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Do the Most Important Artists Make the Most Expensive Paintings?
University of Chicago, Becker Friedman Institute for Economics Working Paper No. 2018-73
28 Pages Posted: 2 Oct 2018
See all articles by David W. Galenson
David W. Galenson
University of Chicago - Department of Economics; National Bureau of Economic Research (NBER)
Date Written: October 2, 2018
Art experts have disagreed about whether art markets accurately reflect artistic importance. This paper uses published surveys of art history to construct a critical ranking of the 30 most important American painters of the last century, then uses auction records to identify the most expensive paintings by these artists. The results show a strong positive association between critical rankings and auction prices: there is a high rank correlation between the critical ranking of artists and a ranking of the artists by their single highest auction price, and the top artists in the critical ranking are highly disproportionately represented among auction sales over $10 million, $30 million, and $50 million. Auction markets do recognize artistic importance: the greatest artists produce the most expensive paintings, and it is their most important works that sell for the highest prices.
Galenson, David W., Do the Most Important Artists Make the Most Expensive Paintings? (October 2, 2018). University of Chicago, Becker Friedman Institute for Economics Working Paper No. 2018-73. Available at SSRN: https://ssrn.com/abstract=3259208 or http://dx.doi.org/10.2139/ssrn.3259208
David W. Galenson (Contact Author)
University of Chicago - Department of Economics ( email )


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Joan Fontaine: The Oscar-Winning Star and Her Notorious Sister Act
The Oscar-winning star, and half of Hollywood's most famous sister feud, leads a parade of celebrities who have died near Christmas
By Richard Corliss Dec. 25, 2013
Celebrity deaths can come in dribbles or deluges. Around Christmas it can get downright blizzardy for Deceased Artists — the phrase that Media Funhouse's Ed Grant applies to the late great. Among the lights that failed on previous Dec. 25ths: Charles Chaplin, W.C. Fields, Dean Martin, James Brown and Ann (Detour) Savage. And on Christmas Eve: actors Jack Klugman and Charles Durning (both last year), Peter Lawford, Rossano Brazzi and Toshiro Mifune; playwrights John Osborne and Harold Pinter; composer Bernard Herrmann.
For millions of children, Christmas morning is a time for seeing what Santa brought them. For me, as TIME's self-designated obituarian, it's checking for any famous figures who have, to invoke the Monty Python Parrot sketch, "kicked the bucket, shuffled off this mortal coil, rung down the curtain and joined the bleedin' choir invisible." So far, it's been a quiet Christmas, but stay tuned. Zsa Zsa Gabor could go any year now.
Entertainers never seize our retrospective emotions so firmly as when they have just died; the chorus of their greatest praise comes when they can no longer hear it. In this year's weeks before Christmas, I bid adieu to classy actress Eleanor Parker, to Peter O'Toole, ever glamorous as David Lean's Lawrence of Arabia, and to Al Goldstein, old-school pornographer and pioneer cable-TV host. But others deserved elegies. Tom Laughlin, dead at 82, starred in Robert Altman's first film, The Delinquents (1957), and created the mythic Native American Vietnam vet Billy Jack — a left-wing, do-gooder Rambo — in four movies he wrote, directed, starred in and, in some cases, distributed. Laughlin, who also ran for U.S. president three times, leaves behind his co-star Delores Taylor, his wife of 59 years.
Ray Price, the country singer who brought country music out of Appalachia by adding strings, died at 87 this week. Straddling several generations — he filled in for Hank Williams at a performance on New Year's Day 1954 after the country legend had died, at 30, early that morning — Price hired future Country Hall of Famers to play in his band (Willie Nelson, Roger Miller) and other to write songs for him (Kris Kristofferson's "For the Good Times"). He was not just the bridge from raw country to pop country; he was the whole, long highway.
I should have found time to write about Richard Heffner, 89, the public intellectual who began his half-century talk show, The Open Mind, in 1956; edited A Documentary History of the United States and co-authored the interview book Conversations With Elie Wiesel; was the first general manager of New York City's WNET, the station that kickstarted PBS; and for 20 contentious years (1974-94) chaired the movie studios' ratings board. I was sorry to learn of the deaths of Lord Infamous (Rocky Dunigan), the Mephis rapper of the group Three 6 Mafia, at 40 after a heart attack; of the young-adult author Ned Vizzini (Kind of a Funny Story), by his own hand at 32; and of Seattle film critic Jeff Shannon, who battled quadruplegia all his adult life and finally succumbed at 54.
And Joan Fontaine, the Hollywood star who died Dec. 15 at 96 at her Carmel, Cal., home. Like O'Toole, she burst into fame in a great director's movie that won the Academy Award for Best Picture. Fontaine's vehicle was Rebecca, the 1940 Alfred Hitchcock film about a nameless young woman recently married to a domineering gentleman (Laurence Olivier) who cannot forget his first wife: Rebecca, the unseen force that haunts her widower and his fragile new wife. Fontaine played virtually the same role the following year as Cary Grant's wife in Hitchcock's Suspicion, and this time she won the Oscar as Best Actress. She was just 24.
Fontaine occasionally played schemers: the Victorian belle who cheats on both her husband and her lover with a wealthy older man, and throws poison into the stew, in the 1947 Ivy; and the breaker of finer hearts in Nicholas Ray's 1950 Born to Be Bad. But her specialty was playing the gracious soul, quiet and querulous, unsure of her allure. Her small, sad smile and delicate gestures made her ideal for romantic heroines slow to realize their own strength. Opposite Orson Welles in Jane Eyre and Louis Jourdan in Letter from an Unknown Woman, she created indelible portraits of shy girls who summon the resources to match the whims of imperious men.
Born Joan de Beauvoir de Havilland in Tokyo, Fontaine grew up in the shadow of her older sister Olivia de Havilland, herself a two-time Oscar winner for To Each His Own in 1947 and The Heiress in 1950. Olivia had more Oscars, but Joan got hers first. It was part of their lifelong rivalry — Hollywood's most notorious sister feud. Legend has it they had not spoken in perhaps 60 years. Maybe the legend was true.
Their mother Lillian had been a scholarship student at the Royal Academy of Dramatic Art and assistant to the legendary actor-manager Herbert Beerbohm Tree. As Lillian Fontaine she graced a half-dozen films in the mid-'40s, including Joan's Ivy (where she enters in the seventh minute). The girls' father, Walter de Havilland, taught law at Waseda University in Tokyo and, according to many accounts, couldn't keep his hands off women. He patronized Tokyo's red light district took the family housemaid as a mistress — in the family house. That outrage spurred Lillian to demand a divorce and move her daughters to San Jose, Cal., in 1920. They took up residence at the Vendome Hotel, where the bilingual girls chatted in "babyish Japanese." Lillian married Gorge Fontaine, manager of a department store.
Fontaine was even less fit a husband and father than de Havilland had been. Joan, who at the age of three scored 160 on an IQ test, "was commonly seen sitting in a graveyard," Higham writes, "leaning against a tombstone and reading romantic verse." Fontaine used this moony solemnity against her: "When Joan started biting her nails, he dug a grave in the garden and showed it to her; as she screamed with fear, he told her he would throw her into it if she continued." Their stepfather also "showed a perverted streak… interfering with their genitals in the bathtub…" Answering her father's call, Joan returned to Tokyo, where Walter had married the housemaid. "He often let his incestuous feelings for Joan be known," Higham writes. "On one occasion, when his wife was sick, he even suggested that Joan go to bed with him." Joan demanded to be sent back to California; in return, Walter cut off her and Olivia's allowance.
Olivia had luck to match her acting gift and her beauty — dark to Joan's blond. As a teenager she replaced Gloria Stuart as Hernia in Max Reinhardt's Los Angeles production of A Midsummer Night's Dream, and played the same role when Warner Bros. filmed it. In her next film, Captain Blood, she starring with Warners' dashing Tasmanian devil Errol Flynn. Joan's job was driving Olivia to the studio. Fontaine claims she recommended de Havilland for the role of Melanie in Gone with the Wind. Never appearing together in a film, they were famously ferocious competitors. In 1947, when Olivia won her first Oscar, Joan was backstage. She extended her hand in congratulations; Olivia turned away.
Shortly before her death, Fontaine told Scott Feinstein of The Hollywood Reporter that the feud has little basis in reality, that the sisters visited each other at their respective homes in Manhattan and Paris. Yet in a 1978 People interview, when asked how she would like to die, she answered: "At age 108, flying around the stage in Peter Pan, as a result of my sister cutting the wires. Olivia has always said I was first at everything – I got married first, got an Academy Award first, had a child first. If I die, she'll be furious, because again I'll have got there first!"
Joan got their first. Now Olivia, apparently going strong at 97, can enjoy life until she follows her sister — some time near Christmas.
The rest of you: stay alive this holiday season and through a bright 2014.

redpajama_set_name: RedPajamaCommonCrawl


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Crespi d'Adda egy munkásfalu Olaszországban, melyet a 19. században alapítottak. A terület Capriate San Gervasio településhez, Bergamo megyéhez, (Lombardia) tartozik. 1995 óta az UNESCO világörökség listáján szerepel.

