that had a hold on Florida politics."
"Debbie Mucarsel-Powell is one of the most partisan members of Congress who would rather support the socialists in Washington and her Ukrainian oligarch-connected husband than the people of South Florida," National Republican Congressional Committee spokeswoman Camille Gallo said in a statement.
Robert Powell worked as general counsel for some companies that were partially owned by Ukrainian businessman Igor Kolomoisky, whom the Miami Herald reported in 2018 has been accused of bribery, murder, and embezzlement. When the issue came up in the last campaign, Mucarsel-Powell said they've never had any contact with Kolomoisky.
Photo by Eva Marie Uzcategui/Bloomberg
Carlos Gimenez, mayor of Miami-Dade County, speaks during a news conference at a new self-swab Covid-19 drive-thru testing site on July 27.
'Married President Trump'
Gimenez's response to the Covid-19 crisis in his role as mayor has also become an issue in the campaign. While his handling of the virus was initially well-received, his shift toward Trump's pandemic "playbook" by hesitating to impose a mandatory mask mandate and shutting down the county again after reopening won't play well in the district, said Elnatan Rudolph, a political consultant who has worked on GOP campaigns.
Rudolph added that Gimenez throughout his career has been a more moderate and pragmatic politician. In 2016, Gimenez publicly said he would vote for Clinton.
"In his desire to move to the right and be accepted as a Republican, which he's not, and be more like Trump and DeSantis, he didn't listen to everything he should've done with the pandemic," Rudolph said, referring to Florida Gov. Ron DeSantis (R). "I'm not sure he gets high grades like he did at first."
Mucarsel-Powell's campaign manager Andrew Markoff said Gimenez has become the "face of economic collapse, an out of control pandemic, and death."
The president endorsed Gimenez in January, hours after he announced his candidacy, predicting on Twitter he "will win big." Trump's endorsement could be a disadvantage for Gimenez, said Mike Hernandez, a political consultant who has previously worked for both candidates. Part of the reason Curbelo lost to Mucarsel-Powell in 2018 was independent voters' dislike of Trump, Hernandez said.
"(Gimenez) has married President Trump in a district that he is incredibly unpopular in," Hernandez said, "and there's no way out of it."
Savage said the mayor has always had a good working relationship with Trump, and Gimenez is "comfortable making decisions knowing he won't make everybody happy all the time." He said Gimenez has relied on his past experiences as a firefighter and city manager, and has been listening to experts on his handling of the pandemic.
Expensive Miami Market
Mucarsel-Powell has raised $3.9 million and had $2.8 million in cash on hand as of July 29, while Gimenez raised $1.2 million and had $882,000 on hand. That fundraising advantage is significant in the expensive Miami media market.
"Not only is it one of the most expensive, you have to do bilingual ads," Rudolph said. "If you spend money on English TV, you have to spend just as much, if not more, on Spanish TV. So it's costing you double."
Hernandez said Gimenez already has strong name recognition in the district, and positive media coverage from the pandemic could benefit him.
But he said Mucarsel-Powell's fundraising will allow her to spend so much more on advertising and urge more turnout from Democrats. He said while it will be a close race, the district is still shifting Democratic with more young Hispanic voters.
To contact the reporter on this story: Samantha Handler in Washington at shandler@bloombergindustry.com


Away Home Kamloops' ED Katherine McParland and vice president Rocky Hunter.
Pamela Tulliani with Nick and Clark Saxby getting set up for the night.
More than 75 people taking part in the annual Camp Out to End Youth Homelessness fundraiser, organized by A Way Home Kamloops.
This year's event took place on Friday night at Mastermind Studios in Southgate, with more than $40,000 raised going toward A Way Home Kamloops Safe Suites, a youth housing project with wraparound support services from various community organizations.
For more information on the project, visit awayhomekamloops.com.


As numerous reports attest to — the refugee crisis is likely the worst humanitarian crisis of our time. The news is rife with images of people fleeing conflict in water-logged rafts, overcrowded into camps, and waiting at border lines.
Yet what has been greatly under-reported is the devastating impact mass displacement has had on the very real threat to the future stability of the world: the education crisis.
The numbers underline the gravity of the situation: only 50 percent of refugee children have access to primary education, compared with a global average of more than 90 percent, according to a UNHCR report last year.
Higher education is firmly linked to increased opportunity, strengthen economic development and safer communities — making it vitally important in places of conflict. Most critically it offers young people hope toward a better future.
Which is why Global Citizen is partnering (alongside seven other organizations) with the Institute of International Education (IIE) in their new initiative: the IIE Platform for Education in Emergencies Response (IIE-PEER). IIE-PEER is an online clearinghouse specifically built to connect displaced Syrian students with opportunities to continue their education in safety around the world.
One student who very much understands the importance of this program is Nour Mounajed. Nour qualified to be one of only 15 women selected for the "100 Syrian Women, 10,000 Syrian Lives" program led by Jusoor and IIE (so called for the estimated multiplication effect that supporting 100 women through their education will have on their communities), and has spent the last year working on a Masters degree at Harvard University.
Not only that, but Nour's chosen subject, as a mother of three who witnessed the devastating impact of war on her home country of Syria is: education.
Global Citizen caught up with Nour to find out how the past year had gone, what she was planning to do with her degree and her hopes for the future.
How long have you been in the US? And when did you leave Syria?
I've been in the States for seven months — I left Syria in 2012 from Damascus.
The main reason I left was because of the kids. It was becoming too stressful for me as a parent. I know many, many people are still living there and I cannot tell you how much I admire their strength for doing that every day.
At one point, things calmed down a little bit in Damascus — they've been up and down but at least the year we spent, which was in 2012, there were quite a few times that were really scary.
For example, the kids would take the bus on their way to school and there'd be a huge bomb, and it took 20 minutes to know if the kids made it to school or didn't make it to school. And it became too much for me to be on my toes all the time.
And actually, many people at the time thought it would be a matter of months — no one thought it would be years. So in 2012, we made the decision to leave for a few months to Egypt — because it was cheap and easy to get a visa there — and then come back, nothing planned, nothing long-term, no big idea.
