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European Commission
Press Release
Brussels, 5 July 2013
Commission seeks public input on European approach to business insolvency
The European Commission launched a public consultation today on a European approach to business failure and insolvency, which puts the focus on helping sound businesses to survive and honest entrepreneurs to get a second chance while protecting creditors' right to get their money back. Procedures should be speedy and efficient, in the interest of both debtors and creditors, and should help safeguard jobs, help suppliers to keep their customers, and owners to retain value in viable companies. Disparities between national insolvency laws can create legal uncertainty and an unfriendly business environment. Key issues are the time required to discharge a debt, the conditions for opening proceedings, the filing of claims and the rules for restructuring plans. These can have an adverse effect on cross-border investment. Today's consultation will seek the views of stakeholders on how best to respond to these challenges.
"Europe needs a 'rescue and recovery' culture for viable businesses and individuals in financial difficulties," said Vice-President Viviane Reding, the EU's Justice Commissioner. "Honest business owners should get a second chance, so that insolvency does not become a life-sentence if things go wrong. SMEs, which are the backbone of our economy, are particularly important. I count on everyone interested to participate actively in this consultation so that we can gather the best ideas about how to respond to the challenge of business insolvencies."
The proposed modernisation of the EU's Insolvency Regulation – which dates from 2000 – is a key first step to bringing EU insolvency law into the 21st century (IP/12/1354, MEMO/12/969). At the same time, it is clear that a revision of the Regulation cannot by itself address the underlying challenges to cross-border insolvencies, such as the sometimes marked differences in national insolvency laws. That is why the Communication that the Commission adopted in parallel with the Insolvency Regulation proposal in December 2012 launched a reflection process on a new EU approach to business failure in light of diverging national insolvency rules. Today's consultation is the follow-up to this.
One of the questions raised in the consultation is the need to harmonise the "time to discharge" (the time it takes to close down a failed business), which is often regarded as crucial for the opportunity to restart a business. Currently, the time it takes to close down a failed business varies significantly throughout the EU, ranging from four months in Ireland to over six years in the Czech Republic and in some countries failed entrepreneurs cannot obtain discharge at all.
The consultation also addresses the rules governing the exercise of the profession of liquidators. Furthermore, stakeholders are asked whether the existing rules concerning directors' duties and liability in case of insolvency create problems in practice and if rules should be introduced at EU level to ensure that fraudulent managers who have been disqualified in one country are prevented from managing a company in another country. Finally, the consultation asks whether the legal uncertainty arising from the different conditions under which an act of an insolvent debtor which is detrimental to their creditors can be avoided before national courts has created problems in practice
This consultation is open until 11 October 2013 and can be accessed here: http://ec.europa.eu/yourvoice/ipm/forms/dispatch?form=InsolvencyTwo
Businesses are essential to creating prosperity and jobs. But setting one up – and keeping it going – is tough, especially in today's economic climate.
Figures reveal how urgent it is to take action in this field: half of all businesses do not survive the first five years of their existence. An average of 200,000 firms go bust in the EU each year, resulting in 1.7 million direct job losses. A quarter of these bankruptcies have a cross-border element.
As the European response, the Commission presented in December 2012 a package of measures to modernise insolvency rules (IP/12/1354, MEMO/12/969). The main elements of the package were a proposal to amend the Insolvency Regulation and a policy Communication on "A new European approach to business failure and insolvency".
European Insolvency Law is laid down in Regulation (EC) No 1346/2000 on insolvency proceedings (the "Insolvency Regulation"), which has applied since 31 May 2002. The Regulation contains rules on jurisdiction, recognition and applicable law and provides for the coordination of insolvency proceedings opened in several Member States. The Regulation applies whenever the debtor has assets or creditors in more than one Member State.
For more information
Public consultation on a new European approach to business failure and insolvency
European Commission – Insolvency proceedings:
Homepage of Viviane Reding, Vice-President of the European Commission and EU Commissioner for Justice:
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i don't celebrate the Valentines Day....but in my country is same with Britain ,..........in Albania is celebrated by all couples.....................
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Do the preparation task first. Then read the article and do the exercises to check your understanding.
February 14th is traditionally a celebration of love, so how do people in the UK mark the occasion?
Sending a Valentine's card to a loved one is a custom that started more than a century ago. Couples give cards to each other, but it is also traditional to send an anonymous card to anyone you secretly love. This practice is particularly common in schools and can be a source of great amusement and embarrassment as everyone tries to work out who sent a card and who has a secret admirer! Valentine's Day symbols on cards include hearts designs, doves, and the figure of the winged angel, Cupid.
The card might also feature a poem. Probably the most famous lines from a Valentine's Day poem are:
Roses are red, violets are blue,
Honey is sweet, and so are you.
There are some ironic variations on this poem such as:
Roses are red, violets are blue,
You look like a monkey and smell like one too!
Millions of people use digital means of creating and sending Valentine's Day greeting messages such as e-cards, social media posts or printable greeting cards. Texting your Valentine message is an easy (although maybe not very romantic!) option. Valentine's Day text messages include:
As well as cards, February 14th is also a day for giving gifts. Traditionally, men give chocolates or flowers to their wives or girlfriends. There are lots of websites offering advice on which Valentine's present to buy. Popular choices include red roses, a box of chocolates, romantic songs, a phone, jewellery, a photo frame or perfume.
Some people think that flowers and chocolates seem a little ordinary and so they look for a more unusual gift. For romantic people with plenty of cash, these gifts could include a helicopter flight, a balloon ride, the chance to test-drive a Ferrari or an all-day session at a health club.
Others, however, complain that Valentine's Day is yet another opportunity for ever-increasing commercialism. For true romantics, the gift of love is enough. Valentine's Day is a popular day to either propose or get married – you'd never forget your wedding anniversary!
We asked a few Brits about their views on Valentine's Day and here's what they said. Following the tradition of Valentine's Day, all the comments below are anonymous.
Love it! I always send cards to my mates – and my boyfriend, of course.
I would prefer to go without it. I don't want gifts or money or anything like that. Knowing that I'm appreciated and that I can make others happy is good enough for me.
I got a card last year – but I still don't know who sent it. I haven't got a girlfriend.
My mum sends me a card every year. It's sort of sweet but I think I'm getting a bit old for that now.
It's just really humiliating. I've never received a Valentine's card in my life. A day to switch off my phone because I know I'm not going to get any messages.
My boyfriend sent me a text message last year on Valentine's Day. I suppose that's kind of romantic but I'd rather have a present or even a real card.
I sent an anonymous Valentine's card to a boy in my class when I was younger. Then my brother told him it was from me. I was so embarrassed!
I'm single so it's definitely irritating. It's too commercial anyway.
If a girl likes me I don't need to waste money sending her a card just because it's a tradition.
Chocolates, roses, cards, presents. Bring it on! I love Valentine's Day!
Do you celebrate Valentine's Day? If not, is there a similar day in your country?
i don't celebrate the Valentines Day....but in my country is same with Britain ,..........in Albania is celebrated by all couples.....................
I'm from Montenegro. In my country, we celebrate Valentine's day, but on our language it's called St. Triphun ( Sveti Trifun ) and Valentine's Day ( Dan Zaljubljenih).
It's very important day for me now!
When you find true love, you don't care so much about Christmas, New Year or Easter, you care about Valentine's day!
I love it!
We celebrate it in my school, all wearing yellow t-shirts while sleeping and red sweaters on 14th of February. :D
I hope, I haven't made many mistakes, sorry!
Best wishes, Vasilije
Hello.. I'm from Albania. Here we also celebrate Valentine's Day.. It's the same as in Britain. All the lovers share with each-other different gifts to show their love.. such as chocolates.. flowers teddy bears etc.. but i think that the most important thing is to be near the one you love that special day :)
Hi everybody! I'm from Russia. I'm not Russian but we also celebrate this day (especially in school). There are "Angels of Love"(they're schoolgirls/boys dressed as angels) greet at the doors of our school in every Valentine's Day. We have such postmen for anonymous scholboys and girls)
here in albania we gave our boyfriends gifts an kissssssss
Hi everybody
I live in Egypt and we celebrate Valentine's day . Actually we don't celebrate it in 4th of November . In my opinion Valentine days not only for lovers . It's for friends too , so happy valentine days my friends .
Hello! I`m from Ukraine. We celebrate Valentine`s Day here. It`s very happy and romantic day for everyone. Also many peoples get cards, roses, candies and gifts))))))))
In my country South Korea, we celebrate Valentine's Day.
We give chocolates,cards and roses to person we love.
A lot of people propose to people they love that day.
I really love Valentine's Day. Yummy chocolates!!!!!!! Love it so much!!
I think Valentine"s Day is the most ramantic day among the whole year.
How cool!! A day for every lovers!!!!!!
in albania we gave our boyfriends gifts to told each other love
Although I was raised in England my family came originally from Argentina. I'm not sure if it was really celebrated there but it has never really been a big thing in our family. It is really a shame because I love receiving cards with beautiful quotes on Valentines Day. I would love be able to give them although I am never sure what to write. Thankfully there are lots of internet searches that help. Maybe it does not really matter where you come from so long as you embrace the feeling and fun of valentines day now.
I'm from India..and we also celebrate Valentine's day..but personally I have no valentine..so I feel dumb for this day
in our country Kazakhstan we celebrate Valentine`s day too.We give to each other diferrent presents like love cards,flowers and anything else!
In Montenegro lovers celebrate this day.It`s such a nice day :D
I'm from Kazakhstan. In my country some people celebrate Valentine's day, but it's not an official holiday. So we have a simmilar day in 15th of April - it called Kozy Korpesh-Bayan Sulu. It called so because of two lovers. It is very interesting story about them, about their unhappy love. In general, i love these holidays, and I try to celebrate both of them))
In Ukrain we celebrate Valentine"s Day too. It is one of hte most romentic days.
I live in Serbia. We celebrate Valentine's Day. The lovers celebrate this and they gives a presents. Usually the lovers goes to the restaurant and they have a romance night.
I live in Colombia and we don't celebrate Valentine's day. We celebrate love and friendship in September playing Secret Friend (In this game we exchange papers and then give a present to the person whose name we got.), and in other countries it is called Secret Santa
in Denmark were are celebrating all the Amerikan tradition so here in Denmark were are celebrating Valentines day!!!
