Patients who leave the hospital against medical advice and/or self-initiate discharge may be at a greater risk of suicide in the year following discharge compared with those who leave the hospital under different circumstances, suggests a report published today in Psychiatric Services in Advance.
Using national Veterans Affairs (VA) administrative data, Natalie Riblet, M.D., M.P.H., and colleagues identified all inpatient discharges and discharge types from 2001 through 2014. The authors used mortality and cause of death data from the Centers for Disease Control and Prevention Death Index to calculate suicide risk within the 12 months following the first discharge in patients. They then compared suicide risk following regular discharge versus irregular discharge from both general medical and psychiatric wards, adjusting for age, gender, and VA facility.
Among the 5,051,051 discharges from general medical or psychiatric wards included in the analysis, 103,995 (2.1%) were documented as being irregular. A total of 666,674 deaths occurred within 12 months of the index discharge date, 5,337 of which were caused by suicide.
Overall, suicide risk was two times higher following irregular discharge compared with regular discharge. However, additional analysis revealed that "suicide risks did not differ significantly between patients who were regularly versus irregularly discharged from a psychiatric ward," the authors wrote. In contrast, patients leaving general medical wards following an irregular discharge had three times the one-year risk of suicide compared with patients who were discharged in a regular fashion.


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Sniper (1993)
Tom Berenger as Tom Beckett
Billy Zane as Richard Miller
J. T. Walsh as Chester Van Damme
Action, Adventure, Drama, History, Mystery, Thriller, War
Rated R For Strong Violence and Language
"Sniper" expresses a cool competence that is a pleasure to watch. It isn't a particularly original film, but what it does, it does well. We've seen so many bad movies about guys walking through the jungle with rifles that it's interesting the way this one grabs us through its command of the locations and its storytelling skill.
The plot could have been borrowed from a Western. Tom Berenger plays a hired gun who knows the terrain well. Billy Zane is the hotshot kid who is an expert marksman but has never killed a man.
They're hired as a team, to knock off some bad guys, and along the way a rivalry develops, until at the climax their very lives are threatened.
The movie gives this formula a modern political touch by making both Berenger and Zane U.S. military men, acting under secret orders from the National Security Council. Their assignment is to knock off Panamanian leaders the NSC doesn't like, in order to change the outcome of approaching elections. The film's director, Luis Llosa, is a Peruvian, and perhaps this subplot about Yankee imperialism was appealing to him, but the movie simply accepts the illegality of the assignment and gets on with it.
Berenger is a skilled veteran (he could be the character he played in "Platoon," 20 years down the road). Zane, however, outranks him - and is determined to take command of their little mission, even after it becomes clear he could not find his way out of the jungle with a trail of jelly beans behind him. It's up to Berenger to introduce him to the dangers of the jungle - animal, insect and human - and eventually they penetrate to the secret rendezvous where their targets will be meeting.
Any movie set in a forest or jungle has trouble convincing us the characters have actually gone anywhere, since all of the trees look the same. Llosa does a good job of making the terrain look real and keeping us oriented, so that we can halfway follow the strategy - until an ending so drenched in rain we have to take him on faith.
It is, I admit, strange that "Sniper" is so blase about the fact that the heroes are murderers. After all of those angry Costa-Gavras thrillers about corrupt American death squads, here is a movie that pragmatically assumes that's the way the world turns.
Because the movies are such a curiously voyeuristic form, we identify with the heroes and accept their actions, at least as long as the film runs. Afterward, there are some hard questions to answer, but not about Llosa's filmmaking skill.
Lightening the Load: Miles Teller, Jason Hall & Adam Schumann on "Thank You For Your Service"
by Nick Allen
"Evil Against Evil": The Fascinating Incoherence of American Sniper
by Niles Schwartz
Thumbnails 1/19/15
by Matt Fagerholm

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text:
Citizen science: One family's search for answers in their genes
KT Pickard
In April, we celebrate Citizen Science Month, World Autism Day, and National DNA Day. In this guest blog post, all three events come together as KT Pickard, father of a young woman with autism, shares his family's story of personal genomics and citizen science.
This past Sunday was National DNA Day, which commemorates the discovery of DNA's double helix in 1953 and the publication of the first draft of the human genome in 2003. Events on National DNA Day celebrate the latest genomic research and explore how those advances might impact our lives. Last year, I wrote a playful article for DNA Day that investigated whether genetics is truly like finding a needle in a haystack. This year, our family is honored to share our story and ideas with you.
Our family's DNA odyssey
My wife and I have a young adult-aged daughter who is on the autism spectrum. We first discovered that our daughter had autism when she was eight years old. As we struggled to understand autism and what it meant for our family, we learned that autism is uniquely expressed: Meeting one person with autism means that you have met one person with autism.
Long fascinated with genomics, my wife and I wondered how our DNA may have contributed to her condition, and we set out to learn all that we could. It was the beginnings of this diagnostic odyssey that gave expression to my second career as a citizen scientist. My professional background in supercomputing, software engineering, and medical imaging were a good start to apply scientific principles and gain insights.
We began our journey by talking with our family doctor, then my wife and I had our whole genomes sequenced through the Understand Your Genome project. Later, we crowdsourced the sequencing of her genome and began looking for genetic clues. By applying trio analysis to our family data, we discovered some preliminary findings: Our daughter has deletions in the NRXN1 gene and in a large region of chromosome 16, which have been found to be widely associated with developmental issues including autism. It looks like my wife and I have each contributed some variant alleles, but we are being careful about interpreting these findings because our WGS data and our daughter's were processed through different pipelines, which could lead to inconsistent results.
Trio analysis of the NRXN1 locus shows a compound heterozygous deletion, with each parent possibly contributing one allele (visualization by VarSeq from Golden Helix).
To continue our journey, I want to reprocess our family's WGS data with the latest GATK Best Practices, in the hope that this will give us a consistent baseline. I came across Terra through the book Genomics in the Cloud, which I picked up to help me learn more about GATK. I led an online book club in early 2021 based on the book, and subsequently moved our WGS data to the Terra platform. Now I am using the GATK Whole Genome Analysis Pipeline in Terra to reprocess our data. Working with Terra has been challenging, but highly satisfying because it provides access to industry standard genomics tools.
From personal genomics to citizen science
My family's main goal with this project is to make meaningful discoveries about the genetic basis of our daughter's autism. In 2015, genetics could explain the heritability of autism spectrum disorder in approximately 1 in 5 cases. Amazingly, that number has increased to 4 in 5 cases today.
Our daughter (who drew this image) is on the left. At the time, she represented the 1 in 5 people whose autism could be explained by genetics.
Yet there is more to be gained. Although whole genome sequencing may not provide directly actionable results for autism itself, WGS can make a huge difference for parents who discover a comorbid, but treatable condition. By sharing our data and our findings with others, we can accelerate medical knowledge.
A growing number of projects offer opportunities for non-scientists to contribute in various forms to the advancement of biomedical research. In U.S. healthcare, one of the largest citizen science projects—All of Us—seeks one million people to share their unique health data to speed up medical research. By creating a national resource that reflects and supports the broad diversity of the U.S., the goal of All of Us is to advance precision medicine for all.
We have enrolled in the All of Us project and are looking forward to doing our part. I find it inspiring that this is something we can all contribute to, as citizens, even those of us who are not researchers.
At its core, citizen science is a collaboration between scientists and those who are curious and motivated to contribute to scientific knowledge. As our family's odyssey unfolds, I like to reflect about what I see out here on the bleeding edge of research, and how it could be applied to improve outcomes for patients in the real-world.
In community practice, many medical providers have limited knowledge of autism. Due to a lack of effective data sharing and awareness, an undiagnosed person with autism who walks through the door of a hospital may appear like a rare disease patient. A clinician evaluating them would miss out on a huge amount of valuable context. How could we improve the system so that clinicians could more effectively recognize the underlying context of that person's condition? We can address some of these issues with machine learning, but that requires pooling together huge amounts of data, and much of that data is difficult to access.
As a citizen scientist, I see an enormous opportunity to combine research data with real-world data and evidence across healthcare delivery organizations. Common ontologies and interoperability standards are making it increasingly easy to pool de-identified datasets to test hypotheses on synthetic data—realistic-but-not-real data—to gain insights. A recent "call to action" encourages citizen scientists to evaluate the utility of this method precisely because data can be shared without disclosing the identities of anyone involved. Done ethically and responsibly, this synthetic DNA approach has the potential to accelerate autism research and deliver new benefits to patients.
This is the perspective I have gained from my journey so far. By asking questions and continuing to discover more about what our genomes contain, I have been fortunate to learn much about scientific principles, bioinformatics, and a bit about the genetic basis of autism. Although it is at times a challenging road, I have found that the path of personal genomics and citizen science is a satisfying way to find answers to the questions that my family faces. I hope this story will inspire others to explore, and perhaps let researchers and clinicians see patients and their families as potential collaborators in the quest to understand complex conditions like autism.
Analysis, Community, Genomics, Guest Author, Medical and Population Genetics, Most Popular, Testimonials


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Віталій Сергійович Ажнов ( 1993) — український актор театру і кіно.

Життєпис
Народився 27 квітня 1993 року в м. Калуш Івано-Франківської області.

У 2015 році закінчив Київський національний університет театру кіно та телебачення ім. І. К. Карпенко-Карого (майстерня Дмитра Богомазова). З 2015 року викладає сценічну мову в театральному університеті.

З 2015 року — актор Київського театру «Золоті ворота».

З 2018 року у театрі ім. І. Франка.