A falu minden bizonnyal a legérdekesebb példája a munkásfalvak olaszországi megjelenésének. A hely eredeti, szinte tökéletes állapotban maradt fenn.

A gyárat és a falut a 19. század végén – az olaszországi ipari forradalom kezdete idején – építette a Crespi család, akik eredetileg gyapot-pamut manufaktúrákat vezettek. A felvilágosult tőkés vállalkozók a kor szociális doktrínáinak, szociológiai áramlatainak köszönhetően munkásaiknak olyan életkörülményeket kívántak biztosítani, mellyel megóvják alkalmazottaik egészségét, biztosítják megélhetésüket, segítik családtagjaikat. A Crespi család a munkásoknak nemcsak házakat, lakásokat építtetett, hanem templomot, iskolát, kórházat, közösségi házat, színházat, közfürdőt is. A munkás-települést 1878-ban alapították az Adda folyó partján Lombardiában, a gyár tulajdonosainak paternalista gondoskodása munkásaik fölött a nagy gazdasági világválság idejéig tartott, a munkásvárosban végig a Crespi társaság oltalma, gondoskodása alatt éltek az iparosok és családtagjaik.

Napjaikban a faluban nagyrészt az egykori munkások leszármazottai élnek, a gyár 2004-ig továbbra is üzemelt, és továbbra is pamut-textil termékeket állított elő.

További információk
 Villaggio Crespi (Crespi munkásainak világa)
 Crespi d'Adda

Kulturális világörökségi helyszínek
Olaszország világörökségi helyszínei

redpajama_set_name: RedPajamaWikipedia


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Shaq's Enormous Waterfront Florida Mansion Sells for $11 Million (PHOTOS)
Alicia Selin Published: November 5, 2021
Christian Pestana Rodriguez via Realtor.com / Joe Scarnici, Getty Images for Turner Sports
Shaq's enormous waterfront mansion in Florida has an indoor basketball court and an aquarium room — as well as a lucky new owner!
Legendary basketball player Shaquille O'Neal has finally sold his customized mansion in Florida, which features a pool known as Shaq-apulco. The waterfront mansion is located near Orlando, Fla. and sits in a gated, private golf club community.
The Kazaam star has moved to Atlanta, where he is currently filming a show on TNT called Inside the NBA.
Look Inside Shaq's Enormous Waterfront Florida Mansion
There are so many grand features inside Shaq's waterfront mansion in Florida. The 31,000 square foot home has a custom rock waterfall, a basketball court and an aquarium room. See photos, below!
Inside Martha Stewart's Former $16.5 Million East Hampton Home
Take a look inside of Martha Stewart's $16.5 million home of 30 years. The cottage was built in 1873 and sold for nearly ten times what Martha paid.
Source: Shaq's Enormous Waterfront Florida Mansion Sells for $11 Million (PHOTOS)
Filed Under: Shaq, shaquille o'neal
Yung Gravy Says He Took Martha Stewart on a Date: 'A Step Ahead of Pete Davidson' (EXCLUSIVE)
Jax's Viral Hit 'Victoria's Secret' Packs a Powerful Message for Anyone Who Struggles With Body Image (EXCLUSIVE)
Lizzo Explains Why It's Important to Show Up for Queer and Marginalized Fans: 'I Identify With So Many People' (EXCLUSIVE)
How Camila Cabello's First Breakup Turned Into 'Hard-Earned Wisdom' (EXCLUSIVE)


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Wildstone - British Columbia - Canada
Wildstone Golf Course,
950 Wildstone Drive,
Cranbrooke,
British Columbia,
V1C 0B4,
190 miles S of Canmore
Chris Andrews
Gary Player, Jeff Lawrence
Dave Linardic
Write a review for Wildstone
Jeff Lawrence, a former associate of Tom Fazio, laid out the Wildstone Golf Course which opened for public play in 2011. Working for Black Knight Design, a subsidiary of Gary Player Design, the architect fashioned the first Canadian layout that Gary Player's company had ever constructed.
Situated in south east British Columbia, not too far from the tourist town of Kimberley, Wildstone stretches to 7,127 yards from the back tees, with fairways set out amongst the rolling highlands above Cranbrook, where stunning views of the Rocky Mountains dominate proceedings from start to finish.
Set within a large residential development, the course is configured with five par fives, the best of which is also the shortest at the 505-yard 9th, where pine trees split the fairway to give approach options. On the back nine, the 174-yard 16th is the second of consecutive par threes, with water to the front and right of the green adding pressure to the tee shot.
Reviews for Wildstone
Description: Jeff Lawrence, a former associate of Tom Fazio, laid out the Wildstone Golf Course in 2011 when he was working for Black Knight Design, a subsidiary of Gary Player Design... Written by: Top100 Aggregated Rating Rating: 7 out of 10 Reviews: 1
Leland Chumik
My favorite course in the cranbrook area. It's newer, and when you show up, you can tell that they invest most of their money in the quality of the golf course. It is fun to play and is in mint condition
See other reviews from Leland Chumik


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Felix & Matthias Gephart have been guests in Essen for Hafendampf several times; that is why I know about the brothers and their work. My Berlin friend has found a work of them at the former RAW property in Berlin-Friedrichshain in July and I have to confess that I again have changed the colour of my face to green! See for yourself!


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Angell, Herbert Raleigh (1893–1992)
by Nancy Poundstone Opdyke
This article was published online in 2020
Herbert Raleigh Angell (1893–1992), plant pathologist, was born on 21 August 1893 at Old England, Manchester, Jamaica, son of Jamaican-born parents Charles Angell, pen keeper, and his wife Rose Edith, née Sconce. In World War I he served in the British West Indies Regiment. Commissioned on 31 May 1917 and promoted to lieutenant (November 1918), he was posted to the 7th Battalion, which was deployed to the Western Front (1917) and Italy (1918). In 1921 he moved to Montreal, Canada, where he studied agriculture at Macdonald College (McGill University), Montreal Island, (BSA, 1925). Nicknamed 'Herbie,' he was photographer for the college magazine and president of the literary and debating society. After graduating, he moved to the United States of America to continue his studies at the University of Wisconsin, Madison (MS, 1926; PhD, 1928). His doctoral thesis, 'Purple Blotch of Onion (Macrosporium porri Ell.)' was published in a shortened version in the Journal of Agricultural Research (1929).
Angell was appointed senior plant pathologist in 1928 in the newly established Council for Scientific and Industrial Research, Canberra. On 30 October 1930 at Scots Church, Sydney, he married Rosalind Kate Ramsay, a librarian. His research interests were broad; they included the relationship of protocatechuic acid (a type of phenolic acid) with disease resistance in onions, and the control of soft rot (also known as black rot) in pineapples by benzoic acid. In 1929 and 1930 he visited Papua to investigate a disease of coconuts. His most notable work was research (1928–38) into the fungistatic properties of vapours of benzol in the control and prevention of downy mildew (blue mould) in tobacco, a disease that threatened the viability of the industry in Australia. The method he developed was to grow seedlings under tents, using benzol as a mist, but only at night. He found that the vapour prevented development of the disease. According to Angell, before his research, Australian tobacco was unpopular and difficult to market because it had an unpleasant odour. In the course of his research he developed a close relationship with growers, even learning some Italian to communicate directly with many of them. Before he could complete his work on tobacco he was directed to undertake research into take-all of wheat, a plant disease caused by a fungus. However, following requests from the tobacco industry, Angell, who was the only person with the knowledge to introduce growers to the practical application of his research, was allowed to return and finish his work. His solution was described as a 'boon to Australian tobacco growers, [which] … with modifications, was adopted worldwide' (Technology in Australia 1788—1988 2001, 43).
In 1934 Angell published the results of his research into the early symptoms of flag smut in wheat in the Journal of the Council for Scientific and Industrial Research, and in 1939 he was appointed OBE for his work on downy mildew in tobacco. Promoted to principal plant pathologist in 1940, his research focused on browning of flax (1945), seedling blight of peas and poppy (1949–54), and brown rot of stone fruits (1949–55). He retired in 1958 but at the request of his employers continued research for another four years, with his wife working as his associate. His hobbies were gardening and mechanical pursuits that included reassembling a T-model Ford, grinding telescope lenses, and casting parts from scrap aluminium that he melted in an open-hearth fireplace in his dining room. Having had a long interest in pottery he produced stoneware that was fired with sump oil in a downdraft kiln he had constructed. He died on 18 March 1992 when visiting his daughter at Mornington, Victoria, and was cremated. His wife, their son and two daughters survived him. A street in Banks, Australian Capital Territory, is named after him.
Research edited by Brian Wimborne
Angell, Herbert Raleigh. Interview by Judy Cannon, 20 February 1986. Sound recording. National Library of Australia
McCarthy, G. J. 'Angell, Herbert Raleigh (1893–1992).' Encyclopedia of Australian Science. Last updated 20 February 2010. Accessed 6 January 2019. http://www.eoas.info/biogs/P000020b.htm. Copy held on ADB file
Technology in Australia 1788–1988. Melbourne: Australian Academy of Technological Sciences and Engineering, 1988
is appointed OBE, Sydney Morning Herald, 2 January 1939, p 7
Angell, Herbert Raleigh
Nancy Poundstone Opdyke, 'Angell, Herbert Raleigh (1893–1992)', Australian Dictionary of Biography, National Centre of Biography, Australian National University, https://adb.anu.edu.au/biography/angell-herbert-raleigh-28254/text35956, published online 2020, accessed online 16 January 2021.
Manchester, Jamaica
Mornington, Victoria, Australia
Officer of the Order of the British Empire
Angell Place (Conder, ACT)
CSIRO (plant industry)