Here in albania it is also celebrated. Even in here girls and boys give to each-other presents to show their love. Shops al over the towns are decorated with ballons and cupid hearts and there is red colour everywhere.
yes it's very true paula. i agree with you at all
Valentine's Day is celebrating in Montenegro too. But we rather call it St. Trifun's Day. The tradition is like in the UK: the guys or the girls give presents or carts to the people they have deep feelings for.
Two years ago I got a card from a guy who liked me. There was a poem in it. I was surprised, because I have never gotten such a type of card before. Last year I felt so lonely 'cause no one thought about me. :'-(
Wow. So many people from Montenegro. (Including me, of course...) . Anyway... I don't celebrate it...
Hi I'm from Montenegro too.
I'm from Montenegro too.. but I live in Ulcinj.. and people here don't celebrate Valentines day..
Honestly I would like to celebrate it.. even if this isn't a celebration for Muslims.. :D
I am from Yemen . and I am Musilm too. I want to know more tradition about UK culture.
Hi! I'm from Montenegro too :) where are you from? I mean,what city?
I'm from Italy and we celebrate Valentine's Day. I don't really love. It reminds me that I'm alone. But it's the most romant day for me :)
Here in Romania the Valentine's Day is a little bit exaggerated. There are enormous hanging heart balloons everywhere, red hearts and Cupidos on the shopwindows. The magazines are full with Valentine tests and ideas. But I still like it, because it's good to know that you are loved.
Valentine´s day is actually a very beautiful tradition brought by I do know who. Its purpose was to remind people to take notice of people you like and care about them. Making people know that they are precious for us makes them happy. But do we really need to get presents, gifts or treats to feel loved? I know it´s nice and lovely but wouldn ´t we appreciate other ways of saying "I love you"than getting a bear with such words printed on it? For example, just simple words coming out of our beloved ones´mouth, their hug or smile. Don´t be so materialistic. The best things in the world are those you can not touch but only feel with your heart.
Anyway, why should we express our attention and love to beloved ones only on this very day? If we love or like someone we should let them know it every single day of our lives and not just on the Valentine´s day.
As a result, people who are not seeing anyone or are single at that time might feel blue, sad and this just makes them feel even worse.
As far as I am concerned, I like this custom, but I see it much more as a commercial tradition than a celebration of love.
People think buying expensive things means giving love and the more expensive the more love you give.
There are many inventive companies which know how to play with people´s minds and therefore expolit it.
Don´t get tricked by them, but rather call your sweethearts out and take them for a long walk. :)
Valentine´s day in Slovakia is not so popular but also not so unpopular. Those who have partners celebrate it and those who don´t have anyone hate it. So, it´s hard to say.
Here in Albania the Valentine's day is in 14th..
I don't know if it is same at different countries...
© British Council The United Kingdom's international organisation for cultural relations and educational opportunities. A registered charity: 209131 (England and Wales) SC037733 (Scotland).


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"Eyesore" it is!
I resent the characterization of the owners of All Things Pawssible as innocent victims of a bumbling city bureaucracy [News, "Dogged daycare," July 31] (http://www.readthehook.com/93811/newsbiz-dogged-daycare-play-yard-shut-down). I further protest the characterization of the home of Sarah and Ernest Terrell as the "lone dwelling" in an industrial zone.
The Terrells are, in fact, part of a neighborhood: the Rose Hill neighborhood.
Quite recently, businesses have begun aggressively encroaching upon residential blocks in Rose Hill, causing much grief for property owners who've lived 40-50 years in the same spot. City officials should be applauded, not criticized, for looking closely at the Terrells' situation and trying to improve it.
The fact is, Rose Hill has always been mixed use, but all mixed uses are not created equal. The word "immaculate" does not spring to mind when looking at the play yard at All Things Pawssible from the vantage point of the Terrells' beautifully constructed deck– though "eyesore" does.
The area is ungraded and covered with unsightly brown mulch. Add in the thought of animal waste oozing onto one's yard, flooding one's basement, or being diverted, untreated, into the storm drains (and hence the city water system), and frankly, the city's caution seems justified.
The owners of All Things Pawssible have lots of defenders. They should; they provide a much-needed service. But no one's asking these defenders the most important question: Would you want that business next to your home?
To anyone who'd seen the view from Sarah Terrell's porch, the answer would have to be a resounding no!
Kendra Hamilton
President, Rose Hill Neighborhood Association


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Dear SOS: I get to Denver a few times a year, and at Elway's restaurant in the Ritz-Carlton, they serve a terrific charred roasted red pepper soup. Can you get the recipe?
Dave Wohl
Sudbury, Mass.
Dear Dave: Elway's was happy to share its recipe for this flavorful (and completely vegan) charred red pepper soup. I liked adding a little crumbled fresh cheese for garnish (which makes it not vegan but still vegetarian), but you can top it however you wish.
Elway's charred red pepper soup
Total time: 1½ hours
Servings: 6 to 8
Note: Adapted from Elway's at the Ritz-Carlton Denver.
4 red bell peppers
5 tablespoons vegetable or canola oil, divided
Salt and freshly ground pepper
2 onions
3 large carrots, chopped
3 large cloves garlic, coarsely chopped
1 (28-ounce) can whole peeled tomatoes
6 cups water
3 tablespoons sherry vinegar, or as desired
1. Heat the broiler. Core the bell peppers and place them, skin-side up, on a baking sheet. Drizzle over 2 tablespoons oil and season with a generous pinch of salt and pepper. Broil the peppers until they are well-charred but not burnt, 3 to 5 minutes depending on the heat of the broiler.
2. Heat a grill or grill pan over medium-high heat until hot. Meanwhile, peel the onions and halve them lengthwise. Coat the onions with 1 tablespoon canola oil and season with a pinch each of salt and pepper. Grill the onions on all sides until well-charred. Set aside until cool enough to handle, then trim off the root end and coarsely chop the onions.
3. In a large, heavy-bottomed pot, heat the remaining 2 tablespoons of oil over medium-high heat until hot. Add the onions and stir around the pot for a minute to soften, then add the carrots. Cook, stirring occasionally, until the onions are translucent. Stir in 2 teaspoons of salt and one-half teaspoon of pepper, along with the garlic. Continue to cook, stirring frequently, until the carrots are softened slightly and the garlic is aromatic, 1 to 2 minutes.
4. Stir in the tomatoes and peppers. Cook, stirring frequently, for about 10 minutes to develop the flavors. Add the water and bring the soup to a simmer. Reduce the heat to a gentle simmer, loosely cover the pot and cook for 45 minutes.
5. Blend he soup using an immersion blender, or in batches using a stand blender, and strain. Add the sherry vinegar, and season to taste with salt and pepper. This makes a generous 2 quarts soup.
Each serving: 136 calories; 2 grams protein; 13 grams carbohydrates; 3 grams fiber; 9 grams fat; 1 gram saturated fat; 0 cholesterol; 7 grams sugar; 165 mg sodium.


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‹ ›
We provide the ideal solution for people living abroad for at least 1 month: superbly furnished and equipped apartments, conveniently located in the most central districts.
Infinitely more comfortable than hotels or guest-houses, your own apartment will allow you to experience life as an inhabitant rather than as a tourist.
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We know neighborhoods - from the older and more established to the just developing, from close-in to the neighborhoods that are more secluded.


text:
On This Page
Public Health Statement for 2-Hexanone
September 1992
This Public Health Statement is the summary chapter from the Toxicological Profile for 2-hexanone. It is one in a series of Public Health Statements about hazardous substances and their health effects. A shorter version, the ToxFAQsTM, is also available. This information is important because this substance may harm you. The effects of exposure to any hazardous substance depend on the dose, the duration, how you are exposed, personal traits and habits, and whether other chemicals are present. For more information, call the ATSDR Information Center at 1-888-422-8737.
This Statement was prepared to give you information about 2-hexanone and to emphasize the human health effects that may result from exposure to it. The Environmental Protection Agency (EPA) has identified 1,177 sites on its National Priorities List (NPL). 2-Hexanone has been found in at least 15 of these sites. However, we do not know how many of the 1,177 NPL sites have been evaluated for 2-hexanone. As EPA evaluates more sites, the number of sites at which 2-hexanone is found may change. This information is important for you to know because 2-hexanone may cause harmful health effects and because these sites are potential or actual sources of human exposure to 2-hexanone.
When a chemical is released from a large area, such as an industrial plant, or from a container, such as a drum or bottle, it enters the environment as a chemical emission. This emission, which is also called a release, does not always lead to exposure. You can be exposed to a chemical only when you come into contact with the chemical. You may be exposed to it in the environment by breathing, eating, or drinking substances containing the chemical or from skin contact with it.
If you are exposed to a hazardous chemical such as 2-hexanone, several factors will determine whether harmful health effects will occur and what the type and severity of those health effects will be. These factors include the dose (how much), the duration (how long), the route or pathway by which you are exposed (breathing, eating, drinking, or skin contact), the other chemicals to which you are exposed, and your individual characteristics such as age, sex, nutritional status, family traits, lifestyle, and state of health.
1.1 What is 2-hexanone?
2-Hexanone, also known as methyl n-butyl ketone or MBK, is a clear, colorless liquid with a somewhat sharp odor. The liquid form can easily evaporate into the air as a vapor. It is a waste product of wood pulping, coal gasification, and oil shale operations. 2-Hexanone was formerly used in paint and paint thinner and in various chemical substances. However, since it was found to have harmful health effects, it is no longer made in the United States, and its uses have been restricted. There are no known major natural sources of 2- hexanone in the environment. When 2-hexanone is released to rivers or lakes, it dissolves very easily, and it may evaporate into the air in a few days. We do not know if 2-hexanone binds to soil. When 2- hexanone is released to the water, air, or soil, it is probably broken down into smaller products, possibly within a few days.
1.2 How might I be exposed to 2-hexanone?
You can be exposed to 2-hexanone if you live near an industry or hazardous waste site that releases the liquid into wastewater or the gas form into the surrounding air. These industries include coal gasification plants, oil shale operations, and wood pulping mills. We have no information on background levels of 2-hexanone in the environment.