Ролі в театрі
 Київський театр «Золоті ворота»

 Київський театр «Актор»
  — «Схід — Захід» за дитячою драматургією; реж. Влада Бєлозоренко — Кіт
  — «Вишневий сад» за п'єсою Антона Чехова; реж. Слава Жила — Лопахін Єрмолай Олексійович, купець

 Національний академічний драматичний театр імені Івана Франка
 (введення)  — «Кайдашева сім'я» за повістю Івана Нечуя-Левицького; реж. Петро Ільченко — Лаврін
  — «Буна» Віри Маковій; реж. Давид Петросян — Петро
 2018 — «Коріолан» за п'єсою Вільяма Шекспіра; реж. Дмитро Богомазов — 2-ий городянин, 1-ий Еділ, Посланець
  — «Лимерівна» за однойменною п'єсою Панаса Мирного; реж. Іван Уривський — Карпо
 2019 — «Тев'є-Тевель» за п'єсою Григорія Горіна; реж. Дмитро Чирипюк — Мотл
 2019 — «Снігова королева» за казкою Ганса Крістіана Андерсена; реж. Давид Петросян — Ворон та Білий Ведмідь
  — «Украдене щастя» за п'єсою Івана Франка; реж. Дмитро Богомазов — Олекса Бабич, Парубок
 2020 — «Сірано де Бержерак» за п'єсою Едмона Ростана; реж. Юрій Одинокий — Привид театру
  — «Співай, Лоло, співай» Олександра Чепалова за мотивами роману «Вчитель Гнус, або Кінець одного тирана» Генріха Манна і художнього фільму «Блакитний ангел»; реж. Дмитро Богомазов — Ломан, гімназист
 2021 — «Безталанна» за однойменною п'єсою Івана Карпенка-Карого; реж. Іван Уривський — Дем'ян
  — «Калігула» за п'єсою Альбера Камю; реж. Іван Уривський — Калігула
 2022 — «Кар'єра Артуро Уї, яку можна було спинити» за п'єсою Бертольта Брехта; реж. Дмитро Богомазов — Артуро Уї, ватажок гангстерів

Фільмографія
  — Незламна
  — Пригоди S Миколая — Іржавий
 2019 — Гуцулка Ксеня — Юро
  — І будуть люди — Максим (Володька) Твердохліб
 «Янголи»
  — Кава з Кардамоном — Маркіян
  — «Малевич»; реж. Дар'я Онищенко — Казимир Малевич

Нагороди
 Диплом за кращу чоловічу роль на Міжнародному фестивалі театральних шкіл у Пекіні «GATS» (2013),
 Гран-прі Всеукраїнського конкурсу читців «Лесині джерела» (2013),
 преміант Київського міського голови (2014).

Примітки

Уродженці Калуша
Актори XXI століття
Українські актори
Актори за алфавітом
Персоналії за алфавітом
Народились 1993
Народились 27 квітня
Актори Національного академічного драматичного театру імені Івана Франка

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Sealed road frontage. Historic old farm only 10 minutes from Goulburn. River frontage western side of road divide. Original old stone and brick 2 bedroom cottage with semi modern kitchen and bathroom, dining with gas heater in fireplace and lounge room leading to verandah. Needs work. Adjoining weekender style 2 bedroom addition, basic kitchen, bathroom and large living area, owner built. Non compliant. Power, water and internet connected. One bedroom clad cottage with secure fenced yard. Kitchen with slow combustion stove, small living area and tiled bathroom with toilet, vanity and shower. Separate laundry/storage. Large adjoining colorbond weatherboard family room with wood heater and built in storage. Great for the extended family or holiday accommodation. Large machinery shed with concrete floor and power. Original unequipped shearing shed, mainly timber, corrugated iron and slab construction. Corrugated iron storage shed with concrete floor and various farm buildings. Original orchard with small green house. Undulating to hilly unimproved grazing land. Steep section at rear on basalt hill. Seasonal creek and access to Wollondilly river and small productive river flat. Enjoy fun weekends swimming or fishing in the large water holes on the river. Some income potential from livestock. Great location. Huge potential. Delayed settlement approx 6 months.
Open for viewing Saturday 10th, 17th and 24th November, 10:00-11:00am or by appointment. Bidding from $550,000.


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World Extreme Cagefighting: The AAA Affiliate of the UFC?
By E. Spencer Kyte (espencerkyte@mmaratings.net)
A few weeks back we discussed the merits of a merger between the UFC and the WEC.
Since then, UFC President Dana White has gone on record saying that the bantamweight and featherweight divisions of the WEC would be brought over to the big stage, providing champions like Mike Thomas Brown and Brian Bowles with a chance to perform on an even bigger stage.
Over this past week, MMA Junkie ran a poll to gauge fans interest in such a move, with roughly 60% voting in favor of the two companies pooling their resources.
But an all-or-nothing move isn't the only solution.
Every major sport has a minor league system of one kind or another, so why should Mixed Martial Arts be any different?
Though it wasn't all that long ago that the welterweight through light heavyweight divisions of the WEC were eliminated, perhaps a return to the ways of old makes the most sense moving forward.
Young and inexperienced baseball players work their way up the ladder, proving themselves at various levels of competition before being given the opportunity to play in the big leagues.
Returning the WEC to "minor league status" would afford the UFC the time to let young fighters grow and develop without the immense pressure that comes with competing on the biggest stage of them all.
Such a move would also give the UFC a destination for struggling established fighters as well. Instead of having to severe ties with someone like Thales Leites, a stint in the WEC to rebuild his confidence would be available in the same way an NHL team might send a player on an AHL vacation when they're struggling with the big club.
Furthermore, this type of move would eliminate one of the chief concerns of those opposed to the idea of a merger, the need for the UFC to trim their roster.
No one likes to see people given the pink slip, and while a full merger would inevitably result in a number of fighters being shown the door, re-introducing a full compliment of weight classes to the WEC would actually create more jobs.
Returning the WEC to a complete collection of weight classes would also create more championship belts, as well as some interesting promotional and scheduling opportunities.
With a new collection of titles to showcase, Fight Night cards could become a joint venture between the UFC and WEC, with WEC title fights serving as headliners, instead of being forced to accept Nathan Diaz versus Melvin Guillard atop the marquee.
This would give the UFC the opportunity to showcase the top competitors in the WEC before bringing them over to compete on the big stage.
Instead of bringing in complete unknowns and needing to build them up to the viewing public, fans would already be familiar with the fighters, making the Dana White's job of selling the fighters as legitimate contenders far easier.
The fighters would not be the only people with increased opportunities.
More fighters would mean more fights, and more fights require more referees and judges.
Just as players work their way up the ranks in other sports, so too do officials and move of this nature would provide a smaller stage that can be used to further train the non-fighting fight participants as well.
There is no substitute for experience, for both fighters and officials alike, and being able to gain said experience out from under the spotlight of the UFC could prove beneficial in the future.
Instead of throwing fighters fresh off The Ultimate Fighter into the shark-infested waters of the UFC, many could be given an opportunity to establish themselves in the WEC.
Fans would have household names they recognize and a reason to tune it to events, while the fighters themselves could compete against opponents of similar skills and abilities, unlike the tough tasks facing some of the recent TUF tournament winners.
Season 9 lightweight winner Ross Pearson faces Aaron Riley, a veteran with 40 professional fights under his belt.
Honestly, can anyone see "The British Bulldog" coming away victorious against his more-seasoned counterpart?
If a move like the one proposed here were to take place, the young and inexperienced Brit could face the likes of Shane Roller, Marcus Hicks or Phil Cardella by beginning in the WEC.
While all three are still stiff tests in their own right, Pearson would surely have a greater chance at emerging with a win than he currently has now heading into UFC 105.
The same goes for Season 8 lightweight winner Efrain Escudero, who makes his debut in seven days time against American Top Team veteran Cole Miller.
As Junie Browning found out, scoring a win over Miller is much more difficult than defeating "Diamond" Dave Kaplan.
With the amalgamation of the bantamweight and featherweight divisions into the UFC etched in stone, the question of what to do with the WEC as a whole remains.
Should the company be completely combined with the existing organization or would a return to the WEC of old make more sense moving forward?
Both choices have merit, and only time will tell which path Dana White and company will go down.
My opinion is outlined above, now I want you to tell me what you think.
Eric Kamander September 12, 2009 at 3:21 PM
I tend to think this type of merger would have lots of logistical problems centered around the limited pay scales available to the WEC.
The whole reason to merge the promotions is to 1) make more money off the talents of the lighter weight fighters, and 2) like-wise pay same fighters more money thus solidifying Zuffa as the place to be for lighter weight fighters.
If they don't pursue this course, it will only be a matter of time before promotions such as Strikeforce and Bellator FC are offering feather and bantam weights more money than the WEC can afford.
So while a merger makes sense, keeping the WEC around as a feeder promotions only has one-way limited potential. Fighters like Thales Leites will never take the pay cuts necessary to fight in the WEC. They will be better off moving to Strikeforce ala Fabricio Werdum. Similarly TUF veterans will not find the WEC as appealing as taking their new found fame elsewhere.
E. Spencer Kyte September 12, 2009 at 3:40 PM
I don't necessarily understand the "more money than the WEC can afford" angle, as the WEC is owned by Zuffa they have the deepest pockets in the business.
Would they be willing to up the pay scales is another question altogether.
For me, I see the potential of two-way contracts like they have in many sports; you make X if you fight in the WEC and Y if you fight in the UFC.
I don't think the only reason to merge the two should be about making more money off the already established talents the WEC has.
For me, this move would be about creating a place for fighters to grow and develop as much as it would be about cashing in on the names of guys like Brown and Faber right now.
If I were coming of TUF and had the choice between a two-way contract with the Zuffa organizations or a little more money with Strikeforce or Bellator, I take the Zuffa deal, as it's they're the best in the business.
But that's just me...
Additionally, this is exactly what I was hoping for in writing this piece... discussion!
My understanding is that the financials are handled separately. The WEC has to make enough money to pay it's fighters and can't borrow from the profits of the UFC.
Yes, the two run separately financially, but if Zuffa decides to make a move such as the one I'm suggesting, including the inclusion of WEC fighters on Fight Nights and Spike programming, more money will be available to the WEC.