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After three centuries of demographic expansion during the High Middle Ages, by 1300, Europe was showing signs of overpopulation. To produce the food needed to sustain its people, Europeans brought marginal land under cultivation. The Great Famine of 1315, which struck northern Europe, was the worst famine in centuries, killing perhaps 5 to 10 percent of the population in affected areas. Famine and food shortages may well have paved the way for the Black Death by making Europeans more susceptible to disease.
The Black Death struck Europe in full force in the spring of 1348, following existing commercial networks. Mediterranean Europe, nearly all of France, and southern England were affected by the end of 1348. Germany and nearly the whole of the British Isles were affected by the end of 1349. Scandinavia and eastern Europe were affected by the end of 1350. Russia was affected by the end of 1351. At that point, the Black Death vanished across Europe.
Contemporaries most often sought to explain the Black Death in theological or astrological terms, and the remedies they devised reflected those explanations. Many interpreted the Black Death as divine punishment for human sins; as such, it could be warded off only through penance for those sins. (For example, those Christian clerics and Jews whose failings had brought about divine punishment).
The standard figure given for the Black Death's mortality rate is one in three--that is, between 1347 and 1351, one-third of Europe's people died. This figure should be regarded as a minimum, though; detailed local research has led some historians to conclude that a more accurate mortality rate would be about one in two, with lower mortality rates in northern Europe and even higher ones in Mediterranean Europe.
Although the Black Death of 1347 to 1351 was of the greatest psychological consequence, subsequent outbreaks (the first came in 1361) were just as demographically important, driving the European population lower sti1l. Depopulation affected both town and countryside. For example, the population of Florence dropped from 120,000 in 1338 to 38,000 in 1427. And thousands of villages were abandoned entirely in the Late Middle Ages, probably because the Black Death had reduced their populations to unsustainably low levels. Europe's population in 1450 was probably 60 percent lower than it had been in 1300, and it may not have reached the level of 1300 again unti1 1600.
Depopulation was economically beneficial to some Europeans and economically disastrous for others. After the Black Death, vacant land was readily available and labor was scarce. As a result, wages shot up (often tripling or quadrupling within a few years of the Black Death's arrival), while land values plummeted, rents dropped, and food prices generally dropped. These trends favored the poor, whose purchasing power increased markedly. Those who were wealthy found themselves at a relative disadvantage.


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Galerie Hofburg Artists
Bruno Lipp was born in 1951 in Hindelang in the Allgäu. At the age of 12, the enthusiastic boy already knew how to carve pleasing figures from simple pieces of wood.
Due to the far-sightedness of his parents, who probably recognised their son's talent early on, Bruno Lipp was fortunate enough in his early youth (at the age of 15) to enjoy an apprenticeship as a wood sculptor at the carving school in Elbingenalp/Tyrol with Prof. Rudolf Geisler-Moroder between 1966 and 1970. This also laid the foundation for Bruno's artistic career.
Bruno Lipp then pursued sculpture with Prof. Walter Ritter at the Art School of the City of Linz. Since 1971 he has been a member of the Eferdinger Künstlergilde.
In 1973-1976 the artist devoted himself to studying sculpture at the Linz Art School with Prof. Erwin Reiter. Subsequently (1976-1978) he taught at the carving school in Elbingenalp for a period of 2 years.
Since 1979 the artist has been working as a freelance sculptor. For 10 years he restored many churches and art-historical buildings together with his wife Elisabeth – who is also an academic artist.
He organises various exhibitions at home and abroad. In his modesty, the artist does not even list them. However, this in no way diminishes his great renown.
Bruno Lipp's technique:
This is the so-called plate technique, which was already used by the ancient Etruscans.
The artist's talented hands model the half-moist clay slab. This process requires enormous concentration and precision, because the more reduced the forms, the more precise the execution must be in order to bring expression and movement into the work of art.
The unique pieces are then painted with egg tempera; the artist rarely uses special wax paints.
The delicate, modern shapes and forms, reduced to a minimum, only come into their own through the candlelight in an interplay of light and shadow. Thus, through their radiance, they convey a sense of security and love of life from faith.
Lipp Bruno – Angel
Lipp Bruno – Holy Family
Lipp Bruno – Nativity scene
KOMPATSCHER KUNST-GALERIE-ART | Jakob Kompatscher | Hofgasse 5, I-39042 BRIXEN/BRESSANONE (BZ) | Phone: +39 0472 832080 | E-Mail: info@kompatscher.eu | VAT 01248150219
Art Shop & Frames KOMPATSCHER: T. +39 0472 832 215
Art Gallery: Mo-Tu 10-13 / 15-18 / We closed: creativeday Th-Sa 10-13 / 15-18 and on request
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We are excited to announce that once again this year, Tucson Subaru has selected Youth On Their Own as a partner for the annual Subaru Share The Love Event. Sponsored by Subaru of America, the Share the Love Event has raised over $140 million dollars nation-wide for select national and local charities. In 2018, Tucson Subaru donated over $78,000 to YOTO, which helped provide a monthly stipend to Tucson homeless youth who earned good grades and had good school attendance. This year, Tucson Subaru will donate $250 to YOTO for every vehicle sold or leased. YOTO is honored to be selected one of this year's Share The Love charities, and looks forward to working with Tucson Subaru to support more homeless youth.