2-Hexanone has been found as a natural substance in foods such as cheese, nectarines, nuts, bread, and chicken muscle. We do not know the levels of 2- hexanone in these foods. 2-Hexanone has been found in milk and cream at levels up to 0.018 ppm (0.018 parts of 2-hexanone in one million parts of liquid). These levels are far below the levels that have caused harmful effects in animals. It has also been found in drinking water and soil near hazardous waste sites. Exposures at these sites may take place if you drink the contaminated water or bathe in it, if you get contaminated soil on your skin, or if you breathe the contaminated air.
1.3 How can 2-hexanone enter and leave my body?
2-Hexanone can enter your body when you breathe its vapors, eat food or drink water that contains it, or when you come in contact with it through your skin. When 2-hexanone is breathed in, about 75% of it is taken up and remains in the body unchanged or as a breakdown product for an unknown length of time. If it enters the body by mouth, about 65% of the chemical leaves the body slowly (in about a week), either unchanged or as breakdown products, in the breath and urine. The rest may either stay in the body or may leave the body slowly through the breath or urine. One of the breakdown products, called 2,5-hexanedione, may be responsible for the harmful effects on the nervous system (see Section 1.4). When 2-hexanone gets in through the skin, some leaves the body through the lungs and urine within a few hours. We have no information on how much stays in the body or for how long. If you live or work near a hazardous waste site, you may be exposed to 2-hexanone in the air that you breathe or in the water you drink or bathe in, if it contains small amounts of this chemical.
1.4 How can 2-hexanone affect my health?
The most important health concern for humans from exposure to 2-hexanone is its harmful effects on the nervous system. These effects were seen in workers who were exposed to 2-hexanone for almost a year. The major effects were weakness, numbness, and tingling in the skin of the hands and feet. Similar effects were seen in animals that ate or breathed high levels of 2-hexanone; these effects included weakness, clumsiness, and paralysis.
We do not know whether 2-hexanone can cause cancer or birth defects. In one study, when pregnant rats were exposed to 2-hexanone in the air, fewer offspring lived after birth, and those that did survive had low birth weights.
Many of the studies in which the health effects of 2- hexanone in humans or animals were reported did not use pure 2-hexanone. Therefore, we do not know whether the results were caused by 2- hexanone itself or by the other chemicals in the mixture.
1.5 Is there a medical test to determine whether I have been exposed to 2-hexanone?
Tests can be used to find out whether you have recently been exposed to 2-hexanone. The tests measure levels of 2-hexanone or its breakdown products in blood or urine. These tests require special equipment and are done in a special laboratory, so they are usually not available in a doctor's office. However, these tests cannot be used to predict whether harmful effects will occur.
1.6 What recommendations has the federal government made to protect human health?
The federal government has set certain regulations and guidelines to help protect people from the possible health effects of 2-hexanone in the workplace. The Occupational Safety and Health Administration (OSHA) has set a limit of 5 ppm (5 parts of 2-hexanone in 1 million parts of air) as an average exposure level to this chemical over a 40- hour work week. The American Conference of Governmental Industrial Hygienists (ACGIH) has made the same recommendation. The National Institute for Occupational Safety and Health (NIOSH) recommends an even lower limit, 1 ppm, as an average exposure during a 10-hour period.
Agency for Toxic Substances and Disease Registry (ATSDR). 1992. Toxicological profile for 2-hexanone. Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service.
Where can I get more information?
If you have questions or concerns, please contact your community or state health or environmental quality department or:
For more information, contact:
Agency for Toxic Substances and Disease Registry
Division of Toxicology and Human Health Sciences
1600 Clifton Road NE, Mailstop S102-1
Atlanta, GA 30333
Phone: 1-800-CDC-INFO · 888-232-6348 (TTY)
Email: Contact CDC-INFO
ATSDR can also tell you the location of occupational and environmental health clinics. These clinics specialize in recognizing, evaluating, and treating illnesses resulting from exposure to hazardous substances.
- Page last reviewed: January 21, 2015
- Page last updated: January 21, 2015
- Content source: Agency for Toxic Substances and Disease Registry


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State - INACTIVE
Modesto Police Department Crime Map (April 12-May 9)
Here is a look at Modesto Police Department crime maps and statistics from April 12-May 9 Click on the crime to see if one occurred in your neighborhood.
Robbery: Defined as a theft from a person or location by means of force or fear. Typically, this would be a suspect armed with a gun or a knife or by using some force to complete the crime. In essence it means someone gave up their own personal property because they feared for their life or injury.
Vehicle Burglary: Requires forced entry into a vehicle (window smash, lock pick etc)
Residential Burglary: A crime where someone entered a residence to commit a theft or any other felony. It does not require forced entry.
Commercial Burglary: Same as above but the crime occurred at a commercial establishment.
Auto Theft: A crime where someone takes a vehicle without permission.
Auto Theft Recovery: Location a vehicle was recovered. It could have been occupied or simply found on the street or alley. The vehicle may have been originally reported to a different law enforcement jurisdiction but the map will reflect all recoveries not just those originally reported stolen to MPD.
For more information on reporting a crime, go to the Modesto Police Department web site or Crimestoppers.
Source: Modesto Police Department


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Fleur Alston - Kit and Caboodle 8
Fleur Alston - Kit and Caboodle 8
C-type print, 15x15cm, edition of 15
The central point of a Mandala is the space within which the viewer can identify themselves, in the centre of the circle is the highest deity or abstract concept. By placing a dead animal in that centre there is a possibility to shake some individuals perception of themselves and their whole, instinctively at a sub-conscious level. Something could be understood and revealed in that experience of the unfolding pattern. As the Mandala is an integral part of our collective consciousness that is shared by every person who ever lived. The varying reactions to my work make me believe that people are now so far removed from the cycles of the living world that they either fear that unknown exponentially or crave intimate knowledge of it and seek a return to a stronger connection with the natural cosmos. In some ways my work is exploring this disorder and departure from nature. I believe that our wholeness with the natural world around us is broken and by looking at the Mandala we can open our minds to what it actually means to be a human, as part of a natural environment, at this time in our history.


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Tuesday, July 17, 2012
An Interview with Marvin Kaplan - Part Four
4 comments:
This whole interview was a joy to read. Thanks for sharing.
- July 17, 2012 at 9:00 AM
Thanks alot Kliph, again this might be my favourite one of yours and that is saying alot.
Only thing is that I don't remember Gale Gordon being in "It's a Mad Mad Mad Mad World"
What kills me about the extra segment on the DVD it's so much Jerry Lewis and he only had a few seconds in the movie.
I always felt Marvin was a great, underrated actor- now getting to read what a nice person he is, added to it.
- July 17, 2012 at 1:41 PM
A fantastic raconteur. And of course he just appeared in L.A. on a panel before the showing of a 70mm copy of "It's a Mad Mad Mad Mad World" that I'd kill to see, and there's a great youtube clip from that appearance with Kaplan saying exactly what he did about the film in this interview.
- July 17, 2012 at 4:38 PM
Mr. Kaplan exemplifies the two qualities most underrated in Hollywood: decency and sanity.
- July 25, 2012 at 10:43 AM


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A warm welcome to all current and future Shenzhen expats! If you want to get in touch with others in the same boat as you then you're in the right place; InterNations brings together all you lovely Shenzhen expats! When you join our community you have access to the advice, experience, and more importantly friendship, of hundreds of expatriates just like you. Whether you're considering working in Shenzhen and want practical advice on making the move, or you're already settled and looking to meet like-minded people, InterNations is your go-to site for everything you could need. We understand the worries, and many questions, you as an expat will have, but with InterNations you can rest assured knowing that you have an entire community of people who are always ready and waiting to help.
With InterNations, you never stand alone. Whether you move to another city or go on a business trip somewhere — our communities are there to make you feel at home wherever you are in the world. Feel free to also check out our other vibrant InterNations Communities like the ones in Brussels or Singapore.
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Voted one of the most liveable cities in China, Shenzhen is a modern, fast-growing city where you will find all of the home comforts that you could wish for. Shenzhen is a hugely popular destination with domestic tourists thanks to its fashionable shopping malls, restaurants, and bars, as well as its large number of parks and attractive beaches. The city is also home to many theme parks including Window of the World, the Folk Culture Village, and Minsk World, so during your downtime you will have plenty of options for day trips. If you're a sports lover then there is something for you too, the city boasts two stadiums, one of which is home to the local football team, Shenzhen Ruby FC. Getting from A to B is simple as the city has a fantastic metro system which offers fast, reliable and cheap transportation. Shenzhen's location is perfect if you're hoping to explore more of China; it's very close to Hong Kong and also has excellent links to Guangzhou, Shanghai, and Beijing.
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Remember me? McKinney scrambles to connect with voters
Chris Christie won't be making any cameos.
Not at this diner, where one of the regulars is lost in a bowl of chocolate ice cream and this week's issue of The Weekly Standard.
And not for John McKinney, the Republican who is running for governor without the party endorsement or the out-of-state fundraising fireworks.
Instead, on a quiet Tuesday morning, retiree Frank Perillo motioned for McKinney, the state Senate minority leader, to approach the lunch counter at the Plaza Diner in Shelton.
He was eager to size up McKinney, who treated Tuesday's special election in the 122nd House District as an opportunity to connect with and, if not, remind Republicans of his mere presence in the race and his vision for a Connecticut of spending cuts and renegotiated labor contracts.
It was a welcome entree for a candidate virtually written off by the national party and standard bearers such as Christie, who stumped the previous day in Greenwich for the GOP's endorsed front-runner, Tom Foley.
"Is there a primary?" Perillo asked Tuesday, telling McKinney, "I was a Republican once."
With exactly three weeks until GOP primary voters choose their nominee, McKinney was scrambling.
The day began with the debut of a biting television ad aimed at Foley and ended with McKinney celebrating the somewhat belated opening of his Fairfield campaign headquarters, which occupies the same building used by his late father, Stewart B. McKinney, in his final congressional run.
"It is surprising to see the state party sort of take a partisan position in a primary," McKinney said. "I've never seen it before."
State GOP Chairman Jerry Labriola Jr. maintained he is a neutral broker in the primary tussle.
"I have not made any personal endorsements to date," Labriola said. "However, our party gave a strong endorsement to Tom Foley at our state convention in May. We are taking reasonable steps to put our party in a position for victory in the fall by building out our ground game infrastructure, which will benefit either candidate should they become the nominee."