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Q: Kotlin: idiomatic way to map a list of objects to a list of values from one field? I map a list of objects to a list of the value of one of the fields of the objects.
In this case, the variable languages is a List<Language> where the class Language has a field code that is of type String:
val languageCodes:List<String> = languages.map { language -> language.code }

Is there a more idiomatic and/or concise way to do this (except to omit the type of the list, which I left in this example for the sake of clarity)?

A: You more or less have it. To make it a bit clearer, you could eliminate language in favor of it, the default name of a single argument to a lambda:
val languageCodes = languages.map { it.code }

But what you have would work just as well, and is probably about as clear (this is subjective). You'll notice I named my val "languageCodes", as your reuse of languages wouldn't compile.

A: I found about the it keyword that refers to an implicit single parameter of a lambda:
val languageCodes:List<String> = languages.map { it.code }

Pretty neat!
Quoting the official doc:
It's very common that a lambda expression has only one parameter.
If the compiler can figure the signature out itself, it is allowed not to declare the only parameter and omit ->. The parameter will be implicitly declared under the name it.

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Wolfram Research, Inc. je americká softwarová společnost, jejímž hlavním produktem je programovací jazyk Wolfram Language a sada softwarových nástrojů umožňujících práci s tímto jazykem. Nejvýraznějším takovým nástrojem je výpočetní systém Mathematica, ačkoli v současnosti (rok 2022) společnost nabízí různé produkty cílené na různé typy zákazníků. Společnost založil v roce 1987 se svými spolupracovníky Stephen Wolfram, jenž je dosud též jejím výkonným ředitelem.

Produkty

Prvním a hlavním produktem společnosti byl po dlouhá léta výpočetní systém Mathematica. Spolu s uvedením její verze 10.0.0 však došlo k zavedení dalších produktů a především k přejmenování produktů stávajících, což vedlo k rozporuplnému přijetí mezi uživateli, které je ještě umocněno tím, že firma nepopisuje zřetelně rozdíly mezi svými produkty, které v některých případech působí duplicitně. Aktuální seznam všech produktů společnosti lze nalézt na jejích oficiálních stránkách. Níže je podán pouze jejich výběr.

Mathematica a deriváty

Mathematica

Jedná se o historicky nejvýraznější softwarový produkt firmy, jenž slouží k provádění výpočtů v závislosti na příkazech zadávaných v jazyce Wolfram Language vyvíjeném toutéž společností. Do verze 10.0.0 označovalo jméno Mathematica jak vývojové prostředí, tak samotný jazyk, více viz oddíl "Pojmenování" v článku Wolfram Language. Nástroj rozšiřující základní funkčnost Mathematicy pro distribuované výpočty se nazývá gridMathematica a inzeruje se odděleně.

Wolfram|One
Wolfram|One je v současnosti (rok 2022) hlavním produktem firmy, určeným pro nové uživatele, kde je přitom zdůrazňováno propojení s cloudem. Interně je však tento produkt patrně ekvivalentní Mathematice. Cloudová služba společnosti se jmenuje Wolfram Cloud a umožňuje ukládání dat spolu s vývojem a prováděním kódu ve Wolfram Language na vzdáleném serveru. A to buď přímo z desktopového prostředí či prostřednictvím webového vývojového prostředí s omezenější funkčností.

Wolfram Engine
Wolfram Engine je patrně výpočetní jádro, na kterém je vybudována Mathematica a podobné produkty. Namísto notebooků lze k jádru přistupovat přes příkazovou řádku pomocí programu WolframScript.

Wolfram Player
Wolfram Player je okleštěná forma Mathematicy, v níž je znemožněno vyhodnocování kódu a notebooky tak lze otevřít pouze pro čtení s velmi omezenou možností interakce. Jednou z povolených interakcí je například otáčení 3D grafiky. Tento produkt je stáhnutelný zdarma.

Wolfram Workbench
Wolfram Workbench je plug-in do vývojového prostředí Eclipse, které umožňuje vývoj kódu ve Wolfram Language. Toto prostředí, primárně určené pro vývoj programů v jazyce Java, poskytuje rozšířené možnosti pro ladění a profilování kódu. Až do verze Mathematicy 13.0.0 byl Workbench jedinou možností pro koncové uživatele, jak vytvořit interaktivní dokumentaci pro svůj kód.

Wolfram|Alpha

Wolfram|Alpha je online služba pro rozpoznávání příkazů zadaných v přirozeném jazyce, kde je na základě dotazu položeného v angličtině vrácen interaktivní výpis výsledků. Tyto výsledky mohou mít například formu dat či spočteného matematického výrazu.

System Modeler

Wolfram System Modeler je grafické programovací prostředí pro modelování a simulaci komplexních systémů zahrnujících mechanické, elektrické, termální a jiné součásti. Jedná se o implementaci modelovacího jazyka Modelica.

Další projekty

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Every time I hear stories about people who lived over 100, the first question I ask myself is: Is it really attainable? Or is it just one of those stories that are yet to be discovered? At this day and age, most young people are now susceptible to chronic diseases such as Diabetes, Hypertension, and Imminent Heart Attacks, just to name a few. Due to sedentary lifestyle and obesity, the life expectancy of people is now shorter than before.
However, as proven by people who lived over 100, also known as, centenarians, longevity is attainable.
Centenarians, as the name implies, are people who lived 100 years and beyond. Scientists have discovered places such as Sardinia Italy, Islands of Okinawa Japan, Loma Linda California, Nicoya, Costa Rica, and Ikaria, Greece, where centenarians are active and healthy! As a matter of fact, there's a village in Bapan, China, called "The Longevity Village," that shows significant number of residents, who prove, it is possible.
Further and as affirmed by United Nations, 2.2 million people are expected to live over 100 years by 2050. Another fact was written by Wikipedia, an estimated 316,600 centenarians were recorded last 2012, worldwide.
Now that we know it is possible, the next question is, how do we that? These 8 tangible secrets to attaining longevity will surprisingly change your perception in life.
No, it's not about exercise nor genes. Know these 8 secrets to living longer from people who lived over 100 years.
Just because you're not in the gym, doesn't mean you're already compromising your health. For centenarians, like Benjamin Goldfaden, 100 (September 16, 1913 – March 25, 2013), who played basketball during 1940's, keeping an active lifestyle means constantly moving. Cardiologist John Day, M.D., of Intermountain Healthcare in Utah, proved it as well when he visited The Longevity Village in 2013. He noticed that apart from being a rural place, most of them are NOT exercising but are physically active doing routines like farming, walking to reach places, etc. Keeping an active lifestyle isn't a secret. After all, it is a must to further life expectancies.
In this rat-race world, everyone's time is compromised by bills and other major responsibilities. In short, life is now shorter – but NOT for these centenarians. For them, spending time with the family is pivotal. Another study done by Harvard Health Department affirmed that having quality relationships matter. It was also proven that if someone is happy and content with their respective families and/or relationships, they have lower risk of cardiovascular diseases compared to low-satisfying relationships, such as bad marriages, etc.
Often heard but hardly practiced. However, Anthony Mancinelli (101), tagged as the oldest barber in the world and Ebby Halliday (103 last March 2014) proved that having a pair of healthy lungs can be a stepping stone to longevity!
Fun fact: Jeanne Calment (February 21, 1875 – August 4, 1997) of France, who lived 122 years, was an exemption! She started smoking at the age of 21 and stopped at 117 years old. She must be the real wonder woman, you think?
Being sociable doesn't necessarily mean being at a bar drinking with friends. Their definition of it is having a strong social relationship with friends by going out for a walk, small talks over a cup of coffee, etc. As witnessed by Dr. Day, people in the Longevity Village are actively participating in their community and family by sharing their expertise when it comes to family and child rearing. A data collated from one study which examined 309,000 people show that lack of social interaction increased the risk of premature deaths up to 50% or similar to someone smoking 15 cigarettes a day and has an inactive lifestyle. Therefore, having a sense of purpose is a significant reason for attaining longevity. Thus, stay sociable, reach out and stop being a hermit.
Sadly, stress has now become a part of our lives. It is colloquially known to be the culprit on developing chronic diseases. However, according to Dr. Leila Denmark (February 1 1898 – April 1, 2012), a Pediatrician, also tagged as the oldest and longest medical practitioner in America, who lived for 113 years, eating right and doing what you love is the essential key to longevity! It may be easier said than done, but obviously, it can be done!
"Laughter keeps you healthy," said Bel Kaufman, 101 (May 10, 1911 – July 25, 2014), an American Teacher and Author of the bestselling novel, Up The Down Staircase. Amazing as it is, laughter is indeed and proven as the best medicine. After all, it's free and inexpensive. So, forget breaking wind, laugh out loud if you can!
Has been the mantra of these centenarians. Although, some didn't follow this rule, but the majority of them did. The secret to their longevity is keeping a well-balanced meal every day.
Carpe Diem, or "seize the day" as what all of them said. They just took it one day at a time and enjoyed every moment of it. The simpler your life is, the better. Samuel Ball, who lived for 102 years, had a simple yet powerful advice: Find a good partner, enjoy two scotches a night and lastly, be easy going. If you follow his advice, you will definitely embrace longevity.
Secrets are meant to be kept, but, if it will promote longevity, more should be revealed. These secrets are not hard to do, after all.