I've always loved this Lou Reed quote. It reminds me that the stories in my head just don't cut it, that if I want them to count, they've got to hit the page and come alive for people other than myself.
It also reminds me that the work is important, and hard. There's an element of expression, as an act, that is sort of like giving birth: you've got to give into it and see it through.
But if expression were really as hard as giving birth, would Lou Reed have made 20 solo albums (and so much more)? I think not.
You could really get their attention and really take them someplace. You're joining the voice in their head with your voice - there's no one else there.
Expression is risky and challenging, but if there is a payoff, this is it.
So how does one hold on to such a standard and be prolific too? For Lou Reed, the strategy was simplicity, but also honesty. He didn't rely on fancy musicianship or big words. He preferred two or three chord songs, and the truth (sometimes the hard truth) of his own life.
I shoot up because when the drug begins to flow, I really don't care anymore.
An extreme example, perhaps, but powerful in its honesty and simplicity.
It's worth mentioning that the focus statement is a big part of journalism - radio journalism in particular (my first love). There are many varied and valid approaches to the focus statement, which can be easily explored on Google. The critical bit is to keep it short and nail down what you really want to say.
Figure out how you hide as a writer. As in speech, there are word or phrases that we may use regularly (and sometimes too regularly) to avoid getting straight to the point. Or you might be using that language to highlight aspects of yourself or your story you are more comfortable with. Try to decide when those phrases/words are actually necessary and limit your use of them.
Don't just know your facts, check your facts. If you are writing about your area of expertise, or your own life, it might be easy to assume you know what happened. You might be right, for the most part, but there will always be grey areas and part of the story that deserve more clarity. Be willing to delve into these, and double-check your own truths. By carrying out this exercise, you are likely to find new perspectives and uncover unexpected layers of your story. At worst, you will have put in time that will inevitably pay off when your readers and critics start asking you questions.
Yes, this all takes work and effort, but a lifetime? No.
Just get your words on the page, and know that your story, your truth, is worth the effort.


I've been experimenting with green smoothies for a while now. But I've never been able to get the results I wanted until today. I'm so excited that I wanted to share it with you. I think it was the lemon that did the trick – I've found lemon to be a great flavor enhancer. Plus it helps keep the colors bright.
Blend spinach and blueberries with juice and syrup until pureed. Add in the apples and banana, then re-blend. Add ice until desired thickness.
Note – this smoothie will be fibrous, which I don't mind. In fact I don't even peel my apples. I'm just lazy like that. But you might. I learned that Jamba Juice actually juices their greens and vegetables rather than using whole which is why their smoothies have such a smooth/consistent texture. And why I never achieve similar results. For a smoother version of this recipe, strain the spinach and berry pulp, then use the remaining juice with the peeled apples, banana, and ice.


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Store and organize lots of big items and sporting equipment on the StoreWall Grab Bag – Large.
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Fits on the 12-inch long Universal Hooks (included) and attaches to StoreWall Panels or standard 3 inch on center slatwall panels in which the slot goes straight in and straight up.


Now that your child got accepted to college, you have to begin to plan to pay for it. There is a lot of financial aid available, as well as other money sources out there for college. You will need to look at all options after studying your child's financial aid award.
Look at the financial aid letter very carefully, especially if your child applied to multiple colleges. The letter will spell out where all the aid is coming from, from grants to your pocket. There may even be an itemized list of what the bottom line for school costs are, like room and board. If it does not, you must factor that all in. Beware of the hidden costs of college. The bottom line is the total out of pocket expenses you will on the hook for. One college may be more expensive, but gives more financial aid, making it less costly for you. Others may me cheaper, but don't give as much financial aid, making more money come from your pocket. If you have financial hardships, write the financial aid office explaining this.
You will also need to dissect your family's financial situation. How much can you afford to pay? How much can the student pay? Remember, college will last 4 years, not just this one. Your child may need to work to pay for some of the college expenses.
Do a good scholarship search. Don't pay for one. The high school your child attends will have a full list. Also, look at other places for scholarships. You company may have scholarships for employees. Any clubs or professional organizations you belong to will probably have them as well. Apply for any and all you can.
Talk to the college about financing options. Most colleges and universities have some sort of payment plan. Normally, there is no interest.
Much of college is paid through student loans. All direct loans are now made by the government. Private supplemental and parent loans are also available, like PLUS loans. Private students loans should only be used as a last resort. They do not have the same low interest or repayment plans that direct student loans have.


Founded in 2003 by Steve Couture, Philippe Bégin and Christian Daigle, Frima Studio was born from the desire of three visionaries to craft video games and digital entertainment for all platforms and screens. The name "Frima" is a testament to the studio's humble wintery beginnings, as the apartment in which the trio had set shop was so poorly insulated and heated that both floor and windows were covered in frost. In French, "frimas" means "frost," so they ditched the silent "s" and dubbed the company "Frima." Even though the studio's beginnings were humble, Frima still started out by producing Web games for some of the biggest names in Hollywood, such as Harry Potter and Looney Tunes. With prestigious names like these lining its portfolio, the studio quickly earned a reputation for the quality of its games, both art-wise and technology-wise.
Frima took its first step into the world of multiplayer games in 2007 with the launch of its first massively multiplayer online game: GalaXseeds. The game's success prompted the company to develop more virtual worlds for its partners and clients, which served to propel the studio into an upward spiral at neck-break speed. In 2008, Frima acquired Humagade, a company specializing in the production of games for consoles and mobile phones, allowing Frima to merge the new market of virtual worlds with that of traditional gaming. Ever since, Frima has been able to provide its clients with multiplatform solutions.
In 2011, Frima expanded its already comprehensive line of services with two new business line additions. First, Frima acquired the animation and special effects division of Studio Virtuel Concept (SVC), enlisting the collective talent of a seasoned team of creative minds. This allowed Frima to launch Frima FX and realize its long-time dream of developing entertainment products for the advertising, television and movie markets. Next, Frima acquired Volta, a world-renowned Québec-based studio specializing in concept art. In preproduction, concept art is vital, as it serves to establish a product's visual style and direction. This highly specialized and sought-after field of expertise adds to the services that Frima can provide its partners and clients.
Year after year, Frima continues to strike wide-scale partnerships with giants of the entertainment industry, such as Activision, Adobe, Disney, Electronic Arts, Hasbro, Microsoft, Nickelodeon, Sony, Square Enix, Ubisoft and Warner Bros. Not only does Frima offer a complete and varied line of services, it also produces its own products and intellectual properties. Successes like Zombie Tycoon, Young Thor and A Space Shooter for Free! bear witness to the passion and effervescence that inhabit Frima and its 200 employees.
There are currently no trailers available for Frima. Check back later for more or contact us for specific requests!
There are far more images available for Frima, but these are the ones we felt would be most useful to you. If you have specific requests, please do contact us!


text:
August 14, 2020By No Limit BoråsPressrelease
Complete Line-up – These are the artists for No Limit: Artscape Edition 2020 BoråsNo Limit Borås2020-08-14T09:41:11+00:00
During the No Limit: Artscape Edition street art festival in Borås 17–20 Sept, the line-up will for the very first time solemnly focus on the Swedish artists.
The Swedish street art scene has evolved greatly during the last years and we have many artists who are now established on the highest international level.
The 12 participating artists in this year's No Limit: Artscape Edition represent several different styles and artistic expressions, which creates a very interesting mix.
– Artscape's aim is to present an exciting array of artistic expression in our projects, while at the same time have everything gel as a coherent entity. This year, No Limit takes place in only one area of the city and with only Swedish artists – but within the chosen group the differences and range is massive. Completely different techniques, colour palettes and influences. You will see creations of everything from photorealism, and calligraphy to magical fairy-tale worlds and graffiti-inspired work. Even sculptures and textile art will fit into this year's project, says Daniel Wakeham, Artscape.
– We require the artists we invite to be on a certain level artistically. We also look to mix experience with new talent – showcasing artists at the height of their powers as well as giving an opportunity to tomorrow's stars. We are very pleased with the balance in this year's project and it is a very colourful line-up who will decorate Norrby next month, says Tor Hedendahl, Artscape.
Line-up for No Limit Artscape Edition 2020:
Huge started painting graffiti in the mid '80s and has created murals all over the world, from Stockholm and Paris to Los Angeles and Shenzhen. He's mainly known for his own unique style known as "Helium Graff" – hyper-realistic balloon letters.
https://www.instagram.com/hugeart/
Amara Por Dios
Amara Por Dios is an artist born and raised in Stockholm, who has spent several years living and working in London. Her style is recognizable with her vibrant colors, organic shapes, patterns and faces. In 2014, she was named by The Guardian as one of Britain's top female street artists.
https://www.instagram.com/amarapordios/
Q – an artist from Lindesberg, whose unique characters often share similar features to those you see in comic books and animated films – colourful and expressive. Q is a versatile artist who can be found in a wide range of mediums such as murals, photography, film and sculpture.
https://www.instagram.com/q.bless/
Delicious Brains
Delicious Brains, or Kim Demåne, is a multifaceted artist doing both exhibitions, murals, album and magazine covers and music videos. He has a playful and colourful style with a lot of details and has previously made art work for the worldwide conference TEDGlobal.
https://www.instagram.com/delicious_brains/
Vickan
Vickan is a tattoo and street artist. With her imaginative and characteristic style, she has quickly established herself as a name in the Swedish street art scene. Vickan is originally from Boden and is today based in Stockholm.
https://www.instagram.com/vickanart/
Adam Algotsson, or Appear37, a graffiti artist from Borås who now gets to paint on a large scale on home turf. Adam found graffiti in his teens and got one of his first paid commission from the same police officer who had once arrested him for painting illegally. He has since created over 100 commissioned murals for both businesses and individuals.
Elina Metso
With a background in game development and illustrations, Elina Metso has experience of many different materials and styles, which is reflected in her artwork. She prefers to paint colourful portraits, often of female or androgynous characters.
https://www.instagram.com/metso_art/
Etile uses a style called Calligraffiti, which is a mix between calligraphy and graffiti. He is originally from Karlstad and today works as a tattoo artist in Gothenburg.
https://www.instagram.com/etile1/
Malin Bobeck Tadaa
Malin Bobeck Tadaa is a textile artist and designer who combines traditional textile materials with optical fiber, LEDs and new technology, creating spatial installations that encourage interaction. She is fascinated by the worlds that are hidden from us – worlds in the deep sea, outer space or inside our imagination – which is reflected in her artwork.
https://www.instagram.com/malinbobecktadaa/
Tim Timmey
Tim Timmey is a self-taught multi-talent who creates everything from huge wooden installations to murals and sculptures. His work often depicts colorful animal motifs, three-dimensional patterns and crazy creatures with a great dose of humor.
https://www.instagram.com/timtimmey/
Korallpionen & Rickman
During No Limit: Artscape Edition, artists Korallpionen and Rickman will paint together. In a joint work of art, they combine Rickman's raw and colorful photorealistic style with Korallpionen's colorful flowers, plants and animals.
https://www.instagram.com/korallpionen/
Swedish artists in focus during No Limit: Artscape Edition →
This is Artscape
No Limit: Artscape Edition 2020
Instagram @nolimitboras
Shai dahan