So at what point after leaving Syria did you decide to enroll for the course at Harvard?
Fifteen women were sponsored in the 2016 program and I was one of them. And every year they're planning to sponsor more women.
Did it feel good to be one of the 15?
It felt amazing, as getting accepted to study and then being faced with the possibility of not being able to take up the offer was really frustrating.
Although it was not an easy decision, to decide to only come for one year. All the upheaval has been difficult for my kids. We've had many changes over the recent years.
Why did you elect to study education?
Around 2% of all humanitarian aid is directed to education. And I completely understand that life, food, and shelter come before education in those situations. Even for parents who are living in conflict settings, your priority is not that they get an education, your first priority is for them to be safe so you're afraid to send your kid to school as you're worried whether they will come back or not.
But the thing is when you have conflicts that are more and more protracted and last on average for 17 years, some of the kids spend their entire childhood in conflict situations. So you have to find solutions for these people.
Those kids are the ones who will be rebuilding Syria and that's what makes it more personal and meaningful for me to study education.
And what specifically in education are you studying?
So the program I am on is called Technology, Innovation and Education, which, in a nutshell, explores how technology and media are used to advance education. Plus I have a background in computer science so that program is of relevance to my previous experience.
When thinking about the refugee crisis and refugee education and knowing that there are millions of refugees in neighboring countries with limited capacities unable to attend school due to logistical and practical barriers, I thought that technology might be a way to surpass those barriers.
I took a course in the fall called Education in Armed Conflict with Sarah Dryden-Peterson. I took it as an elective because it's actually not one of the modules on the technology course. And I found it fascinating.
It really changed the way I thought about conflict and the role of education in conflict settings. We always tend to think that education solves problems and it's enough to educate kids to ensure their future. Yet this course introduced new ideas, for example that sometimes education is at the root of the problem of the conflict.
And you must ask certain questions "What is being taught?" "Who is being excluded?" "Who is teaching?" Plus the challenges of providing education to marginalized populations who have absolutely no opportunity of doing anything with that education they are getting.
So what will your next steps be with your degree?
My interest is definitely in working on finding solutions for children in conflict settings. However, precisely how I will do that I have not fully made up my mind yet. I'm developing ideas that I want to take further and am interning with USAID ECCN (Education in Conflict and Crisis).
The last five years have been full of uncertainty for us. Having three kids being uprooted all the time is exhausting. So my priority is having something stable. Knowing that I'm staying in this particular place for at least three to five years and knowing what I'm doing.
While we were in Egypt, every year we weren't sure if we were staying or not. So stability is my priority, in terms of location, job, everything.
Does the currently suspended executive order factor into how you think about you and your family's future?
I am here on a Lebanese passport through my husband, so am not directly affected by the executive order. On the first iteration of the travel ban I was at risk because people with dual nationalities in the banned countries were restricted to enter the US.
However I am all Syrian. That is who I am. So it still affects me.
That said I've only felt welcome during my stay here. I've never at any point felt that there's any negative attitude towards me. But you do keep thinking 'do I want to be in a place where I'm not wanted?' So in that way, it does factor into my thinking.
For my country I dream of a day when this war stops. And the rebuilding starts. I hope for a day when refugees can make it back home. I'm aware that it's a hard and long process and that there's a lot to do.
Is there one thing you want our Global Citizens to take away from this interview?
For me, the war, the displacement, the uncertainty, made me feel that it's important to have a goal and something meaningful to follow.
And I feel that many of us don't have that when you're safe and secure and living everyday life, you don't have the urge to do something or to change things. And I regret it now, I feel that there were many things I could have done before, participating more in the society and the community, trying to make a difference to change the world.


Thelma Irene Mills, 90, of Washington Court House, Ohio passed away Sunday, January 20, 2019, at 4:45 a.m. at her home. She had been in failing health.
Mrs. Mills was born February 27, 1928, in Fayette County, Ohio to Richard Harold and Cora Audra Zurface Orr. She was a graduate of Atlanta High School and had lived most of her life in Fayette and Pickaway Counties.
She was a special accounts assistant at Farm Credit Services for 17 years before her retirement in 1997.
Mrs. Mills was a member of the Sugar Creek Baptist Church.
On July 29, 1946, she married Charles Wesley Mills, Jr. He preceded her in death on February 8, 2009. She was also preceded by an infant son, Richard L. Mills in 1948, and a sister, Louise Dean in 2007.
Mrs. Mills is survived by a son and daughter-in-law, David Joe and Cindy Mills of Washington Court House; a daughter and son-in-law, Lucinda Kay and Russell Smith of Pickerington; five grandchildren, David J. Mills, Jr., Heather Gordon, Zachary Smith and his wife, Melissa, Rusty Smith and Alex Smith; five great grandchildren; Skylar, Bryce and Becka Mills, Owen and Addison Smith; several great great grandchildren; a sister and brother-in-law, Patty and Harrylee Downing of Washington Court House and nieces and nephews.
The funeral will be Thursday, January 24, 2019, at 11 a.m. at the Sugar Creek Baptist Church, 3263 Old U.S. Route 35-NW, Washington Court House, with Mike Henry, pastor at church, officiating. Burial will follow in the Sugar Creek Baptist Cemetery.
Friends may call at the Kirkpatrick Funeral Home in Washington Court House Wednesday from 4-8 p.m.
In lieu of flowers, memorial contributions may be made to Ohio's Hospice of Fayette County, Ohio, 222 West Oakland Avenue, Washington Court House, Ohio 43160.
I will miss you very much. Always was fun staying with you and Uncle Junior when I was a kid. Cinda and I probably drove you crazy but you never complained. You will be greatly missed but I know you are happier now.. See you soon.
I am so sorry I was unable to attend Thelma's funeral. I did not learn of her passing until later today while I was in Columbus. I have very found memories of your Mother. She had such a great smile and brought great food to the church and neighborhood gatherings at Donahue's. She also helped my dad a great deal at the PCA office. She kept him in line. She was a great lady. I couldn't believe she had great great grandchildren. What a blessing. We will miss her. God Bless you and your family.