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A biography of kurt vonnegut jr a contemporary american author
Though reviews were mixed, it quickly became a best seller. Vonnegut recalled the sirens going off whenever another city was bombed. Eternal Returns, or Tralfamadorian Ethics: In addition to promoting the work of Vonnegut, the nonprofit organization served as a cultural and educational resource centre, including a museum, an art gallery, and a reading room.
Rosewater more "a cry from the heart than a novel under its author's full intellectual control", that reflected family and emotional stresses Vonnegut was going through at the time.
This type of alien visitor would recur throughout Vonnegut's literature. Instead of waiting to be drafted, he enlisted in the army and in March reported to Fort BraggNorth Carolina, for basic training. We Are What We Pretend to Be comprised an early unpublished novella and a fragment of a novel unfinished at his death.
He was placed on academic probation in May and dropped out the following January. James, Steven, and Kurt Adams. Another play, Who Am I this Time?
He saw his completed version as partially autobiographical, mourning for his lost America. Like Vonnegut, the main character of Slaughterhouse-Five, named Billy Pilgrim, was present at the bombing of Dresden and has been deeply affected by the experience.
While being moved as a result of Patton's advance, they awoke one morning to find their guards gone. In addition, he suffers from a peculiar condition, of being "unstuck in time," meaning that he randomly experiences events from his past, present, and future. Hoenikker, in addition to the bomb, has developed another threat to mankind, ice-9, solid water stable at room temperature, and if a particle of it is dropped in water, all of it becomes ice Vonnegut called the disagreements "painful", and said the resulting split was a "terrible, unavoidable accident that we were ill-equipped to understand.
There were also mental problems in his wife's family. Vonnegut was also at this time trying his hand at journalism with articles in Esquire, New York Times Book Review, and other places. Thus, they did not teach their youngest son German or introduce him to German literature and tradition, leaving him feeling "ignorant and rootless.
He escapes to a television studio, tears away his handicaps, and frees a ballerina from her lead weights. November 11, — April 11, was an American writer. He described his work there: He later stated that the loss of confidence in government that Vietnam caused finally allowed for an honest conversation regarding events like Dresden.
But in this critical study, Robert Tally argues that Vonnegut himself is actually a modernist, who is less interested in indulging in the free play of signifiers than in attempting to construct a model that could encompass the American experience at the end of the twentieth century.
Burger suggested he quit GE, a course he had contemplated before.
The rigor of writing daily to deadlines helped shape his habits as a writer. This contradiction between the novel's fatalism and its advocacy of human effort is jarring. An Autobiographical Collage of the s.
Before its release Vonnegut noted that Timequake would be his last novel.The bibliography of Kurt Vonnegut includes essays, books and fiction, as well as film and television adaptations of works written by the Indianapolis-born author. Vonnegut began his literary career with science fiction short stories and novels, but abandoned the genre to focus on political writings and painting in his later life.
Kurt Vonnegut is acknowledged as a major voice in American literature and applauded for his subtle criticisms and sharp portrayal of modern society. Kurt Vonnegut Jr. was born on November 11,in Indianapolis, Indiana, the son of a successful architect, Kurt Sr., and his wife, Edith Sophia.
"Kurt Vonnegut and the American Novel is an exciting re-evaluation of this much overlooked author's work. Tally deftly rereads Vonnegut's novels, situating them in an American tradition of fiction that seeks to make sense of the larger American experience. Kurt Vonnegut, Jr., was born in Indianapolis, Indiana, to third-generation German-American parents, Edith (Lieber) Vonnegut and Kurt Vonnegut, Sr.
Both his father and his grandfather Bernard Vonnegut attended the Massachusetts Institute of Technology and were architects in the Indianapolis firm of Vonnegut & Bohn.
Kurt Vonnegut, in full Kurt Vonnegut, Jr., (born November 11,Indianapolis, Indiana, U.S.—died April 11,New York, New York), American writer noted for his wryly satirical novels who frequently used postmodern techniques as well as elements of fantasy and science fiction to highlight the horrors and ironies of 20th-century civilization.
Indiana author connection with Kurt Vonnegut (Indy Style on WISH-TV, 7/12/13) Kurt Jr. and the Vonneguts Rare military training cartoons to be shown at Kurt Vonnegut Memorial Library (Dayton Daily private nonprofit organization The Kurt Vonnegut Museum and Library is proud to partner with the following American Writer's Museum.
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Chaplains are here for patients and families of all faiths. We can contact representatives of most faith communities. The chapel is open for quiet prayer and reflection.
The on-call chaplain is available 24/7 via switchboard.


THE STUDENT MOVEMENT TODAY is the one organised, significant segment of the intellectual community that has a real and active commitment to the kind of social change that our society desperately needs. Developments now taking place may lead to its destruction, in part through repression, in part through what I think are rather foolish tactics on the part of the student movement itself. I think this would be a great, perhaps irreparable, loss. And I think if it does take place the blame will largely fall on the liberal enlightened community that has permitted a situation to arise in which the most committed, sincere, and most socially active of young people are perhaps working themselves into a position at the end of a limb, from which they may be sawed off at great cost to all of us and to society as a whole.
Chomsky, Noam, In Defence of the Student Movement, Australian Left Review, 1(33), 1971, 2-11.


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Meeting Coverage > AAIC
Agent Orange Plus PTSD Equals Extra Dementia Risk
— For Vietnam vets, defoliant exposure "may exacerbate effects of other risk factors."
by Kristina Fiore, Staff Writer, MedPage Today July 21, 2015
WASHINGTON -- Exposure to Agent Orange during the Vietnam War doesn't seem to increase the risk of dementia on its own, but it may exacerbate the effects of other risk factors like PTSD, researchers reported here.
In an analysis of Veterans Affairs data, having been exposed to Agent Orange and having PTSD together was associated with a significantly increased risk of dementia (hazard ratio 1.67, 95% CI 1.27 to 2.18), according to Deborah Barnes, PhD, MPH, of the University of California San Francisco and the San Francisco VA Medical Center.
They reported their findings at the Alzheimer's Association International Conference here.
Some 8% of veterans were exposed to Agent Orange during the Vietnam War, where it was used as an herbicide to clear dense areas of forest.
It comprised two major ingredients: 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T); dioxin was an unintended contaminant, as it's highly toxic and a known human carcinogen.
Many other studies have looked at the health effects of Agent Orange exposure, and there have been mixed results regarding its neurological effects. Some studies found no adverse neurologic effects, while several recent studies have found worse cognitive function with greater exposure. Other studies have shown that verbal memory is the most affected neurocognitive region among Vietnam veterans.
Yet there haven't been any studies specifically looking at the relationship between Agent Orange and the risk of dementia among these veterans, Barnes said.
Consequently, she and colleagues accessed VA electronic medical record data on 46,737 Vietnam veterans over age 55 who had at least one baseline visit and one follow-up visit, and who did not have dementia at baseline.
They looked at Agent Orange exposure alone and in combination with PTSD. They used Fine-Grey proportional hazards models to account for competing risk of death.
Barnes noted that there was a significant difference between the exposed and unexposed populations at baseline. Veterans exposed to Agent Orange were younger and had more comorbidities including diabetes, hypertension, cerebrovascular disease, depression, and PTSD (P<0.001).
In an unadjusted analysis, the researchers found that having been exposed to Agent Orange was associated with an increased risk of dementia (HR 1.31, 95% CI 1.15 to 1.51).
However, that risk disappeared in unadjusted models, Barnes said.
When they looked at PTSD and dementia risk, however, they did find a significant association (HR 1.33, 95% CI 1.10 to 1.62) -- and having both Agent Orange exposure and PTSD together was associated with a larger increase in risk of dementia (HR 1.67, 95% CI 1.27 to 2.18).
"Agent Orange alone doesn't appear to increase the risk of dementia," Barnes said, "but it may exacerbate the effects of other risk factors such as PTSD."
She cautioned that the findings were limited because the researchers weren't able to measure the actual exposure to Agent Orange; they had to rely on patients' own reports of exposure. Future studies should aim for verification of exposure, she said.
Barnes disclosed no financial relationships with industry.
Alzheimer's Association International Conference
Source Reference: Barnes D, et al "Agent Orange exposure and risk of dementia in Vietnam Veterans" AAIC 2015; Abstract O1-09-02.