In San Antonio, Texas, Clark Simson Miller operates through a remote management office that vows to take care of our client base, whether it is comprised of small or larger members. Management partners and communities of all sizes will enjoy the ease of use in the transparent solutions provided by our firm.
We offer a full service that gives clients the ability to obtain accurate reporting at any time through our customized tech solutions. We cater to associations of all sizes and take pride in providing services that are geared to meet your overall needs.
With our accounting tools, you are able to access accurate and timely financial reporting. We also work hard to negotiate rates with vendors when needed in order to reduce your costs overall. We pay your utility bills along with integrating with local financial institutions to obtain payments from your residents directly.
We service the entire San Antonio and surrounding areas with our professional solutions. With our team of qualified professionals, clients get access to 150 years of combined association experience. When you have to turn your accounting and financial functionality over to a professional firm that will always put you first, count on Clark Simson Miller to give you the tools you need to grow and succeed.
San Antonio HOA management services through Clark Simson Miller will give your company the added boost it needs to get the job done. No longer will you have to hire additional accounting personnel and pay exorbitant rates.
San Antonio, Texas is a bustling city full of culture and entertainment. As part of this community through remote management solutions, our team will work with your community, no matter the size, to give you a customized accounting solution.
We prepare your budgets, newsletters, manage payroll, deal with vendors, assist with tax returns, prepare you for audits, provide accurate monthly reporting, and perform collections activity from beginning to end – including referrals for legal action when necessary.
We have designed a custom software solution that is designed to meet the needs of the entire association management industry. Our clients are provided with access to proprietary technology which gives them the ability to access their information at any time they need to. We log all communications with owners, vendors, and residents to ensure that there is a seamless coordination to all of your accounting requirements.
When you outsource your accounting responsibilities to Clark Simson Miller in the San Antonio and surrounding areas, you are getting a financial and accounting service that is unmatched. Our self-managed solutions and those geared toward larger HOA associations provide all of our clients with the ability to better serve their residents.
With us, you can turn your focus on achieving all of your goals, while we handle every aspect of your accounting needs. We aim to provide this service as a less expensive means of giving our clients the accounting support they need at a cost that won't break the bank. Let us focus on your accounting, so that you can focus on your residents.


text:
Q: Htaccess rewrite from one file to another I need to redirect all queries to file 1.php to file 2.php. So I need if somebody will ask 1.php server will give him 2.php. 1.php and 2.php are in the same folder.
RewriteEngine On
RewriteRule ^2.php 1.php

This code in htaccess doesn't work. Where is the mistake?

A: Try this:
RewriteEngine On
RewriteCond %{HTTP_HOST} ^(www.)?[the folder]/1.php$
RewriteRule ^(/)?$ [the folder]/2.php [L]

redpajama_set_name: RedPajamaStackExchange


Dietbald - Name Meaning, What does Dietbald mean?
Dietbald as a name for boys is an Old German name, and the meaning of the name Dietbald is "brave people". Dietbald is an alternate form of Theobald (Old German).
Suggested similar-sounding names are Bernald, Denali, Derald, Derland, Derrall, Derrold (see Darold), Derward, Deshad (see Deshan), Deshal (see Deshan), Deval, Devland, Diarmaid, Diarmuld (see Dermot), Dieter, Dietrich, Donald▼, Dugald, Everald, Fernald, Field, Gerald▼, Gerrald, Jerald▼, Jerrald, Reinald, Reynald, Sewald, Siegwald, Sigwald, Tibold, Tybald and Witold. These names tend to be more commonly used than Dietbald.


text:
<?php declare(strict_types=1);

namespace Limoncello\Tests\Application\ExceptionHandlers;

use Exception;
use Limoncello\Application\ExceptionHandlers\WhoopsThrowableJsonHandler;
use Limoncello\Application\ExceptionHandlers\WhoopsThrowableTextHandler;
use Limoncello\Container\Container;
use Limoncello\Contracts\Application\ApplicationConfigurationInterface as A;
use Limoncello\Contracts\Application\CacheSettingsProviderInterface;
use Limoncello\Contracts\Http\ThrowableResponseInterface;
use Limoncello\Tests\Application\TestCase;
use Mockery;
use Mockery\Mock;
use Psr\Container\ContainerInterface;
use Psr\Log\AbstractLogger;
use Psr\Log\LoggerInterface;
use Psr\Log\NullLogger;

/**
 * @package Limoncello\Tests\Application
 */
class TextExceptionHandlersTest extends TestCase
{
    /**
     * Test handler.
     */
    public function testDefaultExceptionHandler(): void
    {
        $handler = new WhoopsThrowableTextHandler();

        $response = $handler->createResponse(new Exception('Error for Text handler'), $this->createContainer(true));
        $this->assertInstanceOf(ThrowableResponseInterface::class, $response);
        $this->assertEquals(['text/plain; charset=utf-8'], $response->getHeader('Content-Type'));
        $this->assertStringStartsWith('Exception: Error for Text handler in file', (string)$response->getBody());
    }

    /**
     * Test handler.
     */
    public function testDefaultExceptionHandlerDebugDisabled(): void
    {
        $handler = new WhoopsThrowableTextHandler();

        $response = $handler->createResponse(new Exception('Error for Text handler'), $this->createContainer(false));
        $this->assertInstanceOf(ThrowableResponseInterface::class, $response);
        $this->assertEquals(['text/plain; charset=utf-8'], $response->getHeader('Content-Type'));
        $this->assertEquals('Internal Server Error', (string)$response->getBody());
    }

    /**
     * Test handler.
     */
    public function testFaultyLoggerOnError(): void
    {
        $handler = new WhoopsThrowableTextHandler();

        $response = $handler->createResponse(new Exception(), $this->createContainerWithFaultyLogger());
        $this->assertInstanceOf(ThrowableResponseInterface::class, $response);
        $this->assertEquals(['text/plain; charset=utf-8'], $response->getHeader('Content-Type'));
        $this->assertStringStartsWith('Exception:  in file', (string)$response->getBody());
    }

    /**
     * Test handler.
     */
    public function testDefaultJsonExceptionHandler(): void
    {
        $handler = new WhoopsThrowableJsonHandler();