B cell translocation gene 1 (BTG1) has long been recognized as a tumor suppressor gene. Recent reports demonstrated that BTG1 plays an important role in progression of cell cycle and is involved in cellular response to stressors. However, the microRNAs mediated regulatory mechanism of BTG1 expression has not been reported so far. MicroRNAs can effectively influence tumor radiosensitivity by preventing cell cycle progression, resulting in enhancement of the cytotoxicity of radiotherapy efficacy. This study aimed to demonstrating the effects of microRNAs on the BTG1 expression and cellular radiosensitivity.
The human renal carcinoma 786-O cells were treated with 5 Gy of X-rays. Expressions of BTG1 gene and miR-454-3p, which was predicted to target BTG1 by software algorithm, were analyzed by quantitative polymerase chain reaction. Protein expressions were assessed by Western blot. Luciferase assays were used to quantify the interaction between BTG1 3′-untranslated region (3′-UTR) and miR-454-3p. The radiosensitivity was quantified by the assay of cell viability, colony formation and caspase-3 activity.
The expression of the BTG1 gene in 786-O cells was significantly elevated after treatments with X-ray irradiation, DMSO, or serum starvation. The up-regulation of BTG1 after irradiation reduced cellular radiosensitivity as demonstrated by the enhanced cell viability and colony formation, as well as the repressed caspase-3 activity. In comparison, knock down of BTG1 by siRNA led to significantly enhanced cellular radiosensitivity. It was found that miR-454-3p can regulate the expression of BTG1 through a direct interaction with the 3′-UTR of BTG1 mRNA. Decreasing of its expression level correlates well with BTG1 up-regulation during X-ray irradiation. Particularly, we observed that over-expression of miR-454-3p by transfection inhibited the BTG1 expression and enhanced the radiosensitivity. In addition, cell cycle analysis showed that over-expression of miR-454-3p shifted the cell cycle arrest from G2/M phase to S phase.
Our results indicate that BTG1 is a direct target of miR-454-3p. Down-regulation of BTG1 by miR-454-3p renders tumor cells sensitive to radiation. These results may shed light on the potential application in tumor radiotherapy.
Enhancing the radiosensitivity of resistant tumor cells is a common emphasis in the application of clinical radiotherapy . Tumor suppressor genes that regulate cell cycle can alter the radiosensitivity of cancer cells. For example, down-regulation of RB1 correlates with increased apoptosis induced by radiation in human breast cancer cells [2, 3]; the mutations in the p53 tumor suppressor gene enhances the radiosensitivity in oral cavity carcinoma cells ; and P21 deficiency is associated with a radiosensitive phenotype in colorectal carcinoma cells . The RB1, p53 and p21 are tumor suppressor genes and play cruical roles in controlling the progression of cell cycle [6, 7]. These findings indicate that cell cycle regulation genes may be intimately related to radiosensitization, therefore, could potentially be exploited in tumor radiotherapy. In this study, the human renal carcinoma cell, which has traditionally been considered to be radioresistant , was used as experimental model.
B cell translocation gene 1 (BTG1) is another tumor suppressor gene [9, 10]. The BTG1 protein, together with five additional proteins (BTG2/PC3/Tis21, BTG3/ANA, BTG4/PC3B, Tob1/Tob, and Tob2), comprise the BTG/TOB family of anti-proliferative genes involved in the regulation of cell growth . Expression of BTG1 not only inhibits the proliferation of cells but also leads to G1 phase cell cycle arrest in multiple types of cells [12–14]. Some studies have shown that BTG1 is involved in the general processes of cell cycle control and in cellular responses to stress , though a specific role for BTG1 in renal cell carcinoma has not been determined. In consideration of the common physiological function of tumor suppressor genes in controlling cell cycle, we propose that BTG1 may have a similar impact as RB1, p53, and p21 on the radiosensitivity of renal carcinoma tumor cells.
Certain members of the BTG/TOB family are known to be regulated by microRNAs (miRNAs) , which are small non-coding RNA molecules that suppress gene expression via sequence-specific interactions with the 3′-untranslated region (3′-UTR) of their target transcripts . For example, BTG2 was shown to be suppressed by miR-21 ; over-expression of miR-142-5p leads to down-regulation of BTG3; and TOB2 was shown to be a target gene of miR-322 . However, miRNA candidates that target BTG1 have not been identified.
The strategy of using miRNAs as therapeutic targets to enhance cellular radiosensitivity has been discussed before . miRNAs can efficiently modulate tumor radiosensitivity at four aspects containing DNA damage repair, radio-related signal transduction pathways, tumor microenvironment and apoptosis [22, 23]. Recent reports show that miRNAs can effectively influence tumor radiosensitivity by impeding cell cycle progression, resulting in enhancement of radiotherapy efficacy . For example, miR-21 can improve tumor radiosensitivity and promote apoptosis through negatively regulating the CDC25A expression and cell cycle progression . Up-regulation of miR-504 can reduce p53 protein level and affect cell cycle arrest and radiosensitivity mediated by p53 . With these precedents, we tested whether the BTG1 could be regulated by miRNAs upon irradiation and how the cellular radiosensitivity in renal carcinoma cells could be affected by the changes of miRNAs targeting BTG1.
Human renal carcinoma 786-O cells were cultured in RPMI-1640 media (GIBCO, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, MA, USA) at 37°C and 5% CO2. Irradiation was carried out by laboratory X-ray source (RX-650, Faxitron Bioptics, USA) . The dose rate was 0.8 Gy/min (100 keV, 5 mA). Cells were seeded into 12-well plates and grown to <70% confluence at the time of irradiation.
Cells were harvested and fixed as described previously . Prior to analysis, fixed cells were washed twice with PBS, treated with 100 μg/mL RNase A and 50 μg/mL propidium iodide (BD Biosciences, California, USA) for 20 min, and analyzed using FACS Calibur flow cytometry (Becton Dickinson, NJ, USA). Cell cycle distribution was analyzed with the FlowJo software package. DNA content of samples was measured with CellQuest (Becton Dickinson).