McKinney shrugged off being omitted from the invite list for Monday's state GOP fundraiser with Christie and Foley, the party's 2010 nominee for governor.
"I've been working for the party, as I said earlier, for my entire life," McKinney said. "I've not been asked to go solicit Chris Christie or bring other Republican figures to Connecticut."
Joining McKinney on the trail Tuesday was his running mate, David Walker, the U.S. comptroller general under presidents Bill Clinton and George W. Bush.
"He's playing prevent defense when it's about halftime," Walker, a Bridgeport resident, said of Foley.
Foley, a private equity manager from Greenwich, has limited the number of debates he will participate in before the Aug. 12 primary. The strategy puts McKinney, who was first elected in 1998 and is steeped in government policy, at a disadvantage.
McKinney and Walker visited several polling places in the 122nd District, where voters were charged with electing a successor to fill out the term of Republican Larry Miller, who died May 31. The district includes parts of Stratford, Shelton and Trumbull.
In campaign ads, McKinney and Foley have painted each other as clones of Democratic Gov. Dannel P. Malloy. In McKinney's latest entry in the television blitz, he warned that Foley's plans to hold spending flat and not reopen contracts with state employee unions would hamstring the state.
"I don't think Republicans are going to be able to beat Dan Malloy by being vague and defensive," said McKinney, top Republican in the Democratic-dominated Legislature.
Larry Cozzolino, who is not affiliated with a political party and would have to enroll as a Republican to vote for McKinney, said McKinney's conversational demeanor was refreshing.
"I was waiting for the campaign pitch," said Cozzolino, a Clinton resident.
Perillo, the uncle of GOP state Rep. Jason Perillo, of Shelton, said he was open to supporting McKinney, but would have to check his registration.
"You can switch all the way up until Aug. 11," McKinney said.
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void loop(void) {Serial.println(F("Loopy")); Serial.println(SdPlay.isStopped()); if (SdPlay.isStopped()) { SdPlay.setFile("Hum.afm"); SdPlay.play();} while(!SdPlay.isStopped()) { SdPlay.worker(); }}
$ sox inputfile -b 8 -r 64000 -c 1 -t raw outputfile
void loop(void) { Serial.println ("running"); //delay (5); if (!SdPlay.isPlaying() ) SdPlay.play(); // Let the worker work until playback is finished SdPlay.worker(); }
Fantastic library, got it working within few minutes! Is it possible to play Audio in parallel while Arduino does other things? I wasn't able to figure out how. I wanted to see if I can dump WaveShield in favor of this library. WaveShield allows me to play Audio regardless of other things I'm doing, which is essential for my Alarm clock (shows time, blinking dots while playing alarm audio file).I did some testing with SimpleSDAudio and it looks like if delay between calling SdPlay.worker() greater than 5 milliseconds, playback slows down and becomes choppy... Even calling Serial.println ("test") slows down playback noticeably. Here's example of code from loop:Code: [Select]void loop(void) { Serial.println ("running"); //delay (5); if (!SdPlay.isPlaying() ) SdPlay.play(); // Let the worker work until playback is finished SdPlay.worker(); }Let me know if there's a trick to it Maybe buffer can be auto-filled by Interrupt?
Don't quote me on this, but from looking at the code, I think you could do such given that you make a call to the SdPlay.worker() function often enough to keep the buffer filled. I'm not sure how much of the processor is left once playing audio, but it seems you could squeeze in a few other non time dependent things. (Like adding up time or checking for a button and updating its state...simple things)From what I have gathered, the SdPlay.worker() function reads the next sector of the sdcard into a buffer. So if you call it often enough, you should be okay, just don't ask for anything that needs a ton of resources while playing audio.Feel free to correct me if I'm wrong, but this is what I have gathered so far.Edit: Just from playing with the serial I can see what you are saying, but I haven't come up with a solution.Edit2: If you hear the skip when using serial at 9600, move it to a higher speed. I'm running at 115200 just to see, and there isn't a noticeable skip when using the serial commands in the demo.
HiI'm exited about this library. I might have a troublesome SD Card module.I keep getting an error code: 49. I cant find out what code 49 is.If I use Arduino's standard SD example listfiles, I get a correct list of files on the SD card. The SdFat library from http://code.google.com/p/sdfatlib/ also read the SD Card with the same wiring. So the wiring must be correct. And I use SdPlay.setSDCSPin(10); as pin 10 is my CS pin.Then I commented the line: SPSR |= (1 << SPI2X); Same result.My sd module is this one http://www.lipoly.de/index.php?main_page=product_info&products_id=213383 I really hope someone can with this problem.EDIT ----It was not the SD Card Module but the SD Card itself. I tried with another one. Now it works.
Hi, I will try answer some of your questions:@timberwolf9:Why the argument -r 64000? If you use a 16 MHz Arduino, it should be 16 MHz / 256 = 62500. But anyway, the sound is just a little bit to slow then...@bratan & timberwolf9:I will tell you some internal details about my library: The library uses a ring buffer of 1024 bytes when no work buffer is set manually. As the buffer is filled in one sector blocks of 512 bytes each, the buffer became a ping-pong buffer in most cases. With 512 bytes, at 8-Bit/Mono/62.5kHz that is about 8ms time, because the data reading from sd-card also takes some time, your observation of 5ms max delay between worker()-calls is very realistic.
I also thought about calling the worker-function automatically in a timer interrupt. This should be possible, but only if that interrupt is set to non-blocking to guarantee the main audio interrupt the maximum priority. Maybe I will try to implement such in a next version of my lib, so that calling worker() is not necessary then anymore.
To your question about other Audio-Playback-Shields: As far as I have seen it, all other audio-shields contain a dedicated processor that reads the audio-data from storage and feed it to an audio-DAC. For a good sound the DAC must be feed with low jitter at the audio rate of the audio material, otherwise you may recognize audio distortions like slowdowns or stuttering. With a dedicated processor it doesn't matter what your Arduino does while audio is playing, but my library is for all those who don't want to spend extra money to such a thing. SimpleSDAudio get the most out of the AVR's by pushing the Arduino processor strongly to its limits - that's the price you have to pay if you want nice audio from that little thing.


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Articles (ISV)
Browse all publications by
Recent submissions:
Low Resources Machine TranslationCarl, Michael; Maite, Melero; Badia, Toni; Vandeghinste, Vincent; Dirix, Peter; Schuurman, Ineke; Markantonatou, Stella; Sofianopoulos, Sokratis; Vassiliou, Marina; Yannoutsou, Olga (, 2008)[More information][Less information]
Abstract: METIS-II was a EU-FET MT project running from October 2004 to September 2007, which aimed at translating free text input without resorting to parallel corpora. The idea was to use 'basic' linguistic tools and representations and to link them with patterns and statistics from the monolingual target-language corpus. The METIS-II project has four partners, translating from their 'home' languages Greek, Dutch, German, and Spanish into English. The paper outlines the basic ideas of the project, their implementation, the resources used, and the results obtained. It also gives examples of how METIS-II has continued beyond its lifetime and the original scope of the project. On the basis of the results and experiences obtained, we believe that the approach is promising and offers the potential for development in various directions. URI: http://hdl.handle.net/10398/8037
Files in this item: 1METIS-II.pdf (503.5Kb)
Carl, Michael (, 2008)[More information][Less information]
Abstract: One of the aims of the Eye-to-IT project is to investigate the possibility of using eye-tracking devices for detecting situations of targeted help for human translators. A prerequisite for automated assistance in human translation is the understanding and modelling of reading behaviour, the ability to follow human eye movements and to map gaze sample points — the output of eyetracking devices — onto words and symbols fixated. Within the Eye-to-IT project we currently use a so-called "Gaze-to- Word Mapping" (GWM) device (ˇSpakov 2008) that first computes possible fixations from sequences of gaze sample coordinates and then maps the fixations on the words which are likely to be fixated. This paper suggests an alternative framework of a probabilistic gaze mapping model for reading, in which fixations on textual objects are directly computed from the gaze sample points. The framework integrates various knowledge sources with the aim to compute the most likely fixations on words and symbols on the basis of the available data. URI: http://hdl.handle.net/10398/8043
Files in this item: 1CLS.pdf (186.2Kb)
Carl, Michael; Doherty, Stephen; O'Brien, Sharon (Preprint, 2010)[More information][Less information]
Abstract: Eye tracking has been used successfully as a technique for measuring cognitive load in reading, psycholinguistics, writing, language acquisition etc for some time now. Its application as a technique for automatically measuring the reading ease of MT output has not yet, to our knowledge, been tested. We report here on a preliminary study testing the use and validity of an eye tracking methodology as a means of semi- and/or automatically evaluating machine translation output. 50 French machine translated sentences, 25 rated as excellent and 25 rated as poor in an earlier human evaluation, were selected. 10 native speakers of French were instructed to read the MT sentences for comprehensibility. Their eye gaze data were recorded non-invasively using a Tobii 1750 eye tracker. The average gaze time and fixation count were found to be higher for the "bad" sentences, while average fixation duration and pupil dilations were not found to be substantially different between output rated as good or bad. Comparisons between BLEU scores and eye gaze data were also made and found to correlate well with gaze time and fixation count, and to a lesser extent with pupil dilation and fixation duration. We conclude that the eye tracking data, in particular gaze time and fixation count, correlate reasonably well with human evaluation of MT output but fixation duration and pupil dilation may be less reliable indicators of reading difficulty for MT output. We also conclude that eye tracking has promise as an automatic MT Evaluation technique. URI: http://hdl.handle.net/10398/8045
Files in this item: 1SubmissionforMT_dohertyobriencarl.pdf (226.2Kb)
Quantifying alignment units with keystroke dataCarl, Michael (, 2009)[More information][Less information]
Abstract: The paper discusses a method to triangulate process and product data. We suggest converting Translog data into a relational format which contains both process and product data. We outline how this representation allows us to retrieve and correlate the various dimensions of the data more easily. The concept of Alignment Unit (AU) is introduced and contrasted with that of Translation Unit (TU). While AUs refer to translation equivalences in the source and target texts of the product data, TUs refer to cognitive entities that can be observed in the process data. With an (almost) exhaustive fragmentation of the source and target texts into AUs, we are able to distribute and allocate the entire set of keystroke data to appropriate AUs. Using the properties of the keystroke data, AUs are quantified in a novel way which enables us to visualise and investigate the structure of translation production on a fine-grained scale. URI: http://hdl.handle.net/10398/8040
Files in this item: 1keystrokes.pdf (940.0Kb)


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Next month, the General Assembly returns to Richmond, and transportation funding will be on the agenda. Fairfax County continues to consider funding as well. It's an ongoing issue—in large part because the state, which owns nearly every road in the county, has for years under-funded transportation, and more recently has stopped, permanently, all secondary road construction.