        $response = $handler->createResponse(new Exception('Error for JSON handler'), $this->createContainer(true));
        $this->assertInstanceOf(ThrowableResponseInterface::class, $response);
        $this->assertEquals(['application/json'], $response->getHeader('Content-Type'));
        $this->assertStringStartsWith(
            '"{\"error\":{\"type\":\"Exception\",\"message\":\"Error for JSON handler\"',
            json_encode(json_decode((string)$response->getBody()))
        );
    }

    /**
     * @param bool $debugEnabled
     *
     * @return ContainerInterface
     */
    private function createContainer(bool $debugEnabled): ContainerInterface
    {
        $container = new Container();

        $container[LoggerInterface::class] = new NullLogger();

        /** @var Mock $provider */
        $provider = Mockery::mock(CacheSettingsProviderInterface::class);
        $container[CacheSettingsProviderInterface::class] = $provider;
        $provider->shouldReceive('getApplicationConfiguration')->once()->withNoArgs()->andReturn([
            A::KEY_IS_DEBUG         => $debugEnabled,
            A::KEY_APP_NAME         => 'Test App',
            A::KEY_EXCEPTION_DUMPER => [self::class, 'exceptionDumper'],
        ]);

        return $container;
    }

    /**
     * @return ContainerInterface
     */
    private function createContainerWithFaultyLogger(): ContainerInterface
    {
        /** @var Container $container */
        $container = $this->createContainer(true);

        $container[LoggerInterface::class] = new class extends AbstractLogger implements LoggerInterface
        {
            /**
             * @inheritdoc
             */
            public function log($level, $message, array $context = [])
            {
                // emulate such error as no permission to write logs to disk.
                throw new Exception();
            }
        };

        return $container;
    }

    /**
     * @param array ...$args
     *
     * @return array
     */
    public static function exceptionDumper(...$args): array
    {
        assert($args);

        return [
            'some' => 'related details',
        ];
    }
}

redpajama_set_name: RedPajamaGithub


text:
Кула́ши — посёлок городского типа на западе Грузии, расположен в 4 км севернее города Самтредиа.

Население 1702 человек (2014).

История
26 января 1961 года село Кулаши получило статус посёлка городского типа.

В начале 1970-х годов в посёлке проживало более 5 тысяч жителей — преимущественно грузинские евреи, имелось две синагоги. С конца 1970-х началась эмиграция еврейского населения в Израиль, полностью завершившаяся к середине 1990-х.

В посёлке существовали ткацкая фабрика, завод по производству хрустальных изделий и по пошиву рабочей одежды, в настоящее время не функционирующие.

Известные уроженцы
 Гур, Эфраим (р. 1955) — израильский политический деятель, экс-мэр г. Ашдод, бывший депутат Кнессета
 Каладзе, Николай Иванович (1909—1997) — советский военачальник, генерал-майор
 Канделаки, Николай Порфирьевич (1889—1970) — советский скульптор
 Канделаки, Давид Владимирович (1895—1938) — советский государственный деятель, дипломат
 Мирилашвили, Михаил Михайлович (р. 1960) — российский предприниматель
 Стуруа, Иван Фёдорович (1870—1931) — грузинский революционер
 Шенгелия, Георгий Давидович (1908—1981) — Герой Советского Союза

Источники
 БСЭ

Примечания

Населённые пункты Самтредского муниципалитета


Children's Rescue Mission (CRM) is a qualified 501 (c)(3) non-profit, charitable organization - all gifts are tax deductible. Any gift is welcome and appreciated!
toothbrush, toothpaste, soap, comb, etc.
so...how would you like to donate today?


text:
Science + Tech
What Happens When Health Care Becomes a Stock Market Play?
Well Health is a new kind of health-care company in Canada. But some see big risks. Part of a series.
Andrew MacLeod 9 Sep 2020 | TheTyee.ca
Andrew MacLeod is The Tyee's Legislative Bureau Chief in Victoria and the author of All Together Healthy (Douglas & McIntyre, 2018). Find him on Twitter or reach him at
Email address protected by JavaScript
Family doctor Baldev Sanghera says corporations offer the health care equivalent of fast food, but not all patients will notice. 'If the only thing that's been advertised to you is McDonald's, then that's a great meal.' Photo by Maggie MacPherson.
Profit Before Patients? The Tyee's Check Up on Corporate Moves into BC Health Care
Profits Before Patients? The Corporate Push into BC's Primary Care System
Why Are Corporations Moving into Health Care? Doctors Say It's the System's Fault
Corporations Want Your Health Records. Who's Keeping Them Safe?
How BC Can Fix Primary Health Care, With or Without Corporations
(Click here for the complete list of stories.)
Even before the COVID-19 pandemic, Vancouver company Well Health Technologies Corp. was growing rapidly and had ambitious expansion plans.
Some investors saw an opportunity. But other people saw a threat to public health care.
Well had, over a couple of years, acquired 21 primary care clinics in B.C., become the electronic medical record provider to another 1,446 clinics across Canada and dedicated a significant marketing budget to promoting its services.
Well is something new, a Canadian company focused on providing direct health-care services and traded on the stock market.
Investors have been enthusiastic, bidding up Well's stock price. Shares that were worth 25 cents a little more than two years ago had risen to about $2 by early this year. The COVID-19 pandemic brought more interest, and shares topped $6 by Sept. 1.
By September, Well was worth about $790 million and looked set to keep growing.
But what may be good for investors is likely going to be bad for patients, said Marcy Cohen, a community researcher who has worked on issues around primary care and community care for two decades.
"They're obviously trying different strategies about how and where the profit possibilities are," Cohen said. "Because they're a corporation, their responsibility is to their shareholders. They are looking at health care as a revenue generating opportunity, so they're looking and experimenting with all the different ways they might be able to make a profit out of health care."
Private entities have been providing health care in Canada for years, but they have tended to be small and run by physicians who put the priority on patients, not shareholders, she said.
With Well's establishment, Canada is entering a new world of marketing, advertising and corporatization in health care.
Researcher Marcy Cohen says corporate health care brings a focus on shareholders, not patients. Photo by Maggie MacPherson.
"I think it's potentially a huge shift and very problematic and is the reason there are so many mainstream physicians' organizations trying to respond," Cohen said.
Nobody from Well was available for an interview.
Pardeep Sangha, the company's vice-president of corporate strategy and investor relations, asked for detailed questions by email but didn't respond to them by publication time.
Sangha did send a June presentation for investors and reports on the company from three stock analysts, all of which focus on the strength of the company as an investment. There's also a wealth of documents filed with regulators on the company's finances and plans.
According to that financial reporting, Well sees itself as part of the solution to improve health care. "Well aims to positively impact health outcomes by leveraging technology to empower and support patients and doctors," it said in its most recent quarterly report filed May 15.
Well also sees a business opportunity. Health-care delivery in Canada was worth $242 billion in 2017 and 15.4 per cent of that, about $37 billion, was spent on physicians, it told investors.
"The company's overarching goal is to consolidate and modernize primary health-care assets using digital technologies and processes that improve patient experience, operational efficiency and overall care performance," it said. "Well intends to address the issue of fragmentation in the Canadian health-care system which management believes has led to a lack of digitization of primary health-care clinics."
Observers suggest the approach is similar to what corporations have done in other industries, including veterinary hospitals and funeral services.
Well has officially existed since 2010, but it's only since 2018 that it has focused on primary care clinics and related services, discontinuing its previous endeavours Canada Yoga Inc. and Shakti Yoga Apparel LLC.
Well Technologies' stock was trading at about $1.40 the day a pandemic emergency was declared in BC. This week, it's valued at more than $6. Data from TSE.
Company materials highlight the investment Li Ka-shing of Hong Kong, the 35th richest person in the world according to Forbes Magazine, has made in the company.
It also talks about the track record of chairman and CEO Hamed Shahbazi, who previously founded TIO Networks Corp. and grew it into a major provider of bill payment services before a sale to PayPal Holding Inc. in 2017 for more than $300 million.
Since 2018, Well has grown by buying clinics and electronic medical records companies. Well now owns and operates 21 clinics and provides EMR software and services to some 1,500 clinics. It also owns a 51-per-cent stake in SleepWorks Medical Inc., a company in the business of diagnosing sleep disorders and selling the equipment to treat them.
This spring, Well opened a dermatology clinic in North Vancouver under the brand name "Derm Lab," where three doctors will provide medical and cosmetic services.
And in March, as the COVID-19 pandemic arrived in Canada, Well launched VirtualClinic+ to connect patients and physicians using video, phone and secure messaging, allowing patients to avoid sitting in a clinic waiting room.
"These capabilities are designed to support continuity of care, improve accessibility to quality care and ultimately drive better health outcomes," the document said.
Well now has hundreds of people providing care through its VirtualClinic+ program and can handle more than 1,000 appointments a day, which in turn has allowed the company to cut costs and increase its profits.
"Going forward Well anticipates that physicians will be operating in a hybrid model of seeing patients in-clinic as well as using telehealth," the company's quarterly report said. "In this scenario, physicians aren't required to be in the office as much; therefore, each clinic can support more physicians than before."
Well Technologies has expanded by buying established clinics, with plans for more acquisitions across Canada. Photo by Maggie MacPherson.
It announced plans to close a clinic in Surrey and relocate the physicians to other clinics the company owns nearby, "thereby maintaining revenues and increasing the utilization in our other clinics and improving our overall EBITDA [earnings before interest, taxes, depreciation and amortization] profile."
In other words, it anticipated being able to close the clinic and consolidate services in other clinics, each of which would become more profitable.
In the three months that ended in March, Well had $10.2 million in revenue. The majority of that, $6.8 million, came from providing publicly insured services. Well says it pays staff doctors about 70 per cent of the fees received from the province.
Another $1.7 million was paid directly by patients or third parties for services not covered by the province's Medical Services Plan.
Digital services provided another $1.7 million, 10 times higher than a year earlier, the company said.
The company's documents are also clear about the ambitions for continued growth.
It intends to expand its clinics from B.C. into other parts of Canada and to continue expanding its telehealth program "not only as a defensive strategy to mitigate against any potential decreases in its onsite clinical business due to COVID-19 physical distancing requirements, but also as an offensive strategy to grow its share of the expanding telehealth market in North America."
According to the company's documents, there is a "strong pipeline of potential acquisition targets to drive its inorganic growth strategy." Due to COVID-19, it said, it would likely increase its focus on investing in technologies and services that could be provided remotely.
Baldev Sanghera provides community care in Burnaby, including at a physician-owned clinic. A family doctor since 1997, he sees how Well's corporate clinics are already changing the landscape.
"I think they're a very big threat in that they have a lot of marketing behind them," he said, adding neither the public nor physicians have recognized what's coming.
Since buying clinics in the Lower Mainland, Well has laid off frontline staff and sought efficiencies, he said. Physicians working in the clinics have had to focus on seeing more patients, and are generally less satisfied with their work, Sanghera said.
For patients, the care they get is comparable to fast food, he said, though many won't know what they're missing. "If the only thing that's been advertised to you is McDonald's, then that's a great meal."
Ultimately, it's a question of resiliency and how to deliver primary care in a way that puts the needs of the community first, Sanghera said. "The pandemic has shown us having a corporate outlook on community life doesn't give you the tools."
Read more: Health, Coronavirus, Science + Tech
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Jack Bauer Goes Emeril on Cupcakes: Bam!
I don't know about you, but I haven't felt safe since Jack Bauer walked into the sunset on Fox's "24".
Kiefer Sutherland as Jack Bauer in "24"
Sure, the show's creators have brought us another excellent program — maybe even better — with Showtime's "Homeland", but Claire Danes, er, Carrie Mathison, is crazy. I'm not so convinced she's going to be able to save us from total destruction the way Jack always did in 24. SHORT. HOURS.
So what has Bauer been doing in retirement?
In a Super Bowl-worthy commercial, Acer laptops has hired Kiefer Sutherland to go all CTU in a quest to make the best cupcakes.
The ad is filled with "car chases, explosions, the actor's trademark intense gravelly line delivery and … butter-cream frosting," writes AdWeek's David Gianastasio, who says the commercial was created by London-based Mother Advertising.
The commercial is supposed to be selling Acer's new Aspire 35 Ultrabook as a useful tool helping Sutherland pursue his secret dream.
"We all have our hidden passions," says the description on YouTube. "For Mr. Kiefer Sutherland this is something moist and fluffy."
Maybe. Seems to me the only thing moist and fluffy will be the face of the guy driving the cupcake truck after Jack Bauer is done pummeling him for recipe information.
No, much better to see the actor channel his inner Emeril and go "Bam!" on everything in sight. I don't know if it makes me want to buy a new laptop, but it does make me feel safer. And a little hungry for cupcakes.