Western blotting was performed as described previously . Briefly, cells were collected and lysed with RIPA buffer solution (Beyotime, Haimen, China). Supernatants were collected at 10,000 g for 10 min at 4°C. The concentrations of samples were determined using a BCA protein assay kit (Pierce, IL, USA). Equal amounts of protein were loaded onto 10% SDS-polyacrylamide gels for electrophoresis, and proteins were transferred onto PVDF membrane by western blotting. GAPDH was used as loading control. Membranes were then blocked with nonfat milk and incubated overnight at 4°C with the following primary antibodies: anti-GAPDH (1:5,000, Santa Cruz), anti-BTG1 (1:1,000, Abnova, Taipei, Taiwan). Immunological complexes were detected by enhanced electrochemiluminescence (Millipore, Darmstadt, Germany) with either an anti-rabbit peroxidase (1:5000, Santa Cruz, Texas, USA), or an anti-mouse peroxidase (1:4000, Santa Cruz) antibody. The fold changes of protein levels were analyzed by the Image J software.
MicroRNA mimic and negative control mimic were synthesized and purified by RiboBio Co. (Guangzhou, China) . Transfections of the miRNA duplexes were performed with 40–60% confluent cells using Lipofectamine 2000 (Invitrogen, California, USA). The medium was replaced with new culture medium for 5 h after transfection.
Cell viability is calculated as the number of viable cells divided by the total number of cells within the grids on the hemacytometer. Cells which take in trypan blue are considered non-viable. Cells were harvested with trypsin, suspended in PBS and mixed with 0.4% solution of trypan blue stain (Invitrogen) after various treatments. Count at least 500 cells for calculation. The percentage of viable cells = [1.00 - (Number of blue cells/Number of total cells)]*100%.
To evaluate the activity of caspase-3, the caspase-3 activity kit (Beyotime) was used. Cells were collected and lysed with reaction buffer and the total protein concentration is 1-3 mg/mL. In the samples, activated caspase-3 cleaves substrate (Ac-DEVD-pNA) (2 mM) between DEVD and pNA, quantitatively generating pNA that can be detected using an ELISA reader at an absorbance of 405 nm. In the caspase-3 colorimetric calibration, the value of R2 should be greater than 0.999.
The clonogenic assay was conducted as described previously . Briefly, cells were harvested and an appropriated number of cells were seeded onto each of the 60 mm dishes to produce about 50–120 colonies. After 8–10 days incubation, the colonies were washed with 1 × PBS softly, fixed with 70% ethanol for 5 min and stained with 0.5% crystal violet for 3 min at room temperature. Colonies with more than 50 cells were counted.
The 3′-untranslated region (3′-UTR) of human BTG1 transcript was cloned downstream of the luciferase gene between the Xho I and Sal I sites of the pmirGLO dual-luciferase vector (Promega, WI, USA). A pmirGLO dual-luciferase vector containing one mutated seed sequences of miR-454-3p was constructed. The sequencing of constructed plasmids was verified by Shanghai Sangon Biotechnology Co. (Shanghai, China). 1.5 × 105 786-O cells in 12-well plate were co-transfected with 300 ng DNA (pmirGLO-3′ UTR constructs or derived mutants) and 30 nM of either miR-454-3p mimics using transfection reagent Lipofectamine 2000 (Invitrogen). Luciferase activity was measured 48 h later using the Dual Luciferase Reporter Assay System (Promega)  with a Tecan Infinite M200 Pro microplate reader (Tecan, Mannedorf, Switzerland).
For qRT-PCR, total RNAs were extracted from cultured cells using TRIzol Reagent (Invitrogen) according to the manufacturer's protocol. Reverse transcription and quantitative RT-PCR were performed according to the protocol of the qRT-PCR Detection Kit (Promega). All of the stem-loop RT primers were purchased from RiboBio Co. (Guangzhou, China) to detect miR-454-3p or U6. U6 was used as an endogenous control for miRNAs and GAPDH for coding genes. Other gene-specific primers were as follows: BTG1, 5′-TCCATAATCCATCCCCAAGA-3′ and 5′-GGATGCAATCCTGGACATTT-3′, SKA2, 5′-CCGCTTTAAACCAGTTGCTG-3′ and 5′-CTCTGCCGCAGTTTTCTCTT-3′, GAPDH, 5′-GTGGACCTGACCTGCCGTCT-3′ and 5′-GGAGGAGTGGGTGTCGCTGT-3′. Gene-specific primers were synthesized from Shanghai Sangon Biotechnology Co. (Shanghai, China).
All experiments were repeated at least three times, and data were presented as means ± SE. The statistical significance of the results was determined by Student's t-tests using Microsoft Excel (Microsoft Campus, Redmond, WA, USA).
Many tumor suppressor genes are recognized as responders to ionizing radiation (IR) . To investigate whether BTG1 functions as a responder to IR, we examined the protein levels of BTG1 in response to 5 Gy of X-rays in renal carcinoma 786-O cells by Western blot analysis. The protein levels of BTG1 were significantly increased 8 h after irradiation (Figure 1A). We also treated 786-O cells with DMSO and serum starvation to investigate whether BTG1 responds to other types of extracellular stressors and a similar increase in BTG1 levels was observed. Consistent with the results of Western blotting, qRT-PCR showed that the mRNA levels of BTG1 were increased by more than 2-fold upon treatment with irradiation (Figure 1B), DMSO (Figure 1C) or serum starvation (Figure 1D). These results indicate that, like other tumor suppressor genes, BTG1 is up-regulated in response to X-ray. Meanwhile, the BTG1 can also be induced by other sources of extracelluar stressors, including the treatments by DMSO and serum starvation.