Recently, the county undertook an effort to engage and survey its residents to gauge what level of support exists for generating additional transportation funding. The county held a series of meetings in every supervisor's district and an online survey reported that 85 percent of respondents favor additional transportation spending. The survey also showed a reluctance to pay for the spending that so many support. Most respondents favored having developers pay more, and a vehicle rental tax and hotel tax were two other popular items. However, revenue sources that would fall on the population at large, such as a gas tax or sales tax, were less popular, as were user fees such as car registration and titling fees. It seems we want more roads, but for someone else to pay the bill.
Asking developers to pay for transportation makes sense, and we already require significant transportation dollars from them. We also require that funds be contributed for schools and parks, and we impose stringent, expensive environmental and stormwater retention standards. We also ask developers to build housing affordable to the lower and middle class, so all people can find appropriate housing. We could excuse developers from these various requirements solely in favor of transportation funding, but that would limit our ability to accomplish other important goals.
And if we impose too many costs on developers, commercial developers in particular, we run the risk of them building elsewhere or not at all, leaving the county unable to provide the same level of services residents have come to expect without increasing residential property taxes. This is because commercial development, unlike residential development, generates more revenue than it consumes in government services. This revenue permits the county to provide significant government services without placing the whole burden on the homeowner.
In our county discussions, some assert only "the state" must pay for transportation, as if "the state" is some outside source as well. In reality, who is "the state" if not us? The fact of the matter is that whether increased transportation funding comes through the state or the county, or through user fees such as tolls, the ultimate source of the money is the same—the people of Fairfax County. And we all know that whenever a statewide funding formula is concerned, we receive back from Richmond only a fraction of what we contribute.
Do we want to pay for transportation improvements, or are we going to tolerate increased congestion and declining road maintenance? It's a fair question and a stark choice. But beware of those who say you can have good things without paying for them. There is no free lunch on transportation. If we don't want to pay, that's ok—but then the responsibility for the results lies with us as well.


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i've read speculation about the man down being someone hurt. but what if just refers to the fact that the losing team votes out a man first. someone makes the comment that we are a "man down" from the other team? *(^
fwiw~ found at sucks Published by: Carlos - Julio 29, 2005 04:57 A.M. the film of the series of Guatemala of the survivor is around extreme in the next week. The CBS is very contented how it has been. We have heard that five are lazy with the past only gone before were called Marjorie. They are beginning now to clean upon the village of the equipment. # Published by: Almagimo - Julio 31, 2005 01:04 p.m. the program of Guatemala of the Survivor finishes finishing film in date yesterday. There is portion of the enthusiasm of the equipment of the demonstration and the CBS is very contented with the new survivors. The cleaning now goes ignition and we told on a rise from the tourism the area in a moment. The final survivor to win is a named woman Lynda. # Published by: Almagimo - August 5, 2005 11:28 A.M. www.guate360.com/blog/archivo/000599.php
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A, Immunoblots showing the protein expression of tumor protein p53 (TP53), B-cell CLL/lymphoma 2 (BCL2), and vinculin (VCL) in MEG-01 cells transfected with microRNA 15a (miR-15a), microRNA 16 (miR-16), their combination, or their antisense oligonucleotides. Cotransfection of miR-15a and miR-16-1 was performed at the same concentration of oligonucleotides per each; therefore, the total amount of transfected microRNAs was doubled with respect to the other lanes. VCL is the normalization standard used to normalize the amount of proteins loaded to each well. The numbers above the blots indicate the intensity of the band expressed as a ratio "gene product (TP53 or BCL2)/VCL" and normalized to "scrambled." B, Immunoblots showing the protein expression of TP53, cyclin-dependent kinase inhibitor 1A (p21, Cip 1) (CDKN1A), BCL2 binding component 3 (BBC3), BCL2, zeta-chain (TCR)–associated protein minase 70 kDa (ZAP70), and VCL in primary B-cell CLL cells of 3 patients with CLL with a homozygous 13q deletion. Primary leukemic cells were stably infected with a lentiviral vector expressing miR-15a (LV- miR-15a), a lentiviral vector expressing miR-16-1 (LV- miR-16), or an empty lentiviral vector (LV-Empty). VCL is the normalization standard used to normalize the amount of proteins loaded to each well. The numbers above the blots indicate the intensity of the band expressed as ratio "gene product (TP53, CDKN1A, BBC3, BCL2 or ZAP70)/VCL" and normalized to "LV-Empty."
A, Luciferase reporter assay (as means [error bars indicate 95% confidence intervals] of experiments conducted in sextuplicate) in cells cotransfected with wild-type tumor protein p53 (TP53) 3′-UTR (TP53 wt) and microRNA 15a (miR-15a) or 16 (miR-16). Luciferase activity normalized to scrambled; RLU indicates relative light units. TP53 del indicates deletion of miR-15a/miR-16 binding site on TP53 3′-UTR; TP53 mut indicates mutation of miR-15a/miR-16 binding site on TP53 3′-UTR. P values calculated for miR-15a and miR-16 vs scrambled; values were statistically significant (P < .05) for TP53 wt comparisons only. B, Expression of miR-15a, miR-16, and TP53 messenger RNA (mRNA) in Tet-Off miR-15a/miR-16-1 –inducible HeLa cells as detected by quantified real-time polymerase chain reaction. Results presented as means (error bars indicate 95% confidence intervals) of experiments performed in triplicate. P values calculated for the cells in the presence of doxycycline (indicates reduced expression of the miR-15a/miR-16-1 cluster) vs the cells in the absence of doxycycline (indicates increased expression of the miR-15a/miR-16-1 cluster); all values were statistically significant (P < .05).
A, Promoter luciferase assay in tumor protein p53 (TP53)-null H1299 cells reported as means (error bars indicate 95% confidence intervals) of experiments performed in sextuplicate. The indicated TP53 binding site (BS) numbers correspond to those shown in red in eFigures 9 and 12. Control indicates the results obtained using the promoter vector with no binding site cloned in it. BAX indicates BCL2-associated X protein; Chr, chromosome. P values were calculated for TP53 vs empty for each group; all values were statistically significant (P < .05). B, Quantified real-time polymerase chain reaction (error bars indicate 95% confidence intervals) for microRNA 15b (miR-15b), microRNA 16 (miR-16), and microRNA 34b (miR-34b) performed on MEG-01 cells 24 hours after transfection with empty or TP53-expressing vectors. P values were calculated for TP53 vs empty for each group; all values were statistically significant (P < .05).
A, Northern blots showing messenger RNA (mRNA) expression of microRNA 16 (miR-16) and immunoblots showing the protein expression of tumor protein p53 (TP53), B-cell CLL/lymphoma 2 (BCL2), caspase 3, apoptosis-related cysteine peptidase (CASP3), and vinculin (VCL) in K562 leukemic cells 24 or 48 hours after transfection with an empty or TP53-expressing vector (TP53). VCL is the normalization standard used to normalize the amount of proteins loaded to each well. The numbers above the blots indicate the intensity of the band expressed as ratio "gene product (TP53, BCL2 or CASP3)/VCL" and normalized to "empty." B, Immunoblots showing the protein expression of TP53 and VCL, and the Northern blots showing mRNA expression of miR-16 and RNA, U6 small nuclear 1 (RNU6-1 ), after doxorubicin-induced TP53 activation in MEG-01 cells. VCL and RNU6-1 are the normalization standards used to normalize the amount of proteins and RNA loaded to each well, respectively. The numbers above the blots indicate the intensity of the band expressed as ratio "TP53/VCL" or " miR-16/RNU6-1 " and normalized to untreated cells (left panels) and to anti-CTRL treated cells (right panels).
Luciferase reporter assay (as means [error bars indicate 95% confidence intervals] of experiments conducted in sextuplicate) in MEG-01 cells cotransfected with wild-type zeta-chain (TCR)–associated protein kinase 70kDa (ZAP70) binding site for microRNA 34 (miR-34) family (wt) and miR-34a, miR-34b, or miR-34c. Luciferase activity normalized to scrambled; RLU indicates relative light units. ZAP70 del indicates deletion of miR-34 binding site on ZAP70 coding region; ZAP70 mut indicates mutation of miR-34 binding site on ZAP70 coding region. P values calculated for miR-34a, miR-34b, and miR-34c vs scrambled; values were significant (P < .05) for ZAP70 wt (wild-type) only.
A novel pathogenetic model for chronic lymphocytic leukemia (CLL) showing a pathway of microRNAs and protein coding genes that are involved in the development of CLL. The microRNA 15a (miR-15a)/microRNA 16-1 (miR-16-1) cluster, the microRNA 34b (miR-34b)/microRNA 34c (miR-34c) cluster, and the genes tumor protein p53 (TP53), B-cell CLL/lymphoma 2 (BCL2), myeloid cell leukemia sequence 1 (BCL2-related) (MCL1), and zeta-chain (TCR)–associated protein kinase 70kDa (ZAP70) are the main partners in this model. mRNA indicates messenger RNA.