On the 30th November 2019, the Design and Technology Association partnered with The Design Technology & Engineering department at Highgate School in London, to organise the 'Approaches to Design Conference' mainly for teacher educators but some A-level D&T students from Highgate were also able to attend and benefit from some of the practical workshops.
A big thank you to our speakers from Arkwright Engineering Scholarships, Smallpeice Trust and The Engineering Development Trust with keynotes from Dyson and Loughborough University. There were a choice of practical workshops such as; User Centred Design, Pneumatics and Electronics. The aim was to improve practice and provide excellent and inspiring Design Technology & Engineering teaching.
Quote from one of the A-level D&T students, who attended: It was the best day ever, the highlight was listening to Hugo Wilson's story and experiences. It has really inspiring me and was very encouraging to hear what we are learning in the D&T department at Highgate, is being used in Industry today.
Quote from Andy Thomson, Head of D&T – "Since starting at Highgate, I have also aimed to ensure DTE students are inspired and prepared for participation in tomorrow's rapidly changing world. To have a very successful, former DTE student come back and talk with such expertise and passionate towards Design Technology was so amazing and confirms that what we are teaching in the department is relevant and inspiring.
Quote from another D&T teacher delegate– "was a very inspiring conference, it was great to collaborate and share ideas with other educators. It is amazing to see Design take so many different forms from User-centred design at one end, to electro-pneumatic circuits on the other. Good time was had by all.


text:
Dear Mary, I Heard You...
It's always refreshing to hear the truth about the music business from someone you admire as an artist. And Mary Gauthier is one of those rare artists who is as honest about her career as she is in her songwriting (check this epic post out, if you missed it).
"Make 'em laugh, cry, dance and they will give you money," she said matter-of-factly during her interview at the CD Baby DIY Music Conference in Nashville. During her candid keynote, she shared wisdom about everything from songwriting to the music business.
Here were a few of the gems...
"Everything rests on the song"
"Connecting hearts is what it's about"
"Songwriters are dealing with alchemy."
"We are called to do this for different reasons. But, I do believe it's a calling."
"The focus on fame is a distraction."
"We get paid to say the thing that most people would pay not to say."
And here's a bit of video to learn from as well..
Segment 1: How does Mary manage her career?
Segment 2: How artists should think about 'old' albums?
Segment 3: A Song before it's time...
And I made a couple of my favorite quotes from the conference into 'Mary memes' - please share on social media!
in Music Industry Experts, Hit Songwriters, Indie Music Tips


text:
.class final enum Lcom/google/common/collect/CustomConcurrentHashMap$Strength$3;
.super Lcom/google/common/collect/CustomConcurrentHashMap$Strength;
.source "CustomConcurrentHashMap.java"

# annotations
.annotation system Ldalvik/annotation/EnclosingClass;
    value = Lcom/google/common/collect/CustomConcurrentHashMap$Strength;
.end annotation

.annotation system Ldalvik/annotation/InnerClass;
    accessFlags = 0x4008
    name = null
.end annotation

# direct methods
.method constructor <init>(Ljava/lang/String;I)V
    .locals 1

    .prologue
    .line 319
    const/4 v0, 0x0

    invoke-direct {p0, p1, p2, v0}, Lcom/google/common/collect/CustomConcurrentHashMap$Strength;-><init>(Ljava/lang/String;ILcom/google/common/collect/CustomConcurrentHashMap$1;)V

    return-void
.end method

# virtual methods
.method defaultEquivalence()Lcom/google/common/base/Equivalence;
    .locals 1
    .annotation system Ldalvik/annotation/Signature;
        value = {
            "()",
            "Lcom/google/common/base/Equivalence",
            "<",
            "Ljava/lang/Object;",
            ">;"
        }
    .end annotation