BTG1 is induced in response to various extracellular stressors in 786-O cells. (A) Western blot assay of BTG1 at indicated time points after treatment of human renal carcinoma 786-O cells with 5 Gy of X-rays, DMSO (1.6%, v/v), and serum deprivation. GAPDH is used as a loading control. Fold changes of protein levels represent the average of three separate blots. (B-D) BTG1 mRNA levels were monitored by qRT-PCR at 0, 4, and 8 h after treatment of 786-O cells with (B) 5 Gy of X-rays, (C) DMSO (1.6%, v/v) and (D) serum starvation. Values were normalized to GAPDH. Results represent means ± SE of four independent experiments.
Because BTG1 is involved in the regulation of cell cycle progression and responds to IR, we examined the effects of BTG1 expression on the radiosensitivity of renal carcinoma 786-O cells. We transfected 786-O cells with the BTG1 expression vector pcDNA3.0-BTG1 in order to up-regulate the protein levels of BTG1. The efficiency of transfection was confirmed by Western blotting (Figure 2A). The protein levels of BTG1 increased notably at 24 h after transfection and lasted for at least three days.
Tumor cell sensitivity to IR correlates with BTG1 levels. (A) Western blot showing the up-regulation of BTG1 protein in 786-O cells after transfection with the BTG1 expression vector pcDNA3.0-BTG1 as compared to the control pcDNA3.0 vector and untransfected (blank) cells after 24, 48 and 72 h. GAPDH is used as loading control. Fold changes of protein levels represent the average of three separate blots. (B) The trypan blue exclusion assay was used to assess cell viability in 7860-O cells without transfection or after transfection with pcDNA3.0 vector or pcDNA3.0-BTG1 vector at 48 h after X-ray exposure. (C) The changes of caspase-3 activity of the three group of 786-O cells was analyzed by caspase-3 activity assay kit at 24 h after X-rays. (D) Survival fractions of cells transfected with the control pcDNA3.0 vector and pcDNA3.0-BTG1 vector in response to 0, 5 Gy of X-rays were measured by colony formation assay. (E) Photographs of colonies formed by 786-O cells after various treatments. The data (means ± SE) are representative for at least five independent experiments.
To estimate radiosensitivity, the cell viability was assessed by trypan blue dye exclusion assay . In this study, transfection of 786-O cells with pcDNA3.0-BTG1 significantly increased cell viability after radiation exposure (Figure 2B). To further confirm the effects of BTG1 on radiosensitivity, we measured the activity of caspase-3 (see Methods) which is an acknowledged effector among the caspase family members involved in apoptosis . In order to more accurately characterize the changes of caspase-3 activity among all treatments, we drew standard curve and tested the caspase-3 activity at 24 h after irradiation (Additional file 1A and B). At 24 h after exposure to 5 Gy of X-rays, compared to transfection of pcDNA3.0, treatment of 786-O cells with transfection of pcDNA3.0-BTG1 caused a significant decrease in caspase-3 activation (Figure 2C). A colony formation assay was performed to determine the cellular radiation sensitivity. The survival fraction of 786-O cells transfected with pcDNA3.0-BTG1 without irradiation was significantly lower than that transfected with pcDNA3.0, due to the fact that BTG1 is an anti-proliferative gene. It is worth noting that there was no statistically significant difference in the survival fraction of cells exposed to 5 Gy of X-rays, when the group transfected pcDNA3.0-BTG1 was compared with the pcDNA3.0 group (Figure 2D and E). We also used flow cytometry to quantify changes in the sub-G1 peak at 48 h. The peak of transfection of BTG1 expression vector was decreased upon exposure to IR compared to the untransfected (Blank) group and the pcDNA3.0 group (Additional file 1C). These results suggest that BTG1 expression protects cells from the cytotoxic effects of IR.
To further examine the role of BTG1 in radiosensitivity of 786-O cells, we used siRNA oligonucleotides targeting BTG1 to specifically knock down BTG1 expression. Transient transfection of BTG1 siRNA efficiently inhibited BTG1 expression in 786-O cells at 24, 48 and 72 h (Figure 3A). Cell viability assay showed that a treatment with BTG1 siRNA before irradiation promoted the decrease of cell viability induced by 5 Gy of X-rays (Figure 3B). Caspase-3 activity in cells treated with siRNA against BTG1 was increased by more than 3-fold as compared with cells treated with negative control (NC) group (Figure 3C). The survival fraction was also tested in 786-O cells after exposure to 5 Gy of X-rays. As expected, the colony formation efficiency of cells transfected with BTG1 siRNA was significantly lower than that transfected with its negative control (Figure 3D and E). Furthermore, transfection with BTG1 siRNA led to a specific increase in the sub-G1 peak in 786-O cells at 48 h after IR treatment (Additional file 1D). These results suggest that down-regulation of BTG1 significantly increases cellular radiation sensitivity in 786-O cells.
Down-regulation of BTG1 significantly increases cellular radiation sensitivity in 786-O cells. (A) The down-regulation of endogenous BTG1 protein following transfection with siRNA oligonucleotides against BTG1 (siRNA-BTG1) or nonsense small RNA oligonucleotides (negative control, NC) were compared to untransfected (blank) cells. GAPDH serves as loading control. Fold changes of protein levels represent the average of three separate blots. (B) The percentage of 786-O cell viability after transfection with siRNA oligonucleotides against BTG1 (siRNA-BTG1) or nonsense small RNA oligonucleotides (negative control, NC) was determined by trypan blue dye exclusion assay at 48 h after X-ray exposure. (C) Effects of nonsense small RNA oligonucleotides or siRNA-BTG1 over-expression on caspase-3 activity in 786-O cells without irradiation or with 5 Gy of X-rays. (D) Colony formation assay of 786-O cells transfected with NC or siRNA-BTG1. (E) Representative plates of survival fraction of 786-O cells after various treatments. The data (means ± SE) are representative for at least five independent experiments.