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Fabbri M, Bottoni A, Shimizu M, et al. Association of a MicroRNA/TP53 Feedback Circuitry With Pathogenesis and Outcome of B-Cell Chronic Lymphocytic Leukemia. JAMA. 2011;305(1):59–67. doi:10.1001/jama.2010.1919
© 2019
Author Affiliations: Department of Molecular Virology, Immunology and Medical Genetics, Comprehensive Cancer Center, Ohio State University, Columbus (Drs Fabbri, Bottoni, Valeri, Calore, Alder, Nakamura, and Croce and Mr Adair and Ms Wojcik); Department of Experimental Therapeutics and Center for RNA Interference and Non-Coding RNAs (Mr Shimizu and Drs Spizzo, Nicoloso, Rossi, Barbarotto, Sampath, and Calin) and Department of Leukemia (Dr Keating), University of Texas M. D. Anderson Cancer Center, Houston; Department of Morphology and Embryology, Human Anatomy Section (Dr Barbarotto) and Department of Experimental and Diagnostic Medicine, Interdepartment Center for Cancer Research (Dr Negrini), University of Ferrara, Ferrara, Italy; Institute of Genetics and Biophysics "A. Buzzati Traverso" (IGB), National Research Council (CNR), Naples, Italy (Dr Cimmino); Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori, Meldola, Italy (Drs Fabbri, Fanini, Vannini, Musuraca, and Amadori); Department of Medicine, University of California San Diego, La Jolla (Drs Dell'Aquila, Rassenti, and Kipps); Center for Systems and Computational Biology, Wistar Institute, Philadelphia, Pennsylvania (Dr Davuluri); Department of Internal Medicine, Division of Hematology, Mayo Clinic College of Medicine, Rochester, Minnesota (Dr Kay); and Long Island Jewish Medical Center, New Hyde Park, New York (Dr Rai).
Context Chromosomal abnormalities (namely 13q, 17p, and 11q deletions) have prognostic implications and are recurrent in chronic lymphocytic leukemia (CLL), suggesting that they are involved in a common pathogenetic pathway; however, the molecular mechanism through which chromosomal abnormalities affect the pathogenesis and outcome of CLL is unknown.
Objective To determine whether the microRNA miR-15a/miR-16-1 cluster (located at 13q), tumor protein p53 (TP53, located at 17p), and miR-34b/miR-34c cluster (located at 11q) are linked in a molecular pathway that explains the pathogenetic and prognostic implications (indolent vs aggressive form) of recurrent 13q, 17p, and 11q deletions in CLL.
Design, Setting, and Patients CLL Research Consortium institutions provided blood samples from untreated patients (n = 206) diagnosed with B-cell CLL between January 2000 and April 2008. All samples were evaluated for the occurrence of cytogenetic abnormalities as well as the expression levels of the miR-15a/miR-16-1 cluster, miR-34b/miR-34c cluster, TP53, and zeta-chain (TCR)–associated protein kinase 70kDa (ZAP70), a surrogate prognostic marker of CLL. The functional relationship between these genes was studied using in vitro gain- and loss-of-function experiments in cell lines and primary samples and was validated in a separate cohort of primary CLL samples.
Main Outcome Measures Cytogenetic abnormalities; expression levels of the miR-15a/miR-16-1 cluster, miR-34 family, TP53 gene, downstream effectors cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) and B-cell CLL/lymphoma 2 binding component 3 (BBC3), and ZAP70 gene; genetic interactions detected by chromatin immunoprecipitation.
Results In CLLs with 13q deletions the miR-15a/miR-16-1 cluster directly targeted TP53 (mean luciferase activity for miR-15a vs scrambled control, 0.68 relative light units (RLU) [95% confidence interval {CI}, 0.63-0.73]; P = .02; mean for miR-16 vs scrambled control, 0.62 RLU [95% CI, 0.59-0.65]; P = .02) and its downstream effectors. In leukemic cell lines and primary CLL cells, TP53 stimulated the transcription of miR-15/miR-16-1 as well as miR-34b/miR-34c clusters, and the miR-34b/miR-34c cluster directly targeted the ZAP70 kinase (mean luciferase activity for miR-34a vs scrambled control, 0.33 RLU [95% CI, 0.30-0.36]; P = .02; mean for miR-34b vs scrambled control, 0.31 RLU [95% CI, 0.30-0.32]; P = .01; and mean for miR-34c vs scrambled control, 0.35 RLU [95% CI, 0.33-0.37]; P = .02).
Conclusions A microRNA/TP53 feedback circuitry is associated with CLL pathogenesis and outcome. This mechanism provides a novel pathogenetic model for the association of 13q deletions with the indolent form of CLL that involves microRNAs, TP53, and ZAP70.
Chronic lymphocytic leukemia (CLL) is the most common leukemia among adults in the Western world, with an annual incidence in the United States of approximately 10 000 new cases.1 The clinical staging systems devised by Rai et al2 and Binet et al3 are useful for assessing the extent of CLL in a patient, but they fail to differentiate between the indolent and aggressive forms of CLL. Most typically these forms are characterized by low and high levels of zeta-chain (TCR)–associated protein kinase 70kDa (ZAP70),4,5 respectively. Using fluorescence in situ hybridization (FISH), Döhner et al4 found that chromosomal abnormalities occurred in 82% of cases and included the 13q deletion (55%), 11q deletion (18%), and 17p deletion (7%). Patients with the 17p and 11q deletions experience the aggressive form of the disease, whereas patients with the 13q deletion or with normal cytogenetic profiles experience the indolent form.4,6 The occurrence of common and recurring chromosomal abnormalities4 suggests that these deletions affect thus-far undefined pathways important for the pathogenesis of CLL.
MicroRNAs are small, noncoding RNAs with regulatory functions7; microRNA expression is frequently deregulated in tumors.8 In most CLLs, the expression of the microRNA 15a (miR-15a [GenBank 406948]) /microRNA 16-1 (miR-16-1 [GenBank 406950]) cluster, which maps within a 30-kilobase region of loss9 at 13q14.3 (henceforth designated 13q14), is abolished or reduced.10,11 We have previously shown that the expression of this microRNA cluster is inversely correlated with the expression of B-cell CLL/lymphoma 2 (BCL2 [GenBank 596]), an antiapoptotic gene overexpressed in most CLLs.12 The loss of the long arm of chromosome 11 involves the 11q23.1 region (henceforth designated 11q23), where the microRNA 34b (miR-34b [GenBank 407041]) /microRNA 34c (miR-34c [GenBank 407042]) cluster is located.13 The observation that microRNA 34a (miR-34a [GenBank 407040]), miR-34b, and miR-34c are transactivated by tumor protein p53 (TP53)14 and that the miR-34b/miR-34c cluster maps at 17p suggests the possible existence of a genetic link and significant molecular interactions between the 17p and 11q chromosomal deletions in CLL. At present, it is not known how the 13q, 11q, and 17p deletions contribute to CLL pathogenesis and affect the outcome of patients with CLL.
Blood samples were obtained at the CLL Research Consortium Institutions from 206 patients with B-cell CLL (Table). Written informed consent was obtained from all patients before sample analyses, and the study was in accordance with the approved institutional review board protocols (LAB07-0734 and 2005C0014).
We first evaluated the occurrence of chromosomal abnormalities in 188 consecutive patients with untreated CLL. Then, to test our hypothesis that the miR-34b/miR-34c cluster modulates ZAP70 (GenBank 7535) expression in CLL, we analyzed 18 additional patients with B-CLL who had the 11q deletion alone. Details about the isolation of mononuclear cells are provided in the eMethods. MEG-01, K562, H1299, and A549 cell lines were obtained from the American Type Culture Collection.
Because of the lack of validated CLL cell lines, the megakaryocytic leukemia cell line MEG-01 was chosen because MEG-01 cells (wild-type TP53 [GenBank7157]) carry the 13q deletion, which is the chromosomal abnormality most frequently observed in CLL; thus, this cell line resembles the indolent form of CLL at the molecular level. K562 is a chronic myeloid leukemia cell line, chosen because K562 cells carry a mutated, inactive TP53 and represent a model of chronic leukemia. H1299 is a lung cancer cell line with a homozygous deletion of the TP53 gene, chosen to avoid interfering with endogenous TP53 in our experiments. A549 is a lung cancer cell line (wild-type TP53) chosen as a TP53-expressing control cell line for the H1299 model. HeLa is a cervical cancer cell line chosen because HeLa cells (wild-type TP53) are commercially available for the Tet-Off Advanced Inducible Gene Expression System (Clontech, Mountain View, California) (see eMethods for details). The efficiency of transfection for cell lines and primary B-CLL cells was checked by quantitative real-time polymerase chain reaction (qRT-PCR) analysis (eFigure 1).
Two genes were considered downstream effectors of the TP53 pathway signaling: cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A [GenBank1026], the expression of which is tightly regulated by TP53, which induces a TP53-dependent cell cycle G1 phase arrest in response to various stress stimuli); and BCL2 binding component 3 (BBC3 [GenBank 27113], formerly named PUMA, a p53 up-regulated modulator of apoptosis, which binds to BCL2 and is a proapoptotic gene). Procaspase 3 is the precursor of caspase 3 apoptosis-related cysteine peptidase (CASP3), a key protein involved in apoptosis. The cleavage of procaspase 3 to CASP3 is one of the strongest indicators of apoptosis.
MicroRNAs interact with their target genes by means of a "seed" region sequence between the mature microRNA and the target gene messenger RNA (mRNA). A luciferase reporter assay is performed to demonstrate that the microRNA-mRNA interaction is direct (meaning by complementarity). In this assay, the microRNA binding site on the target mRNA is cloned on a plasmid carrying the gene for luciferase, just downstream of the luciferase gene. Cells are then cotransfected with this plasmid and the microRNA of interest (or a scrambled microRNA as a control). If the microRNA targets the cloned binding site, the overall luciferase reporter activity in the cells cotransfected with the microRNA of interest is reduced. The destruction (and/or mutation) of the microRNA binding site on the target mRNA abolishes the reduction in the luciferase reporter activity, which indicates that the targeting is direct.
A luciferase reporter assay also was used to determine the effects of TP53 on the expression of the microRNAs of interest. TP53, like many other transcription factors, recognizes a specific binding sequence and binds to it. As a result, any gene located downstream of the binding site can be activated or suppressed. The predicted TP53 binding sites were cloned upstream of the luciferase gene in a luciferase-expressing reporter plasmid, and H1299 cells were transfected with this plasmid and a TP53-expressing plasmid or an empty plasmid. The effect (either activation or suppression) of TP53 on the expression of the microRNAs of interest was expressed as increased or decreased luciferase reporter activity of the TP53-treated group vs that of the empty vector–treated group (see eMethods for details).