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Bangor today hosted The Alliance Party's 2008 Conference at The Marine Court Hotel.
Our Party Leader David Ford MLA delivered an excellent speech remarking "I want to bring our agenda into government and deliver in the long-term where the tribal parties have failed".
Deputy Leader Naomi Long MLA stated "The sad reality is that as Sinn Fein and the DUP continue to battle daily with each other, the biggest losers in those battles have been the people of Northern Ireland and the biggest casualty has been public confidence in politics"
Dr Stephen Farry MLA remarked "In another sense, there is growing evidence that those societies that are able to attract and sustain a mixed population tend to be more successful in that talented people are nourished. This truism illustrates the folly of the homophobia from the Iris Robinson's of this world"
Following the conference I managed to watch part of the match between Northern Ireland's gay friendly rugby team The Ulster Titans and Bangor RFC thirds at Uprichard Park, Bangor. It was a great end to the conference which was about making the case for a Shared Future where everyone is valued and allowed to reach their full potential regardless of religion, race, sexual orientation etc.
During the morning session of the Conference I was given the opportunity to speak on a motion calling for publication of the Cohesion, Sharing and Integration Strategy. Below is the speech I delivered.
Conference, it gives me great pleasure to speak to you in support of this motion at what is my first Alliance Party Conference.
Everywhere I go in Northern Ireland I hear the same message. We want change.
The people of Northern Ireland are fed up with bickering and bigotry and want a government with ambition that dares to hope.
We live in a region where only 3% of people would object to having a person of the opposite religion as a neighbour. A region where over 70% of people wouldn't mind if a relative or friend was in a relationship with a gay person.
But yet we have an Executive with Ministers and a First Lady preaching intolerance and prejudice.
People want a change. People want a political system which reflects the new Northern Ireland.
At the heart of the New Northern Ireland must be a society which embraces and celebrates diversity.
Government can help deliver this by providing leadership and a strategy for a Shared Future.
When devolution returned last year we were promised a new bigger and better strategy for a Shared Future but we are still waiting. It's no where to be found.
Maybe CSI should be put onto the case to find the Cohesion, Sharing and Integration Strategy because it seems to be unlawfully at large.
Whilst the Executive dithers and delays The Alliance Party need to continue making the case for diversity.
We need to outline not merely the costs of segregation but rather the opportunities of diversity.
Recent research has proven that cities, towns and regions which embrace and celebrate diversity attract high value knowledge based industries. We need to continually remind people and other political parties that intolerant and prejudicial attitudes don't just harm minorities but also the economy we all thrive off.
We need to end the poverty of aspiration where people fail to hope beyond today, neglect to look to tomorrow and forget that bigotry and division not only damages lives but deters investment and consumes public expenditure.
We must continue spreading the Alliance message of hope and aspiration at every opportunity, whether it be in Council chambers, at the Assembly or maybe at the Executive table.
The people of Northern Ireland are increasingly looking for leadership, hope and change.
Day after day they ask the Executive parties whether they can deliver and the answer is a resounding no.
The only future the Executive parties offer is a mirror image of the past.
To those who ask whether Alliance can deliver my answer to you today is Yes we can.
We'll never get a meaningful shared future policy while the Executive is dominated by parties who don't believe in it. The old Shared Future policy was brought in by Westminister ministers while the Assembly was suspended, I have to say.
Spending more time with the family
An eye for an eye leaves everyone blind
XL have excelled in causing misery and chaos
Helping charities at the press of a button
Time for an end to the circus


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Featured Fiction: Where White Horses Gallop by Beatrice Macneil
Submitted by clelia on October 26, 2007 - 10:37am
By Beatrice Macneil
Key Porter Books, 2007
In the white-shingled houses of Beinn Barra, young men shine their shoes and young girls curl their hair. It's Saturday night, there's a dance in the parish hall, and Benny Doucet is playing. They come from all over Cape Breton to hear "Strings" Doucet play the fiddle.
But it is 1939. England has declared war on Germany. Canada will march beside the mother country. Three friends enlist in the legendary Cape Breton Highlanders: fisherman Hector MacDonald, gifted musician Benny Doucet, and Calum MacPherson, who has been accepted at Dalhousie to study medicine. The three friends sail off to war in November 1941.
The families wait for word of their boys. Gunner MacDonald, a returned man himself, knows only too well what his fisherman son will witness in the trenches of Europe. Joachim and Ona MacPherson seek, and fail to find, solace in each other. Napoleon and Flora Doucet finger their rosary beads at the kitchen table and pray for Benny's safety.
Where White Horses Gallop is a haunting tale of a war where emotional shrapnel riddles the spirit long after the guns a continent away have grown silent.
Beatrice Macneil is the author of the bestselling novel Butterflies Dance in the Dark, winner of the Dartmouth Fiction Award, and the short story collection There is a Mouse in the House of Miss Crouse, which won the Marianna Dempster Canadian Author's Award for Nova Scotia. She has written ten plays, four of which have won awards and two of which have been adapted for Halifax CBC Radio. She is the recipient of the Tic Butler Award for her outstanding contribution to Cape Breton writing and culture, and is the founder of Cape Breton's Reading Ceilidhs. She lives in Cape Breton, Nova Scotia.


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Cracking down on the swim bladder smugglers
Can cooperation between China, the US and Mexico stem the illegal trade in totoaba fish maw?
Zhang Chun	 April 29, 2019
Fish maw for sale (Image: Earnest Tse)
China's craze for the swim bladder of the totoaba, a giant Mexican fish, has driven it close to extinction. Dubbed "aquatic cocaine", trade in the delicacy is banned, but smuggling is still a major problem. There is new hope, however, thanks to increased awareness of the issue in China and better cooperation with both Mexico and the United States, a major transit point.
Since the beginning of 2018, Chinese customs authorities have uncovered five cases of smuggling in totoaba swim bladders, also known as fish maw. Thirty-two people have been arrested and 300 million yuan (US$45 million) worth of contraband seized.
But is this action enough to save the totoaba?
The decline of a species
In China, swim bladder is popular for its believed nutritional and medicinal properties. Sold dried, prices vary widely from a few hundred yuan per kilogramme to hundreds of thousands of yuan, depending on the species of origin. Trade in the maw of most species is not banned – selling the totoaba swim bladder, however, has been illegal since 1976, when the fish was added to the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES).
The swim bladder of the totoaba is particularly prized due to its large size and thickness, as well as its perceived health benefits. It is not the first victim of the Chinese taste for maw. The Chinese bahaba, native to the waters off south-eastern China, has a swim bladder with similar properties. Its popularity led to market prices so high it was known as "golden maw" – by weight, it cost more than gold. This led to overfishing and a rapid decline in the species' population. Although trade was banned on the Chinese mainland in 1989, numbers have continued to fall. In 2006 it was added to the International Union for Conservation of Nature's (IUCN) Red List as critically endangered.
A Chinese bahaba in 2003 (Image: Sadovy & Cheung)
With the bahaba nowhere to be found, China's maw traders turned their attention to the totoaba. But the totoaba was already in crisis – in the early to mid-twentieth century, Mexico exported large quantities of the fish to the United States, where it was prized for its flesh rather than its swim bladder. By the 1970s, overfishing had led to its inclusion on the IUCN Red List. With the increased demand from China, it is now listed as critically endangered.
It is not just the totoaba that has suffered. Overfishing in the Gulf of California has also spelt disaster for a small porpoise similar in size to the totoaba. The vaquita is often accidentally caught in the fine-meshed gillnets used to catch the fish. According to the IUCN, there are currently only 18 adult vaquita still alive.
Exposing the underground trade
The plight of the totoaba and vaquita only came to international attention in 2013, when an alert customs officer triggered the first US seizure of totoaba maw at the Mexican border.
In that year a Mexican vaquita expert published research showing that existing conservation measures had failed to stop the cetacean being killed. In 2014, the Centre for Biological Diversity, a US conservation group, published an open letter calling for a ban on gillnet fishing to protect the critically endangered animal.
That letter prompted the China CITES Endangered Species Scientific Commission to take note of these distant species. "We knew they were on the CITES appendices and that trade was banned. But we hadn't realised the connection with China," said Zeng Yan, assistant to the director of the commission.
When China cracked its first totoaba smuggling case in 2018, it was with remote assistance from the commission. A customs official at Guilin's international airport in south China, unsure of what he had found, sent an image to the commission's experts and, with help from Zeng Yan, identified the contraband as totoaba maw.
"It was only during our first investigation [in 2015] that we realized there was an underground trade… Front-line commercial and customs officials haven't necessarily seen it," said Hua Ning, head of the ecology department at the China office of the Natural Resources Defense Council, an American advocacy group.
A joint enforcement alliance
It is hard for any one country to tackle an illegal supply chain that stretches halfway around the world. It also takes time to set up cooperative mechanisms. It was only in June 2015 that China and the US first had high-level discussions on how to tackle the smuggling.
Investigations by environmental groups helped spur action. In May 2015, Greenpeace reported totoaba maw on sale in a number of shops in Guangzhou and Hong Kong. That year saw the first official investigations, which also found specimens for sale in Hong Kong and the mainland.
At a CITES conference in October 2016, China, the US and Mexico formally proposed to protect the totoaba together.
One of the toughest challenges in policing the trade in totoaba maw is identifying it, especially at a busy customs post. To address this, in December 2016, various government bodies, including China's State Office for the Trade in Endangered Species, and international environmental groups the Natural Resources Defense Council and WildAid, held a workshop in Guangzhou. The event was attended by over 100 people, including representatives from China's customs, fisheries, markets and coastguard authorities.
It showcased technology that can be used to identify totoaba maw and issued a quick visual identification guide to be stored on mobile phones. Representatives from the US Fish and Wildlife Service and the Mexican environmental authorities also shared their experiences of totoaba conservation.
In July 2017, a second workshop took place in San Felipe, Mexico, close to the habitats of the totoaba and vaquita. Both of these training initiatives were key in enabling the Chinese customs authorities to crack their first totoaba smuggling operation the following year.
With better policing, there is a risk that totoaba maw smuggling is now moving further underground, according to Bonnie Tang a campaigner with Greenpeace Hong Kong.
In Hong Kong, only one case of totoaba maw smuggling has resulted in charges since 2015. Minutes taken from a 2017 meeting of the Hong Kong Agriculture, Fisheries and Conservation Department's Endangered Species Advisory Committee show no totoaba swim bladder was found in the region from its first discovery in 2015 to the end of 2016.
But the trade has not stopped. Gary Stokes, former director of Sea Shepherd Asia, relates that two German filmmakers hoping to document the illegal totoaba trade had, on his recommendation, visited Macau. Within four hours they found someone willing to sell them top-grade totoaba maw. They were shown pictures of the goods, which were said to be in Hong Kong, and were told it would be no problem to have them shipped over.
Despite this, Bonnie Tang thinks the increased enforcement by China's customs authorities is sending a crucial signal to both traders and consumers. Greater media coverage is also helping people understand the impact of totoaba swim bladder consumption. Such public awareness is crucial, as online communication and easier travel continue to boost its popularity as a "nutritional" food.
This will no doubt help another species of fish currently threatened by China's hunger for swim bladder, the Nile perch. But for Mexico's vaquita, so close to the precipice of extinction, much more needs to be done.
"We can't save the vaquita just by tackling fish maw smuggling," said Zeng Yan, pointing out that habitat protection is also crucial. "Only they [the countries of origin] can ensure a species is able to survive in its natural habitat," she added.
Zhang Chun is a senior researcher at chinadialogue.
Nile perch threatened in East Africa
China's taste for swim bladder is pushing the species to the brink in Lake Victoria
Geoffrey Kamadi
What We Eat
Pressure mounts to take blue shark off the menu
Blue sharks are still being overfished despite a clamp down on the shark fin trade
Ryan Kilpatrick


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Q: Django Query Filter Null This is my query
count = Attend.objects.filter(date = NULL).count()

This gives me an enter date in correct format error. I am trying to filter for date set as null.
what do i do?