To analyze whether BTG1 can be regulated by miRNAs, we used starBase (sRNA target Base), a miRNA-mRNA interaction prediction software consisting of five different algorithms, to predict the miRNAs which may potentially target the 3′-UTR of the BTG1 mRNA . We selected the miRNAs that were predicted to target BTG1 by four or more algorithms for further analysis. These miRNAs included hsa-mir-130, hsa-mir-301a, hsa-mir-302, hsa-mir-454-3p, and hsa-mir-19b. Next, we constructed a series of pcDNA3.0 vectors that over-express these miRNAs and screened for those that regulate endogenous BTG1 levels at 48 h after transfection in 786-O cells. The BTG1 protein levels in samples transfected with pcDNA3.0-miR-454-3p displayed a significant down-regulation (Additional file 2F), indicating that miR-454-3p can target BTG1. A reproducible repression of endogenous BTG1 protein was also observed for vectors expressing miR-19b and miR-130, suggesting the possibility that multiple miRNAs target BTG1.
To determine whether miR-454-3p directly targets BTG1 via its 3′-UTR, the wild type and a mutant of BTG1 3′-UTR fragment with substitution in the seed region were synthesized and inserted into a luciferase report system (Figure 4A). The activity of the wild type reporter was significantly reduced by miR-454-3p mimic, whereas the miR-454-3p mimic had little effect on the mutant of BTG1 3′-UTR construct (Figure 4B). To further validate these findings, endogenous BTG1 levels were assessed in 786-O cells after transfection with miR-454-3p mimic. The miR-454-3p mimic specifically suppressed both the BTG1 protein and mRNA levels at 48 h post-transfection (Figure 4C and D). These results confirm that BTG1 is a target of miR-454-3p.
MiR-454-3p negatively regulates BTG1 expression. (A) Construction of a vector with either the wild-type sequence of the miR-454-3p binding site within the 3′-UTR of BTG1 (Wt BTG1 3′-UTR) or a mutated seed sequence (Mut BTG1 3′-UTR). The black box shows the predicted miR-454-3p binding site. The seed sequence is shown in red. (B) Luciferase reporter assay results are shown at 48 h following co-transfection of 786-O cells with the Wt BTG1 or Mut BTG1 vectors together with miR-454-3p mimic (miR-454-3p) or nonsense small RNA oligonucleotides (negative control, NC). Results (means ± SE) are representative for three independent experiments. (C) Western blotting was performed at 48 h after transfection with miR-454-3p or NC oligonucleotides. GAPDH was used as loading control. Fold changes of protein levels represent the average of three separate blots. (D) BTG1 expression is regulated by miR-454-3p at the mRNA level. QRT-PCR was conducted to quantify the expression level of BTG1 mRNA at 48 h after 786-O cells were transfected with miR-454-3p or NC oligonucleotides. (E) MiR-454-3p and BTG1 mRNA levels were monitored by qRT-PCR at the indicated time points after irradiation with 5 Gy of X-rays. U6 and GAPDH were used as controls. (F) SKA2 mRNA levels were monitored by qRT-PCR at the indicated times after irradiation with 5 Gy of X-rays. GAPDH was used as a control. The data (means ± SE) are representative for at least four independent experiments with similar results.
To determine whether endogenous miR-454-3p levels are regulated physiologically by IR, we assessed miR-454-3p expression in 786-O cells over a timecourse of exposure to 5 Gy of X-rays. While BTG1 mRNA levels increased, miR-454-3p levels decreased upon treatment (Figure 4E). It is worth noting that the BTG1 mRNA level decreased at 16 h after irradiation, whereas the miR-454-3p level increased.
The miR-454-3p gene is located in the first intron of the host gene, SKA2 (spindle and kinetochore-associated protein 2), which plays a critical role in proper mitotic progression, checkpoint silencing, and timely anaphase onset . To test whether the expression of SKA2 correlates with that of miR-454-3p, SKA2 mRNA was quantified by qRT-PCR. Similar to the results for miR-454-3p, the expression of SKA2 was down-regulated in 786-O cells after IR (Figure 4F), providing further verification of our findings. These results indicate that miR-454-3p regulates the expression of BTG1 through a direct interaction with the 3′-UTR of the BTG1, and that a decrease in its expression correlates with BTG1 up-regulation during IR treatment of renal carcinoma cells.
Our results thus far suggest that down-regulation of BTG1 can enhance the radiosensitivity of 786-O cells and that BTG1 is also a target of miR-454-3p. Consequently, we postulated that miR-454-3p may affect cell radiosensitivity. To assess the effects of miR-454-3p on radiosensitivity, we exposed 786-O cells to 5 Gy of X-rays following transfection with miR-454-3p mimic. Trypan blue assay showed that the cell viability of miR-454-3p mimic transfected group relative to the NC group was reduced by 20% (Figure 5A). Similarly, over-expression of miR-454-3p mimic was associated with more than 4-fold increase of caspase-3 activity at 24 h (Figure 5B). Clonogenicity of 786-O cells transfected with miR-454-3p mimic was significantly reduced after exposure to 5 Gy of X-rays (Figure 5C and D). The sub-G1 percentage increased evidently at 48 h as assessed by flow cytometry (Additional file 1E), indicating that miR-454-3p enhances IR-mediated apoptosis.Because cell cycle control is intimately involved in the induction of apoptosis during IR, we also examined whether BTG1 can regulate the cell cycle. 786-O cells were transfected with control pcDNA3.0 vector or pcDNA3.0-BTG1 expression vector, and the effects of IR on the cell cycle distribution were assessed by flow cytometry after 48 h. The majority of the cells were in the G1 phase prior to IR treatment. Exposure to 5 Gy of X-rays caused a strong G2/M arrest for cells transfected with control pcDNA3.0 vector (Figure 5E and F). Whereas, cells transfected with pcDNA3.0-BTG1 were resistant to IR-induced G2/M phase arrest after exposure to 5 Gy of X-rays. These results indicate that high levels of BTG1 can inhibit IR-induced G2/M arrest.To further understand the role of BTG1 in inhibiting IR-induced G2/M arrest and to assess the effects of miR-454-3p on cell cycle arrest, 786-O cells were transfected with either siRNA oligonucleotides against BTG1 or miR-454-3p mimic. Flow cytometry analysis showed that BTG1 siRNA and miR-454-3p had no significant effect on cell cycle distribution in the absence of irradiation (Figure 5G). Surprisingly, however, BTG1 siRNA and miR-454-3p mimic promoted an IR-induced S phase arrest and abolished the G2/M arrest in 786-O cells exposed to 5 Gy of X-rays (Figure 5H). These results indicate that over-expression of BTG1 overcomes the G2/M arrest induced by IR, but knockdown of BTG1 expression causes a switch of the arrest from the G2/M phase to the S phase. One can easily conjecture about that down-regulation of BTG1 would impede the activation of the G1 checkpoint and lead to damage accumulation during S phase and the consequent triggering of the S phase arrest.