Expression of ZAP70 was assessed by immunoblotting and flow cytometry analysis. Cytogenetic data were available for all 206 patients in this study. The following probes were used to perform FISH analyses: ataxia telangiectasia mutated (ATM) (11q22.3), D13S319 (13q14.3), and TP53 (17p13.1, henceforth designated 17p13). A commercial probe set (CLL Panel; Vysis Inc, Downers Grove, Illinois) was used to perform FISH analyses on peripheral blood samples that had been cultured for 24 hours without stimulation.
Results are presented as means with 95% confidence intervals (CIs). A probability value of P < .05 by 2-sided t test was considered statistically significant. Allelic distributions for the 3 chromosomal abnormalities (ie, 13q, 17p, and 11q deletions) in all patients were tested with a χ2 goodness-of-fit test for compliance with Hardy-Weinberg equilibrium. Relationships between microRNA and TP53 or ZAP70 mRNA expression determined by qRT-PCR were calculated as Pearson correlations. Based on our previous qRT-PCR data for the expression of miR-15a and miR-16,15 we calculated that by using a cohort of 206 patients, the statistical power would be greater than 95% to detect an effect, if one actually exists, with a minimal detectable effect of 9.73 (calculated as the difference between the lowest and the largest detected means) and an α of .05 by 2-sided test (see eMethods for details).
The clinical characteristics of the 188 consecutive patients with untreated B-CLL are shown in the Table. No departures from Hardy-Weinberg equilibrium were detected for any of the 3 chromosomal abnormalities investigated. The 13q deletion was the chromosomal abnormality that occurred most often in patients with CLL and was frequently the only cytogenetic abnormality in these patients, whereas the other 2 most common chromosomal abnormalities in CLL (11q deletion and 17p deletion) were often associated with the 13q deletion, but they rarely occurred together in 1 clone.
Patients (n = 22) with CLLs with a homozygous 13q deletion (13q−/−) had significantly lower expression levels of miR-15a (mean fold induction, 0.04 [95% CI, 0.035-0.045]; P = .03) and miR-16 (mean fold induction, 0.18 [95% CI, 0.16-0.20]; P = .01) than patients (n = 28) with CLLs with normal cytogenetic profiles (defined as no abnormality detected by FISH) (set as fold induction, 1 [95% CI, 0.94-1.05] for both microRNAs). Conversely, patients with 13q−/− CLLs had significantly higher TP53 expression levels than patients with CLLs with normal cytogenetic profiles, both at the mRNA level (mean fold induction, 4.83 [95% CI, 4.36-5.30] vs 1 [95% CI, 0.91-1.09]; P = .03) and at the protein level (mean fold induction, 2.06 [95% CI, 1.98-2.14] vs 1 [95% CI, 0.96-1.04]; P = .04) (eFigure 2). Also, patients with CLLs with a heterozygous 13q deletion (13q+/−) had significantly lower expression levels of miR-15a and miR-16 than patients with CLLs with normal cytogenetic profiles (mean fold induction, 0.48 [95% CI, 0.46-0.50]; P = .03 and 0.64 [95% CI, 0.62-0.66]; P = .04 vs 1 [95% CI, 0.97-1.03], respectively) (eFigure 3A). Patients with 13q+/− CLLs had significantly higher TP53 expression levels than those with CLLs with normal cytogenetic profiles both at the mRNA level (mean fold induction, 2.32 [95% CI, 2.23-2.41]; P = .03 vs 1 [95% CI, 0.97-1.03]) and at the protein level (mean fold induction, 1.48 [95% CI, 1.45-1.51]; P = .04 vs 1 [95% CI, 0.97-1.03]), although TP53 expression in patients with 13q+/− CLLs was not as high as that in patients with 13q−/− CLLs (eFigure 3A and B).
A slight inverse correlation was found between expression of miR-15a/miR-16 and TP53 mRNA (r = −0.31; P < .04) in patients with 13q−/− CLLs compared with patients with CLLs with normal cytogenetic profiles. In MEG-01 cells, TP53 protein expression was highly reduced when the miR-15a/miR-16-1 cluster was overexpressed (eFigure 4). Overexpression of each microRNA of the cluster was associated with reduced TP53 expression (52% for miR-15a and 62% for miR-16) compared with a scrambled oligonucleotide control, and the combination of both miR-15a and miR-16 almost completely repressed TP53 expression (TP53 protein expression was reduced by 82%) (Figure 1A). No TP53 silencing effect was observed with the antisense oligonucleotides. Both miR-15a and miR-16 were found to target BCL2 (Figure 1A), which is consistent with our previous findings.12 In MEG-01 cells stably expressing miR-16, TP53 expression was reduced, as were the protein levels of its downstream effectors CDKN1A, BBC3, and BCL2 (eFigure 5). Additionally, cleavage of procaspase 3 was observed in MEG-01 cells stably expressing miR-15a or miR-16-1, confirming that the miR-15a/miR-16-1 cluster has a caspase-dependent proapoptotic role in this cell line (eFigure 5). No effect on CDKN1A and BBC3 was observed in TP53-negative H1299 cells (eFigure 6), which suggests that the effects of miR-15a and miR-16 on CDKN1A and BBC3 are mediated by their effects on TP53. Conversely, the targeting effect of miR-15a and miR-16 on BCL2 persisted also in TP53-null cells (eFigure 6). These results were confirmed in primary B-CLL cells collected from 3 patients with 13q−/− CLL. When miR-15a and miR-16 were overexpressed in these primary B-CLL cells, the expression of TP53, CDKN1A, BBC3, and BCL2 was reduced both at the protein and mRNA level (Figure 1B and eFigure 7 [left panels]).
A binding site for both miR-15a and miR-16 inside the 3′-UTR (untranslated region) of TP53 was identified using the sequencer software (eFigure 8). A luciferase reporter assay showed that both miR-15a and miR-16 directly target the identified TP53 binding site and significantly reduced the luciferase reporter activity compared with a scrambled oligonucleotide-negative control (mean luciferase activity, 0.68 relative light units [RLU] [95% CI, 0.63-0.73]; P = .02 for miR-15a and 0.62 RLU [95% CI, 0.59-0.65]; P = .02 for miR-16 vs 1 [95% CI, 0.94-1.06] for scrambled). This effect was completely abolished when the binding site was either deleted or mutated (Figure 2A). Lastly, by using a miR-15a/miR-16-1 doxycycline-inducible HeLa cell line when miR-16 was down-regulated in presence of doxycycline, the expression of TP53 mRNA was significantly increased (mean fold induction, 6.39 [95% CI, 5.46-7.32] vs 1 [95% CI, 0.46-1.54]; P = .01) (Figure 2B).
Several TP53 binding sites were found upstream of the 2 homologous miR-15/miR-16 loci located on chromosome 13 (miR-15a/miR-16-1) and on chromosome 3 (microRNA 15b [miR-15b ] [GenBank 406949]/ microRNA 16-2 [miR-16-2 ] [GenBank406951]), which was also analyzed because it encodes for a similar cluster of genes (eFigure 9). Chromatin immunoprecipitation analysis revealed that TP53 directly binds to its predicted binding sites on both chromosome 13 and chromosome 3, both in cell lines and in primary CLLs with normal cytogenetic profiles (eFigure 10 and eFigure 11). A luciferase reporter assay showed that TP53 significantly increased the luciferase reporter activity of all the binding site–containing vectors (Figure 3A). In MEG-01 cells, TP53 transactivation of the miR-15/miR-16 cluster was also confirmed by qRT-PCR (Figure 3B); this transactivating effect also occurred in TP53-mutated K562 cells, in which it was associated with a reduction of procaspase 3 protein levels (Figure 4A). Similarly, doxorubicin-mediated TP53 activation in MEG-01 cells increased the expression of miR-16, an effect that was abolished when TP53 was silenced by an anti-TP53 oligonucleotide (Figure 4B). Although the effect of TP53 on the miR-15/miR-16 cluster was relatively mild (highest fold induction of 2.11 after 48 hours), it still had functional consequences and was associated with 66% reduction of BCL2 protein levels after 48 hours.
Chromatin immunoprecipitation analysis revealed that TP53 binds directly to a pre–miR-34b/miR-34c TP53 binding site on chromosome 11 (eFigure 10 and eFigure 12). A luciferase reporter assay showed a statistically significant transactivating effect of TP53 on the miR-34b/miR-34c binding site (Figure 3A), which was confirmed in TP53-transfected MEG-01 cells, which had increased miR-34b expression levels (Figure 3B). Taken together, these results indicate that TP53 is a positive transcriptional regulator of the miR-34b/miR-34c cluster in leukemic cells, which is consistent with findings observed in epithelial cells.14
Patients with 11q+/− CLLs (n = 23) had significantly lower levels of miR-34b (mean fold induction, 0.37 [95% CI, 0.35-0.39]; P < .01) and miR-34c (mean fold induction, 0.39 [95% CI, 0.37-0.41]; P < .01) than patients with CLLs with normal cytogenetic profiles (n = 28) (set as fold induction, 1 [95% CI, 0.96-1.04] and 1 [95% CI, 0.97-1.03], respectively). Conversely, patients with 11q+/− CLLs had significantly higher levels of ZAP70 than patients with CLLs with normal cytogenetic profiles, both at the mRNA level (mean fold induction, 2.02 [95% CI, 1.98-2.06] vs 1 [95% CI, 0.97-1.03]; P = .01) and at the protein level (mean fold induction, 2.47 [95% CI, 2.43-2.51] vs 1 [95% CI, 0.94-1.06]; P = .005). Patients with 11q+/− CLLs and high levels of ZAP70 experienced poorer overall survival than patients with CLLs with normal cytogenetic profiles and lower levels of ZAP70 (mean, 72.4 [95% CI, 35.4-79.2] months vs 114.7 [95% CI, 98.9-130.5] months; P = .02). A binding site for the miR-34 family was detected in the ZAP70 opening reading frame (eFigure 13A). Reduced ZAP70 expression (both at the protein and mRNA level) was observed in primary B-CLL cells from a patient with an 11q+/− deletion, in which miR-34a, miR-34b, and miR-34c were overexpressed (eFigure 13B and eFigure 14).