A: count = Attend.objects.filter(date__isnull = True).count()


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Apple says they're sorry what you told Siri wasn't private
Apple has never been one for public apologies. Back in 2010, the company came under fire for its defiance during the "antennagate" fiasco. Rather than apologize for a faulty antenna design, Steve Jobs told reporters they were "holding it wrong," inspiring countless memes and criticism in the process.
And for years, the company continued to maintain a boldface against criticism. Despite negative press on manufacturing conditions, build quality and long wait times for support, Apple soldiered on without so much as an apology.
But all that changed in light of the news that Apple, like its industry peers, was recording and transcribing user interactions with Siri. It's issued a public apology for the ordeal and has made some significant changes to its privacy policy in response. Here's what it said, as well as how these changes will affect your privacy going forward.
Apple issues a formal apology for Siri debacle
Apple stunned fans, critics and analysts the world over with a public apology over the revelation that Siri, like Alexa and Google Assistant, had been recording user interactions for transcription.
According to Apple, the company realized that they "haven't been fully living up to [their] high ideals," and stated that privacy is a "fundamental human right" to be respected by their designs and services in the years ahead.
Your daily dose of tech smarts
Learn the tech tips and tricks only the pros know.
In addition to the eloquent statement, the company provided a road map for privacy changes as they apply to Siri in the future. Starting now, Apple will no longer rely on audio recordings to improve Siri by default, and will instead allow customers to opt-in if they decide to participate.
For those who do choose to participate, the company states that only Apple employees will be listening to any recordings and that "accidental recordings" triggered by Siri misunderstandings would be automatically discarded.
What does Apple's apology actually mean for privacy?
Eagle-eyed observers are quick to point out that Apple, despite not listening to literal recordings, would still be relying on some form of transcription in order to "improve" Siri.
Rather than harvest audio clips themselves, Siri will instead convert audio into text for Apple's engineers to look over. This means that, even though your voice isn't audible to the reviewer, they'll still know the contents of what you said.
Of course, considering Siri's skill level at understanding commands, you might not have too much to worry about. She makes enough mistakes as it is.
But therein lies the issue at hand for Apple. Creating voice recognition technology requires a good deal of trial and error, which is why large samples of audio help the software compare and analyze its own templates for course correction. Without samples, the technology would probably be quite a bit further behind.
Still, there is controversy over whether or not any form of audio transcription violates user privacy. The real issue, however, lies in transparency. Had Apple been upfront about its transcription policy, there might have even been an enthusiastic base of willing participants — like there was for the $5 face scanning program from Google.
Nearly every major tech company has faced some kind of data reckoning recently, but it's the responsibility of a corporation to serve the needs of its customers as well as its shareholders. And so far, it looks as if the pendulum is swinging in the direction of privacy again. The powers that be would be wise to listen.
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Robocalls are not only annoying, but they scam Americans out of millions every year. Learn Kim's tricks for stopping them for good in this handy guide.


It's sometimes easy to forget that the Academy Award nominees are movie fans at heart. With the Oscars just days away (coming this Sunday evening), this year's nominees in the acting category -- from Melissa McCarthy and Viola Davis to George Clooney and Meryl Streep -- reminisce about their favorite movie moments of all time.
The answers span genres and decades, from Network's iconic "I'm mad as hell and I'm not going to take it anymore" moment to Goodfellas, The Godfather, Chitty Chitty Bang Bang and The Sound of Music.
For the full videos of 18 out of the 20 acting nominees smiling over their favorite movie moments, head on over to Oscars.com.