MiR-454-3p enhances apoptosis and affects the G2/M arrest induced by IR. (A) The percentage of cell viability of 786-O cells at 48 h after transfection of nonsense control siRNA (negative control, NC) or miR-454-3p mimic (miR-454-3p) was calculated by trypan blue dye exclusion assay with or without exposure to X-rays (48 h after exposure to X-ray). (B) The activation of caspase-3 were tested at 24 h in 786-O cells after transfection with nonsense control siRNA (negative control, NC) or miR-454-3p mimic (miR-454-3p) with or without exposure to X-ray (24 h after exposure to X-ray). (C) The effect of transfection with miR-454-3p mimic on clonogenicity of 786-O cells was characterized by colony formation assay. (D) Photographs of the colony formation of 786-O cells after various treatments. (E and F) 786-O cells were transfected with the pcDNA3.0 vector or pcDNA3.0-BTG1 vector for 48 h were analyzed for cell cycle kinetics (E) without irradiation (0 Gy of X-rays) or (F) with irradiation with 5 Gy of X-rays (5 Gy of X-rays). (G and H) 786-O cells transfected with control siRNA (NC), siRNA oligonucleotides against BTG1 (siRNA-BTG1), or miR-454-3p mimic (miR-454-3p) for 48 h were analyzed for cell cycle kinetics (G) without irradiation (0 Gy of X-rays) or (H) with irradiation with 5 Gy of X-rays (5 Gy of X-rays). The percentage of total cells at G0/G1, S, and G2/M phases were determined. The data (means ± SE) are representative for at least five independent experiments.
In the present study, we identified miR-454-3p as regulator of BTG1 expression based on bioinfomatic software prediction and detailed experimental validation. The expression of BTG1 and miR-454-3p was shown to inversely correlate in 786-O cells upon exposure to environmental stressors, thus supporting a physiological role for miR-454-3p in regulating BTG1 expression. In addition to miR-454-3p, miR-19b was predicted to have two binding sites on the 3′-UTR of the BTG1 mRNA, one of which occupies the same site as the putative miR-454-3p target. A significant repression of endogenous BTG1 protein by miR-19b expression vector was also observed in 786-O cells (Additional file 2F). Therefore, we speculate that more than one miRNA may target BTG1, and these miRNAs may function antagonistically under specific physiological conditions. Future experimentation to address this possibility may unveil a regulation network of BTG1 regulation by miRNAs.
Our results show that the expression of SKA2 is regulated coordinately with the expression of miR-454-3p. Interestingly, miR-454-3p and a third putative BTG1 target, miR-301a, are encoded within the first intron of SKA2, whose depletion affects the cell cycle by inducing a metaphase-like delay . MiR-301a also down-regulates the expression of an inhibitor of NF-κB, Nkrf . NF-κB binds directly to the SKA2 promoter region to activate the transcription of SKA2 and miR-301a and also enhances persistent NF-κB activation to facilitate tumor growth [37, 39], thus suggesting a feedback loop to moderate SKA2 function. It is intriguing that miR-454-3p targets BTG1, which has a strong anti-proliferative ability, and suggesting an additional complexity of the growth regulatory function derived from the SKA2 locus. BTG1 has been suggested to inhibit NF-κB activities . Therefore, complex regulatory loop appears to regulate cell growth inhibition by BTG1, and it is likely that SKA2, NF-κB and multiple miRNAs are coordinated to control the BTG1 expression.
Little is known about the molecular mechanisms of control of the cell cycle by BTG1, and most of the information comes from interaction studies. BTG1 exerts cellular functions by interacting with PRMT1, HOXB9, and hCAF1, which regulate the expression of a number of genes involved in cell cycle control and progression [41–43]. CCNA2 (cyclinA2) physically associates with BTG1  and controls S phase by activating CDK2 kinases to initiate DNA synthesis . Ectopic over-expression of CCNA2 triggers checkpoint response and subsequently increases the S phase population in mammalian cells . We found that siRNA-mediated silencing of BTG1 led to S phase arrest after IR. Additionally, the S phase arrest can also be mediated by miR-454-3p. Furthermore, miR-454-3p is highly expressed in the S phase. The above negative regulation may suggest the association between CCNA2 and BTG1 in the control of the cell cycle progression of S phase. However, our results also show that over-expression of BTG1 in 786-O cells promotes a G1 arrest. Therefore, the link between miR-454-3p, BTG1 and cell cycle is likely complex and warrants further investigation to unravel the network of cell cycle regulators that are functionally associated with BTG1.
In conclusion, our study reveals that BTG1 is a direct target of miR-454-3p. Changes of its expression levels render tumor cells sensitive to radiation due to the role of BTG1 in cell cycle progression. Therefore, down-regulation of BTG1 via miR-454-3p presents a possible strategy to sensitize tumor cells to radiotherapy.
This work was supported by the Major State Basic Research Development Program of China (973 Program, No. 2010CB834201), the National Natural Science Foundations of China (No. U1232125, 31270895, 11335011 and 31370846). We thank Qingxiang Gao and Liang Peng for their kind help of technology of flow cytometry; Torsten Juelich, Wenfei Li for critical reading of the manuscript.
XW, ND, and WH did the most of the experimental work. JH and SX conducted the luciferase reporter assay. HP and JH performed qRT-PCR. JW and XW wrote the paper and designed the experiments. GZ provided intellectual input and helped with experimental design. All authors read and approved the final manuscript.


GB Taelwondo's first ever Olympic gold medallist Jade Jones has had another memorable week after she received an award as taekwondo's Olympic athlete of the year.