No effect on cell growth and cell proliferation was observed up to 72 hours in MEG-01 (ZAP70-negative) cells or in K562 (ZAP70-positive) cells overexpressing miR-34b or miR-34c (eFigure 15). A luciferase reporter assay showed that all 3 microRNAs directly target the predicted region on ZAP70 (mean luciferase activity, 0.33 RLU [95% CI, 0.30-0.36]; P = .02 for miR-34a, 0.31 RLU [95% CI, 0.30-0.32]; P = .01 for miR-34b, and 0.35 RLU [95% CI, 0.33-0.37]; P = .02 for miR-34c vs 1 [95% CI, 0.94-1.06] for scrambled). This effect was abolished when the predicted site on ZAP70 was either deleted or mutated (Figure 5). In K562 cells as well as primary B-CLL lymphocytes, increased levels of ZAP70 mRNA were observed after the miR-34 family was silenced (eFigure 13C and D).
Reexpression of miR-15a and miR-16 in primary B-CLL cells from patients with 13q−/− CLL was associated with a reduction of TP53; reduction of miR-34a, miR-34b, and miR-34c ; and increased expression of ZAP70 protein levels (eFigure 7 [right panels] and Figure 1B).
In this study, we identified a microRNA/protein functional circuitry that likely underlies the pathogenesis and natural history of a major subset of human CLL. This pathway is perturbed at different levels by distinct CLL chromosomal abnormalities, which could explain the occurrence of the same disease but with distinct clinical features. This novel pathogenetic model for CLL is summarized in Figure 6.
In this model, TP53 (located on chromosome 17p) represents the molecular link between the miR-15a/miR-16-1 (located on chromosome 13q) and miR-34b/miR-34c (located on chromosome 11q) clusters. In fact, the tumor suppressor protein TP53 is directly regulated by miR-15a/miR-16-1. The loss of miR-15a/miR-16-1 expression, represented by CLLs with 13q deletions, not only shifts the balance toward higher levels of the antiapoptotic proteins BCL2 and myeloid cell leukemia sequence 1 (BCL2-related) (MCL1), as we have previously demonstrated,12,16 but also toward higher levels of the tumor suppressor protein TP53. Consequently, in patients with CLLs with 13q deletions, while the number of apoptotic cells may decrease because of the increased levels of antiapoptotic proteins, the TP53 tumor suppressor pathway remains intact, thus keeping the increase in tumor burden relatively low (eComments).
This novel finding explains how 13q deletions are associated with the indolent form of CLL, as first identified by Döhner et al.4 Moreover, increased TP53 levels, as found in patients with CLLs with 13q deletions, are associated with transactivation of miR-34b /miR-34c and reduced levels of ZAP70, a tyrosine kinase relevant in the initial step of T-cell receptor–mediated signal transduction.17 Low expression levels of ZAP70 have been found to be positively correlated with survival in patients with CLL,18 further explaining the indolent course of CLL carrying 13q deletions.
Here we showed that, in primary B cells from patients with B-CLL, use of viral infection to restore expression of the miR-15a/miR-16-1 cluster is associated with reduced expression levels of TP53, miR-34a, miR-34b, and miR-34c and increased protein levels of ZAP70. These findings also demonstrate that in primary B-CLLs, restoring the expression of a microRNA cluster (namely, the miR-15a/miR-16-1 cluster) indirectly affects the expression of another family of microRNAs (miR-34 family) by modulating the levels of TP53. Some of our results indicate that these effects also occur in non-CLL leukemic cells (such as the acute myelogenous leukemic cell line K562) and in nonhematologic cell lines (such as H1299, A549, and HeLa cells), which suggests that the proposed microRNA-TP53 loop is relevant to tumor types other than CLL. More studies are necessary to validate this statement.
In conclusion, we found that a microRNA/TP53 feedback circuitry is associated with the pathogenesis and prognosis of CLL. Our findings reveal a new pathogenetic model for human CLL that involves microRNAs (miR-15a/miR-16-1 and miR-34b/miR-34c) and protein-coding genes (such as TP53 and ZAP70) with well-known prognostic significance in CLL.
Corresponding Authors: George A. Calin, MD, PhD, Department of Experimental Therapeutics and Center for RNA Interference and Non-Coding RNAs, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030 (email@example.com) or Carlo M. Croce, MD, Human Cancer Genetics Program and Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, 460 W 12th Ave, Room 1082, Columbus, OH 43210 (firstname.lastname@example.com).
Author Contributions: Drs Calin and Croce had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Drs Fabbri and Bottoni contributed equally to this article.
Study concept and design: Fabbri, Bottoni, Calin, Croce.
Acquisition of data: Fabbri, Bottoni, Shimizu, Spizzo, Nicoloso, Rossi, Barbarotto, Cimmino, Adair, Wojcik, Valeri, Calore, Sampath, Fanini, Vannini, Musuraca, Alder, Davuluri, Negrini, Nakamura, Amadori, Kay, Rai, Keating, Kipps, Croce.
Analysis and interpretation of data: Fabbri, Bottoni, Rossi, Dell'Aquila, Rassenti, Calin, Croce.
Drafting of the manuscript: Fabbri, Bottoni, Spizzo, Amadori, Kay, Rai, Keating, Kipps, Calin, Croce.
Critical revision of the manuscript for important intellectual content: Fabbri, Bottoni, Shimizu, Nicoloso, Rossi, Barbarotto, Cimmino, Adair, Wojcik, Valeri, Calore, Sampath, Fanini, Vannini, Musuraca, Dell'Aquila, Alder, Davuluri, Rassenti, Negrini, Nakamura, Kay, Rai, Keating, Kipps, Calin, Croce.
Statistical analysis: Fabbri, Bottoni, Rossi.
Obtained funding: Fabbri, Calin, Croce.
Administrative, technical, or material support: Shimizu, Spizzo, Barbarotto, Cimmino, Adair, Wojcik, Valeri, Calore, Fanini, Vannini, Musuraca, Alder, Nakamura, Amadori.
Study supervision: Calin, Croce.
Conflict of Interest Disclosures: All authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported.
Funding/Support: Dr Fabbri is supported by a 2009 Kimmel Scholar Award. Dr Cimmino is supported by a fellowship from FIRC (Federazione Italiana Ricerca sul Cancro). Dr Calin is supported by a fellowship from the University of Texas M. D. Anderson Research Trust, a fellowship from The University of Texas System Regents Research Scholar Program, and a grant from the CLL Global Research Foundation. Dr Calin's laboratory is supported in part by the National Institutes of Health, the US Department of Defense, and a Leukemia SPORE Developmental Research Award. Drs Kay and Croce are supported by National Institutes of Health grant R01 CA111953.
Role of the Sponsors: The sponsors had no role in the design and conduct of the study; the collection, management, analysis, and interpretation of the data, or the preparation, review, or approval of the manuscript.
Additional Contributions: We thank Kay Huebner, PhD (Department of Molecular Virology, Immunology and Medical Genetics, Ohio State University, Columbus), for critical discussion and reading of the manuscript and Lionel Santibañez, PhD (Department of Scientific Publications, University of Texas M. D. Anderson Cancer Center), for English editing of the manuscript. The persons acknowledged received no financial compensation for their contributions.
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Is There A Possibility For Brand & Content Collaboration?/
Can we move beyond product placement for a collaboration between those that fund the production and those that create stories? Can it be done without compromising the integrity of the work. Steve Wax and I wrote a blog discussion about this last year and I recently stumbled across this video of Steve and I.


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Teachers and Tutors - Warwick
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Childish Gambino
My hotel cost a kilo, banking like a free throw
Fuck y'all, we A-Team, y'all plan B like Walgreens
Cop that shit and paint it black, niggas want they summer back?
Ha-ha, no way, hublots, Dolce
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She still with her man, but she seeing me tomorrow
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Unnecessary roughness? We just trying to function
Used to sport them Filas cause my cousin pack them heaters
We don't wife 'em like Khalifa, we just party then we leave 'em
Tell 'em that we hittin', we don't really see 'em
My girl look best when she only wear a fitted
Q and 'Bino in the house and you niggas in the senate
Get it?
Four thou' on my fit, that's unnecessary
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That's unnecessary, that's unnecessary
Roll out on a plane, that's unnecessary
'Tussin and champagne, nigga that ain't even necessary
They say don't buy the belt and I got it
They say don't get the Rollie and I bought it
Nigga, I'm balling
My fucking mixtape can get you through college
Getting them dollars
Showing my pearly white teeth, I'm a nigga that's flawless
Came a long way from dumping out Impalas
Same gun and pants, different ski mask from coppers
All of this copper
Knock knock, who could it be? Niggas know I got it on lock
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Been fucking with my side bitch so much my main bitch can't stand it
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Ibiza for a day, that's unnecessary
Shows a hundred K, that ain't even necessary
Black Lip Bastard
Actual natural disaster
I did a song about DMT
Soulja Boy did one right after
3 eyes, these guys ain't seeing me
No radio show up over B.o.B
G-O-D Emcee
I'm the devil with angel wings
Pilot Talk, Curren$y
Stole the show, burglary
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Open condoms & condos
Open bottles of Moet
White with white girls in Eugene
And use to call hoes on 3-Way
And take out to the movies
And now we just do it always
She driving stick in the driveway
Just popped a molly with Molly
She do shrooms every blue moon
Hundred million trillion muthafuckin racks nigga
Pinot grigio the lean'll go in that nigga
Now that's unnecessary
Been a legend since elementary
Ayo, Bino, tell these niggas I ain't no joke
She don't need speech, she ain't giving no throat
You are under my control
Suspender L's but I'm on a roll
Grade A, honor roll
Mayday, Soul!


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'Grimm' Star David Giuntoli Previews the Season Three Premiere
At the end of the second season of NBC's "Grimm," Nick Burkhardt (played by David Giuntoli), a homicide detective who doubles as a supernatural hunter, or grimm, takes an unearthly turn for the worse. Abducted and forced into a zombie, he makes some questionable decisions under the influence of the undead.
"I kind of go berserk," Giuntoli told the Journal.
The season two finale drew an audience of 5.2 million viewers and will continue to remain on Friday nights, serving as a lead in for Jonathan Rhys Meyers' "Dracula."
With the third season of "Grimm" premiering tonight, Speakeasy caught up with Giuntoli during a filming break to discuss "Grimm" and the expanding grey areas Nick will be forced to navigate in the show's junior season.