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Home > Vol 5, No 17 >	Idogawa
Published in Oncotarget V5N17, Sep 15, 2014
Research Papers:
Array-based genome-wide RNAi screening to identify shRNAs that enhance p53-related apoptosis in human cancer cells
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Oncotarget. 2014; 5:7540-7548. https://doi.org/10.18632/oncotarget.2272
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Masashi Idogawa, Tomoko Ohashi, Jun Sugisaka, Yasushi Sasaki, Hiromu Suzuki and Takashi Tokino _
Masashi Idogawa1,*, Tomoko Ohashi1,*, Jun Sugisaka1, Yasushi Sasaki1, Hiromu Suzuki2 and Takashi Tokino1
1 Department of Medical Genome Sciences, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan
2 Department of Molecular Biology, Sapporo Medical University School of Medicine, Sapporo, Japan
* These authors contributed equally to this work
Takashi Tokino, email:
Keywords: p53; shRNA library; apoptosis; PRIMA-1
Received: April 30, 2014 Accepted: July 26, 2014 Published: July 27, 2014
p53 transduction is a potentially effective cancer therapy but does not result in a good therapeutic response in all human cancers due to resistance to apoptosis. To discover factors that overcome resistance to p53-induced apoptosis, we attempted to identify RNAi sequences that enhance p53-induced apoptosis. We screened a genome-wide lentiviral shRNA library in liver cancer Huh-7 and pancreatic cancer Panc-1 cells, both of which resist p53-induced apoptosis. After the infection of adenovirus expressing p53 or LacZ as a control, shRNA-treated populations were analyzed by microarray. We identified shRNAs that were significantly decreased in p53-infected cells compared with control cells. Among these shRNAs, shRNA-58335 was markedly decreased in both cancer cell lines tested. shRNA-58335 enhanced p53-related apoptosis in vitro and augmented the inhibitory effect of adenoviral p53 transduction on tumor growth in vivo. Furthermore, the enhanced apoptotic response by shRNA-58335 was also confirmed by treatment with PRIMA-1, which reactivates mutant p53, instead of adenoviral p53 transduction. We found that shRNA-58335 evokes the apoptotic response following p53 transduction or functional restoration of p53 with a small molecule drug in cancer cells resistant to p53-induced apoptosis. The combination of p53 restoration and RNAi-based drugs is expected to be a promising novel cancer therapy.
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p53 is one of the most important tumor suppressor genes. In approximately half of all human cancers, p53 is inactivated as a direct result of mutations in the p53 gene [1]. Furthermore, mutation or deletion of p53 is related to poor prognosis and resistance to chemotherapy and radiation [2]. Therefore, vector-mediated gene transfer of p53 has been viewed as a potentially effective cancer therapy. However, gene transfer of p53 does not always result in good therapeutic outcomes in all cancers [3, 4]. The inactivation of apoptotic machinery is considered as one of the reasons that p53 transduction fails to induce apoptosis in cancers. In such cancers, p53 status does not necessarily correlate with the responsiveness to chemotherapy [5].
The activation of p53 is induced by a variety of cell stresses, such as DNA damage, oncogene activation, spindle damage and hypoxia. Activated p53 transactivates not only coding genes but also non-coding RNAs, including microRNAs (miRNAs) and large intergenic non-coding RNAs (lincRNAs), many of which are involved in DNA repair, cell cycle arrest and apoptosis [6, 7]. Although the precise mechanisms that regulate whether a cell undergoes cell cycle arrest or apoptosis are unclear, cell cycle arrest induced by p53 is mediated primarily through the induction of the expression of p21 [8], which is involved in restoring genomic integrity by functioning in an anti-apoptotic manner [9]. Thus, the suppression of p53-induced expression of p21 may lead to the preferential induction of apoptosis rather than cell cycle arrest following p53 activation. In our previous study, we constructed a replication-deficient recombinant adenovirus that encoded co-cistronic p53 and artificial microRNAs targeting endogenous p21, resulting in simultaneous p53 expression and the suppression of p21 induction. In colorectal and hepatocellular carcinoma cells, infection with the adenovirus vector augmented apoptosis and suppressed tumor growth compared with an adenovirus that expressed p53 alone in vitro and in vivo [10]. Thus, the combination of p53 and RNAi was shown to be a potential novel cancer therapy.
To discover factors that modulate p53 pathways, we attempted to identify RNAi sequences that enhance p53-induced apoptosis. In this study, we screened a genome-wide lentiviral shRNA library in cancer cells resistant to p53-related apoptosis. We identified specific shRNAs that were significantly decreased in p53-infected cells but not in control cells. These results indicated that specific shRNAs evoked the apoptotic response following p53 transduction or functional restoration of p53 in cancer cells resistant to p53-induced apoptosis, suggesting that the combination of p53 restoration and treatment with specific shRNAs may be an effective cancer therapy.
Genome-wide shRNA screening in p53-resistant cancer cells
To identify shRNAs that can relieve resistance to p53-induced apoptosis in cancer cells, we screened a genome-wide lentiviral shRNA library in liver cancer Huh-7 cells and pancreatic cancer Panc-1 cells, as summarized in Figure 1A and briefly described below. The Huh-7 and Panc-1 cell lines have mutated p53 and a weak apoptotic response following wild-type p53 transduction. The human shRNA library, comprising approximately 200,000 shRNA sequences targeting 47,400 human mRNA transcripts, was packaged into lentivirus particles and pooled. First, we infected the cell lines with the pooled lentiviral shRNA library. Seventy-two hours after lentiviral infection, the cells were infected with adenovirus expressing p53. Forty-eight hours after adenoviral infection, total mRNA was extracted, and mRNA, including shRNA sequences derived from lentiviral vectors, was amplified by PCR. In this shRNA library, each lentiviral vector shRNA sequence corresponds to a probe sequence found on an Affymetrix microarray (GeneChip). Therefore, the shRNA populations can be quantified by cDNA microarray to determine the expression levels of each shRNA; as a reference, the shRNA population in each cell line infected with adenovirus expressing LacZ was also analyzed.
Figure 1: Schematic representation of the array-based shRNA library screening process. p53-resistant cancer cells were infected with a pooled lentiviral shRNA library. After lentiviral infection, the cells were infected with adenovirus expressing p53 or LacZ. The shRNA population was analyzed by a cDNA microarray-based approach.
Identification of shRNA-decreased populations in p53-transduced cells
We identified the shRNAs that were significantly decreased in p53-infected cells compared with control cells (Fig. 2A). Several shRNAs evoked the apoptotic response following p53 transduction in cancer cells resistant to p53-induced apoptosis. In Huh-7 cells, 547 shRNAs were decreased more than 4-fold in cells with p53 transduction compared with control cells. In Panc-1 cells, 1418 shRNA were decreased more than 16-fold in cells with p53 transduction compared with control cells (Fig. 2A, areas encircled by dashed line). Furthermore, 28 shRNAs were commonly decreased in both cell lines (Fig. 2B, Supplementary Table S1). Among these shRNAs, shRNA-58335 in the presence of p53 was markedly decreased (66-fold in Huh-7 cells and 436-fold in Panc-1 cells). Therefore, we further analyzed shRNA-58335.
Figure 2: Analysis of the shRNA library screen in p53-transduced cells. (A) shRNA populations were quantified by cDNA microarray in p53-transduced Huh-7 and Panc-1 cells. The amount of each shRNA in p53-transduced cells was plotted against the amount of the shRNA in LacZ-transduced control cells as log2. In Huh-7 cells, 547 shRNAs decreased more than 4-fold in p53-transduced cells compared with control cells. In Panc-1 cells, 1418 shRNAs decreased more than 16-fold in p53-transduced cells compared with control cells (areas encircled by dashed lines). (B) The overlap of the selected shRNAs in Huh-7 and Panc-1 cells is displayed in a Venn diagram.
Enhancement of p53-induced apoptosis by shRNA-58335
Next, we stably infected lentivirus expressing shRNA-58335 or a control sequence into Huh-7 cells and quantified p53-induced apoptosis by evaluating the sub-G1 population. In these cells, p53 transduction induced a strong apoptotic response in shRNA-58335-infected cells compared with control cells (Fig. 3A, B). Additionally, treatment of adriamycin significantly enhanced the apoptotic response (Fig. 3A, B). Increased caspase-3 cleavage, which serves as another indicator of apoptosis, was also observed by western blotting (Fig. 3C). In colorectal cancer SW480 cells, which also have mutated p53 and a weak apoptotic response following p53 transduction, shRNA-58335 sensitized the p53-induced apoptotic response (Fig. 3D). These results indicated that shRNA-58335 improved the apoptotic response after p53 transduction in other cancer cell lines.
Figure 3: Effect of shRNA-58335 on p53-induced apoptosis. (A) Huh-7 cells were stably infected with lentivirus expressing shRNA-58335 or a control sequence. These cells were then infected with an adenovirus expressing p53 (Ad-p53: +) or LacZ (Ad-p53: -) as a control at an MOI of 200. Twenty-four hours after infection, the cells were treated with adriamycin (0.5 µg/ml) or not. Forty-eight hours after treatment, the cells were analyzed by flow cytometry. The percentage of cells in the sub-G1 phase is indicated. Error bars indicate the S.E. * indicates a p value < 0.05 by a t-test. (B) Representative flow cytometry data in Huh-7 cells infected with Ad-p53. The percentage of cells in the sub-G1 phase is indicated. (C) Under the same conditions used in (A), total cell lysates were extracted and analyzed by Western blot with the indicated antibodies. (D) SW480 cells were infected with lentivirus expressing shRNA-58335 (shRNA: +) or a control sequence (shRNA: -). These cells were then infected with an adenovirus expressing p53 (Ad-p53: +) or LacZ (Ad-p53: -) as a control at an MOI of 200. Forty-eight hours after infection, total cell lysates were extracted and analyzed by Western blot with the indicated antibodies.
Inhibition of tumor growth by p53 transduction with shRNA-58335 in vivo
To confirm whether the improved p53-induced apoptotic response in vitro was correlated with a therapeutic effect in vivo, we examined the tumor suppressive effect of p53 transduction in a xenograft model of tumorigenesis. shRNA-infected Huh-7 cells were injected s.c. into nude mice. When tumor volume reached a consistent size, adenovirus expressing p53 or LacZ was injected directly into the tumor at days 0, 1 and 2 (Fig. 4, arrow). In control shRNA-infected Huh-7 cells, the injection of Ad-p53 did not affect tumor volume (Fig. 4, left). Conversely, in shRNA-58335-infected Huh-7 cells, tumor volume was significantly decreased by injection of Ad-p53 compared with Ad-LacZ (Fig. 4, right). These results suggest that combined treatment with p53 and a specific shRNA may be an effective cancer therapy in vivo.
Figure 4: Therapeutic effect of shRNA-58335 in vivo. Huh-7 cells stably infected with lentivirus expressing shRNA-58335 or control sequences were injected s.c. into nude mice. When the tumor volume reached 100 mm3, adenovirus expressing p53 (Ad-p53) or LacZ as a control (Ad-LacZ) was injected directly into the tumors at days 0, 1 and 2 (indicated by arrows). Ad-p53, open circle; Ad-LacZ, closed circle. The data represent the average volume of three independent tumors injected with adenovirus. The volume of each tumor is expressed relative to the volume at day 0, which was set as 1. Error bars indicates S.E. * indicates a p value < 0.05 by a t-test.
Enhancement of PRIMA-1-induced apoptosis by shRNA-58335
p53 transduction by adenoviral vector is difficult to deliver to cancer tissues in vivo. Previous report indicated that histone deacetylase inhibitors reactivated mutant p53 [12]. Recently, PRIMA-1 was identified as a low-molecular-weight compound that restores the sequence-specific DNA binding and active conformation of mutant p53 proteins [13] and demonstrated positive data in phase 1/2 clinical trials of hematologic malignancies and prostate cancer [14]. Therefore, we replaced adenoviral p53 transduction with PRIMA-1 treatment to activate p53, performed as in Fig. 3A. As observed in Fig. 3A, the combinatorial treatment of PRIMA-1 and adriamycin induced a strong apoptotic response in shRNA-58335-infected cells compared with control-shRNA-infected cells (Fig. 5A, B). Furthermore, we evaluated the apoptotic effect on Panc-1 cells by shRNA-58335 and PRIMA-1. Adriamycin treatment alone did not induce an apoptotic response in Panc-1 cells (data not shown). Therefore, we treated Panc-1 cells with VP-16 (etoposide) and quantified apoptosis by evaluating the sub-G1 population (Fig. 5C). The combinatorial treatment of PRIMA-1 and VP-16 significantly enhanced the apoptotic response in shRNA-58335-infected cells compared with control-shRNA-infected cells. These results demonstrated that shRNA-58335 effectively enhanced the p53-induced apoptotic response not only by p53 transduction but also by the functional restoration of p53 with small molecule treatment.