It completed a hat-trick of honours for the Manchester based 19-year-old who modestly dedicated her award to coach Paul Green.
"I couldn't have done it without PG," smiled the London 2012, -57kg champion. "But even though I have a gold medal, the hard work is still to come.


text:
Longtime Biden Aide Tony Blinken Will Be Secretary of State
GETTING THE BAND BACK TOGETHER
Other Obama-era officials returning to the national-security world include Avril Haines as director of national intelligence and Jake Sullivan as national security adviser.
Spencer Ackerman
Updated Nov. 23, 2020 6:13PM ET / Published Nov. 23, 2020 12:34PM ET
MANDEL NGAN
Antony Blinken, a longtime aide to President-elect Joe Biden, will be secretary of state, the Biden transition announced on Monday.
Blinken's appointment signals the geopolitical course Biden intends to chart in office: a restoration of liberal multilateralism, recentering traditional U.S. allies whom President Donald Trump placed on the margins. The task before Blinken and the Biden foreign policy team is reminiscent of the one shouldered by Hillary Clinton in the first year of Barack Obama's presidency, where Clinton sought to assure foreign allies that Obama would break from George W. Bush's bellicose unilateralism.
Assistance will come from a team that Blinken knows well. Biden's transition unveiled the other senior leaders of the next administration's national security and foreign policy leadership on Monday. They include Avril Haines, a deputy CIA director and deputy national security adviser who was considered Obama's left-most counterterrorism aide, for director of national intelligence; Jake Sullivan as national security adviser, who followed Blinken as then-Vice President Biden's national security adviser; and United Nations ambassador Linda Thomas-Greenfield, a career diplomat who served as assistant secretary of state under Blinken at Foggy Bottom.
John Kerry, Biden's longtime friend and ally, will also return to government next year. Kerry, Obama's final secretary of state, will take a new role as Biden's special envoy for combating climate change. And Alejandro Mayorkas, an immigrant who was deputy secretary for homeland security under Obama, will now be DHS secretary. Mayorkas led DHS's U.S. Citizenship and Immigration Services division and drew heat in that capacity after the agency's inspector general found that he intervened in matters benefitting companies affiliated with high-profile political allies, including former Virginia Gov. Terry McAuliffe and Tony Rodham, the brother of Hillary Clinton. Mayorkas vehemently denied any wrongdoing in the matter and was not sanctioned over the IG's findings.
"I need a team ready on Day One to help me reclaim America's seat at the head of the table, rally the world to meet the biggest challenges we face, and advance our security, prosperity, and values. This is the crux of that team," Biden said in a release announcing his choices.
However, Biden has yet to announce other crucial national-security roles, most importantly his selections for the Pentagon and CIA.
Blinken's appointment comes as no great surprise. He has been at Biden's side for a generation's worth of foreign policy: as a senior aide on the Senate Foreign Relations Committee, as vice-presidential national-security adviser, as deputy secretary of state in Obama's second term, and finally as principal foreign-policy adviser on Biden's campaign. There will be no doubt in foreign capitals that Blinken speaks for Biden, the traditional pitfall for a secretary of state, as Colin Powell learned during the Bush administration.
If there was any surprise in Biden's first wave of foreign policy personnel choices, it might have been Sullivan, whom several people close to the campaign said was also interested in a domestic-policy job.
Like Biden, Blinken is a committed multilateralist experienced in navigating congressional maneuvering over foreign policy, as demonstrated in his role narrowly selling the Congress on the 2015 Iran nuclear deal. During the campaign, Blinken outlined priorities for a Biden presidency, such as a return to the deal and to concerted action on climate change, both of which require marshaling geopolitical coalitions. He's also described rallying allies to counterbalance an assertive China on its economic and military ambitions while working alongside Beijing.
"I think you'd see a Biden administration having reestablished a relative strength in the relationship, then be able to engage China and work with China, in areas where our interests clearly overlap, whether it is again contending with climate change, dealing with global health and pandemics, dealing with the spread of dangerous weapons," Blinken said in July. "We're much better off though, finding ways to cooperate when we're acting from a position of strength than from a position of weakness."
Questions remain about how Biden's restorationist impulses will manifest in a world that looks substantially different than it did before Trump's presidency, as it follows a generation-long decline in American power through a potency-sapping war on terror; an explosion in international oligarchy born of a sharp spike in wealth hoarding by the globe's richest; and the end of America's unipolar moment as the Chinese economy is expected to surpass the U.S.' during Biden's tenure. As well, Blinken and Biden's moves to reenter the Iran deal will face extraordinary headwinds from the traditional U.S. allies they seek to cultivate, such as Israel, Saudi Arabia, and the United Arab Emirates.
As well, progressives have additional questions about Blinken's corporate connections. Along with Michele Flournoy, who is widely expected to become Biden's first defense secretary, Blinken ran the corporate consulting firm WestExec Advisors. Fast Company reported in 2017 that Blinken has advised the tech giants Google and Facebook, which have proven to be disinformation vectors.
Similar concerns from progressives surfaced this summer about Haines, owing to her work for the Trump-aligned datamining firm Palantir; her advocacy of CIA director Gina Haspel, implicated in CIA torture; and Haines' own role in pushing back against declassifications of the 2014 Senate torture report and exonerating CIA officials who spied on Senate investigators. At the same time, Haines consistently advocated for restricting drone strikes at the White House, though progressives have also noted she did not argue for their abolition.
Advisers to Biden's leftward primary rivals have described Blinken as open to their perspectives and have reacted positively to the prospect of his appointment.
"Rebuilding American diplomacy is a hugely important task for the next administration, so having someone who has the president's total trust, as Tony does with Biden, is critical for being our country's top diplomat," said Matt Duss, Sen. Bernie Sanders' foreign policy adviser. "Tony and his team made a point to engage regularly and seriously with progressives during the campaign, identifying some points of difference but also of strong consensus. I think having a secretary of state who can build on that consensus while still listening and engaging on the points of difference, as I think Tony would, will be an important asset."
Additional reporting from Lachlan Markay