Figure 5: Effect of shRNA-58335 on apoptosis with the functional restoration of p53 with PRIMA-1. (A) Huh-7 cells stably infected with lentivirus expressing shRNA-58335 or control sequences were treated with PRIMA-1 (200 µM) and/or adriamycin (0.5 µg/ml). Seventy-two hours after treatment, the cells were analyzed by flow cytometry. The percentage of cells in the sub-G1 phase is indicated. (B) Representative flow cytometry data in Huh-7 cells treated with PRIMA-1 and adriamycin. The percentage of cells in the sub-G1 phase is indicated. (C) Panc-1 cells were stably infected with lentivirus expressing shRNA-58335 or a control sequence. These cells were treated with VP-16 (300 µM) in the presence or absence of PRIMA-1 (200 µM). Forty-eight hours after treatment, the cells were analyzed by flow cytometry. The percentage of cells in the sub-G1 phase is indicated. In (A) and (C), error bars indicate the S.E. * indicates a p value < 0.05 by t-test.
Altered gene expression pattern by shRNA-58335 in p53-transduced cells
According to the NCBI BLAST search (http://blast.ncbi.nlm.nih.gov/), the seed sequence of shRNA-58335 is perfectly matched only to AP2γ (TFAP2C) among all human transcripts in the RefSeq database. AP2γ belongs to the AP-2 family of transcription factors and overexpressed in human breast cancers and the overexpression related with reduced patient survival [15]. A recent report demonstrated that AP2γ is a key transcriptional regulator for maintaining the luminal phenotype in human breast carcinoma [16]. AP2γ is also a direct transcriptional target of p53 [17]. We confirmed a statistically significant but slight decrease in AP2γ mRNA levels by shRNA-58335 (Supplementary Figure S1). However, we were unable to detect AP2γ protein using Western blotting. Therefore, it remains controversial whether shRNA-58335 works through the suppression of AP2γ.
To explore the effect of shRNA-58335 on p53-induced apoptosis, we evaluated gene expression by cDNA microarray analysis in p53-transduced Huh-7 cells. We found that the expression of 350 genes was decreased more than 2-fold in shRNA-58335-infected Huh-7 cells compared with control-shRNA infected cells (Supplementary Table S2). Interestingly, gene ontology analysis revealed that these genes were significantly associated with cell cycle progression and cell division (Table 1). These results indicate that shRNA-58335 evoked an apoptotic response following p53 transduction by modulating the gene expression associated with the process of cell division.
Table 1:Gene ontology analysis of 350 genes decreased by shRNA-58335 in p53-transduced Huh-7 cells.
9.91×10-32
nuclear division
M phase of mitotic cell cycle
organelle fission
M phase
mitotic cell cycle
cell cycle phase
cell cycle process
chromosome segregation
microtubule-based process
spindle organization
microtubule cytoskeleton organization
3.46×10-8
mitotic cell cycle checkpoint
regulation of cell cycle
cell cycle checkpoint
spindle checkpoint
regulation of mitotic cell cycle
mitotic spindle organization
protein-DNA complex assembly
mitotic sister chromatid segregation
sister chromatid segregation
regulation of mitotic metaphase/anaphase transition
mitotic cell cycle spindle assembly checkpoint
negative regulation of mitotic metaphase/anaphase transition
cytoskeleton organization
chromosome organization
cellular macromolecular complex subunit organization
negative regulation of mitosis
negative regulation of nuclear division
microtubule-based movement
DNA packaging
organelle localization
chromosome localization
establishment of chromosome localization
cellular macromolecular complex assembly
nucleosome assembly
regulation of cell cycle process
We demonstrated shRNAs that enhanced p53-related apoptosis by screening a genome-wide shRNA library. Conventional shRNA library screens have shown shRNAs that resist p53-induced apoptosis through the presence of p53-resistant cellular colonies [18]. However, this method is not able to identify shRNAs that enhance p53-related apoptosis because cells that include such shRNAs are lost. Recently, array-based shRNA screening was developed, allowing the direct quantification of the amount of each shRNA. We applied this new method here and successfully identified shRNAs that enhanced p53-related apoptosis.
Although the shRNA library used in this study included approximately 200,000 shRNA sequences targeting 47,400 human mRNA transcripts, the knockdown efficacy of each shRNA has not been validated. Therefore, the screened shRNAs, the sequences of which match specific transcripts, are not necessarily able to effectively knockdown their target genes. In fact, only weak suppression of the TFAP2C gene by shRNA-58335 was confirmed, although the seed sequence of shRNA-58335 completely matched the AP2γ mRNA sequence (Supplementary Figure 1). Furthermore, populations of other shRNAs that target AP2γ mRNA (shRNA-58334, 58336 and 58337) were not significantly decreased in p53-infected Huh-7 or Panc-1 cells compared with LacZ-infected control cells. However, shRNA-58335 effectively enhanced the p53-induced apoptotic response not only by p53 transduction (Fig. 3, 4) but also by the functional restoration of p53 with small molecule PRIMA-1 (Fig. 5). These results may suggest that shRNA-58335 operates as a molecular modulator in p53-related apoptosis through other unknown mechanisms rather than through the knockdown of the TFAP2C gene. Our microarray analysis revealed that shRNA-58335 with p53 transduction modulates gene expression associated with the process of cell division (Table 1), whereas no genes were significantly decreased more than twofold in shRNA-58335-infected cells without p53 transduction compared with control-shRNA-infected cells. These results also support our conclusion that shRNA-58335 does not knockdown any specific genes. However, the molecular mechanism of shRNA-58335-mediated enhancement of p53-related apoptosis is unclear. Further functional analysis should be performed in future studies.
We demonstrated that the apoptotic response induced by adenoviral p53 transduction was enhanced by shRNA-58335 in cancer cells resistant to p53-induced apoptosis (Fig. 3, 4). However, the clinical application of adenoviral vectors is presently limited because adenoviral vectors must be administered locally [4]. Therefore, we replaced adenoviral p53 transduction with PRIMA-1 treatment to reactivate mutant p53 [13] and successfully confirmed the enhancement of an apoptotic response by PRIMA-1 with shRNA-58335 treatment (Fig. 5). PRIMA-1 can be intravenously administered in human clinical studies [14]. Furthermore, non-viral delivery systems, such as nanoparticles, for RNAi-based drug treatments are also being developed and are in various stages of clinical investigation [19, 20].
Most of clinically used chemotherapeutic agents cause DNA damage and target dividing cells in S or M phase. Therefore, chemotherapeutic agents not only kill cancer cells but also injury normal dividing cells, resulting in adverse effects of cancer treatment. In order to protect normal cells from chemotherapeutic agents, pretreatment of drugs which induce cell cycle arrest in normal cells, so-called "cyclotherapy" is considered as an effective therapy [21, 22]. Recent reports suggest that the pretreatment of small-molecule p53 activators such as nutlin-3 protect normal cells from chemotherapeutic agents [23, 24]. The combination of p53 restoration, RNAi-based drugs and p53-based cyclotherapy is expected to be a promising novel cancer therapy in the near future.
The hepatic cancer Huh-7 cell line, the pancreatic cancer Panc-1 cell line and the colorectal cancer SW480 cell line were purchased from the American Type Culture Collection. Huh-7, Panc-1 and SW480 cells were cultured in DMEM, RPMI-1640 and Leibovitz L-15 medium with 10% FCS, respectively. The construction, purification and infection of replication-deficient recombinant adenoviruses containing p53 (Ad-p53) or the bacterial lacZ gene (Ad-LacZ) have been described previously [11].
shRNA library screening
shRNA library screening was performed according to the manufacturer's protocol. Briefly, 3×106 Huh-7 and Panc-1 cells were plated on a 10-cm plate. Twenty-four hours after plating, the cells were infected with lentivirus particles from the GeneNet siRNA Library (System Biosciences). Seventy-two hours after lentiviral infection, Ad-p53 or Ad-LacZ was infected into these cells at an MOI of 50. Forty-eight hours after adenoviral infection, total RNA was extracted with TRIZol (Invitrogen). cDNA was synthesized from extracted RNAs. siRNA inserts were amplified from cDNA using two rounds of PCR with biotinylated primers. PCR products were treated with lambda exonuclease to remove the sense non-biotinylated strand. Ten micrograms of biotinylated samples was hybridized to an Affymetrix GenChip microarray (HG-U133+ 2.0). The obtained data were analyzed with GeneNet siRNA Library Data Analysis Software (System Biosciences).
Establishment of cell lines stably expressing shRNAs
Huh-7 and Panc-1 cells were infected with lentivirus (pSIH-H1-puro) expressing shRNA-58335 (5'-TGGCGGCCCAGCAACTGTGTAAAGA ATCTTCCTGTCAGAATTCTTTACACAGTTGCTGG GCCGCCA-3') or luciferase control shRNA (5'-GTGCGTTGTTAGTACTAATCCTATTTGT GAAGCAGATGAAATAGGGTTGGTACTAGCAAC GCAC-3'). Twenty-four hours after infection, these cells were cultured with media containing puromycin (2 μg/ml in Huh-7 cells and 4 μg/ml in Panc-1 cells, respectively) and resistant cells were selected.
Adriamycin and VP-16 were purchased from Sigma. PRIMA-1 was purchased from Cayman Chemical. The anti-p53 (DO-1), anti-MDM2 (SMP14) and anti-p21 (F-5) mouse antibodies were purchased from Santa Cruz Biotechnology. The anti-actin mouse antibody was purchased from Millipore, and the anti-caspase-3 (8G10) rabbit antibody was purchased from Cell Signaling Technology.
Western blot analysis
Total cell lysate was extracted at 4°C with RIPA buffer (150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS and 50 mM Tris HCl, pH 8.0). The samples were fractionated by SDS-PAGE and transferred onto Immobilon-P membranes (Millipore). Immunoreactive proteins were detected using enhanced chemiluminescence (ECL) (GE Healthcare).
Cells (1×106) were plated on 6-well plates. After infection or treatment, the cells were harvested by trypsinization and pelleted by centrifugation. Pelleted cells were fixed in 90% cold ethanol, treated with RNase A (500 units/ml) and then stained with propidium iodide (50 mg/ml). The samples were analyzed on a FACSCalibur flow cytometer (BD Bioscience). The experiments were repeated at least three times, and 50,000 events were analyzed for each sample. Data were analyzed using FlowJo software (Tree Star).
All animals were maintained under specific pathogen free conditions and treated in accordance with guidelines set by the Animal Care and Use Committee of Sapporo Medical University. To evaluate the effects of treating established tumors, 24 female BALB/c nude mice were injected subcutaneously (s.c.) with 2×106 Huh-7 cells infected with lentiviral shRNA-58335 or control vector into both flanks. When the tumor size reached 100 mm3, the mice received a direct intratumoral injection of 1×109 p.f.u. (in 100 µl of PBS) of the indicated adenovirus a total of three times on days 0, 1 and 2. Three mice were used for each treatment group. Tumor formation in the mice was monitored for up to 2 weeks. The tumor volume was calculated using the equation V (mm3) = a×b2/2, where "a" represents the largest dimension and "b" is the perpendicular diameter.
Huh-7 cells stably expressing shRNA-58335 or control shRNA were infected with adenovirus expressing p53 (Ad-p53) or LacZ (Ad-LacZ) as control at 200 moi. Seventy-two hours after infection, total RNA was extracted by RNeasy mini kit (QIAGEN) according to manufacturer's protocol. Total RNA was labeled with Cy3, hybridized to a microarray (Agilent SurePrint G3 Human GE) and scanned by Agilent SureScan according to the manufacturer's protocols. The obtained data were normalized using Limma (R package). The microarray data were deposited in the NCBI Gene Expression Omnibus (GEO, accession number: GSE56156).
This research was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
The authors have no conflicts of interest to declare.
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