    .prologue
    .line 325
    invoke-static {}, Lcom/google/common/base/Equivalences;->identity()Lcom/google/common/base/Equivalence;

    move-result-object v0

    return-object v0
.end method

.method referenceValue(Lcom/google/common/collect/CustomConcurrentHashMap$ReferenceEntry;Ljava/lang/Object;)Lcom/google/common/collect/CustomConcurrentHashMap$ValueReference;
    .locals 1
    .annotation system Ldalvik/annotation/Signature;
        value = {
            "<K:",
            "Ljava/lang/Object;",
            "V:",
            "Ljava/lang/Object;",
            ">(",
            "Lcom/google/common/collect/CustomConcurrentHashMap$ReferenceEntry",
            "<TK;TV;>;TV;)",
            "Lcom/google/common/collect/CustomConcurrentHashMap$ValueReference",
            "<TK;TV;>;"
        }
    .end annotation

    .prologue
    .line 322
    .local p1, "entry":Lcom/google/common/collect/CustomConcurrentHashMap$ReferenceEntry;, "Lcom/google/common/collect/CustomConcurrentHashMap$ReferenceEntry<TK;TV;>;"
    .local p2, "value":Ljava/lang/Object;, "TV;"
    new-instance v0, Lcom/google/common/collect/CustomConcurrentHashMap$WeakValueReference;

    invoke-direct {v0, p2, p1}, Lcom/google/common/collect/CustomConcurrentHashMap$WeakValueReference;-><init>(Ljava/lang/Object;Lcom/google/common/collect/CustomConcurrentHashMap$ReferenceEntry;)V

    return-object v0
.end method


MiR-155 has emerged as an "oncomiR", which is the most significantly up-regulated miRNA in breast cancer. However, the mechanisms of miR-155 functions as an oncomiR are mainly unknown. In this study, the aims were to investigate the effects of miR-155 on cell proliferation, cell cycle, and cell apoptosis of ERalpha (+) breast cancer cells and to verify whether TP53INP1 (tumor protein 53-induced nuclear protein 1) is a target of miR-155, and tried to explore the mechanisms of miR-155 in this process.
The expression of miR-155 is significantly higher in MCF-7 cells compared with MDA-MB-231 cells. Ectopic expression of TP53INP1 inhibits growth of MCF-7 cells by inducing cell apoptosis and inhibiting cell cycle progression. Overexpression of miR-155 increases cell proliferation and suppress cell apoptosis, whereas abrogating expression of miR-155 suppress cell proliferation and promotes cell apoptosis of MCF-7 cells. In addition, miR-155 negatively regulates TP53INP1 mRNA expression and the protein expression of TP53INP1, cleaved-caspase-3, -8, -9, and p21, and luciferase reporter reveals that TP53INP1 is targeted by miR-155.
TP53INP1 is the direct target of miR-155. MiR-155, which is overexpressed in MCF-7 cells, contributes to proliferation of MCF-7 cells possibly through down-regulating target TP53INP1.
MicroRNAs (miRNAs) are a class of short (21–25 nt), on-coding RNAs, which control their target genes expression at the post-transcriptional level [1, 2]. MiRNAs encoding genes are mostly transcribed by RNA polymerase II by Drosha into short hairpin RNAs, which are then exported from the nucleus, and processed by Dicer to form mature 21–25 nucleotide miRNAs, which are finally transferred to Argonaute proteins in RISC [3, 4]. MiRNAs act on gene expression by binding to the 3'-untranslated region (3'UTR) of the target mRNAs, resulting in mRNA cleavage and/or translational inhibition, thereby down-regulating target proteins expression . In fact, almost 30% of the protein-coding genes are under the regulation of miRNAs, and miRNAs have been implicated in the regulation of various biological functions, including cell proliferation, apoptosis, and cell differentiation . Therefore, miRNA dysfunction may contribute to a variety of human diseases, including cancer . MiRNAs exert their function as oncogenes or tumor suppressor genes depending on their target genes.
MiR-155, located in chromosome 21q21, is encoded with a region known as B cell integration cluster (BIC) gene, which consists of three exons within a 13 kb region . The human BIC gene is activated by promoter insertion and lacks a long open reading frame . MiR-155 represents a typical multifunctional miRNA, which is overexpressed in a variety of human solid tumors such as breast cancer [9–12], lung cancer [13, 14], thyroid tumor , pancreatic cancer [16–19]. These recent clinical pathologic data indicate that miR-155 plays a crucial role in tumor development and tumor diagnosis and prognosis. The available experimental evidences indicate that miR-155 promotes some tumors growth, invasion, and metastasis through inhibits downstream targets such as SHIP1 , C/EBPbeta , and SOCS1 . All these lines of evidence demonstrate that miR-155 functions as an oncomiR in human cancer.
MiR-155 has been found to be up-regulated in breast cancer and is closely related to the status of estrogen receptor (ER) and progesterone receptor (PR) [9, 22], its tumorigenesis role has not yet been defined. In this study, we identified that TP53INP1 is one of the targets of miR-155 in ERalpha (+) MCF-7 cells. Furthermore, we found that overexpression of miR-155 promoted cell proliferation and inhibited apoptosis through the repression of TP53INP1 in MCF-7 cells. In addition, in this study, we indicated that miR-155 significantly repressed cell cycle and apoptosis-related gene caspase-3, -8, -9, and p21 expression. These results revealed that miR-155 is an oncomiR in breast cancer, and miR-155 may serve as a therapeutic option in breast cancer treatment.
Four synthetic, chemically modified short RNA oligonucleotides: miR-155 mimics (miR-155 m, 5'-UUAAUGCUAAUCGUGAUAGGGGUCCCUAUCACGAUUAGCAUUAAUU- 3'), miR-155 mimics negative control (miR-155 m NC, Sense: 5'-UUCUCCGAACGUGUCACGUTT-3'; Anti-sense: 5'-ACGUGACACGUUCGGAGAATT-3'), miR-155 inhibitors (miR-155i, 5'-ACCCCUAUCACGAUUAGCAUUAA-3') and miR-155 inhibitors negative control (miR-155i NC, 5'-CAGUACUUUUGUGUAGU ACAA-3') were purchased from Shanghai GenePharma Co.Ltd. The TP53INP1 overexpression plasmid and control plasmid were purchased from Wuhan Sanying Company and subcloned into pcDNA3. TP53INP1 3'UTR reporter plasmids pYr-MirTarget, pYr-MirTarget-TP53INP1-3U (pMIR-TP53INP1-3U) and pYr-MirTarget-TP53INP1-3U-M (pMIR-TP53INP1-3U-M) were purchased from Changsha Yingrun Biotechnology Co.Ltd.
Human MCF-7, MDA-MB-231 cells were provided by Staff Room of Pathophysiology, Chongqing Medical University and were maintained DMEM (Gibco) or RPMI1640 (Gibco) media supplemented with 10% NBCS (Gibco, New Zealand), penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were maintained in sterile conditions at 37°C in a humidified atmosphere of 5% CO2. Cells were seeded into 6-well plates grown overnight and reached 30-50% confluence before transfection. Transfection of MCF-7 cells with miR-155 m (or miR-155i) or counterpart negative control using lipfectamine™ 2,000 transfection reagent (Invitrogen, USA) according to the manufacturer's instruction. The experiments were classified into three groups, which were miR-155 m (or miR-155i) group, miR-155 m NC (or miR-155i NC) group and control (Con) group. Transfection of MCF-7 cells with TP53INP1 overexpression plasmid or control plasmid using lipfectamine™ 2,000 transfection reagent.
For quantification of miR-155 and TP53INP1 mRNA by real-time PCR, total miRNA, and total RNA were isolated from cultured cells with MiRNA rapid extraction kit and RNApure rapid extraction kit (Bioteke Corporation, Beijing, China) according to the manufacturer's instructions. Reverse transcription was performed using Moloney murine leukemia virus reverse transcriptase (Bioteke Corporation, Beijing, China). MiR-155 was reverse transcripted by looped primers with the sequence of 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACACCCCT-3', while reverse transcription of TP53INP1 mRNA was performed with Oligo dT primers (Bioteke Corporation, Beijing, China). Real-time PCR was performed using the LightCycle RNA amplification kit SYBR Green (Bioteke Corporation, Beijing, China) according to the manufacturer's protocols. U6 snRNA and β- actin served as an endogenous control for normalization. Real-time PCR reactions were performed on a CFX96 real-time PCR detection system from Bio-Rad Co.LTD (America), with cycle threshold values evaluated using the manufacturer's software. The expression of miR-155, TP53INP1 mRNA, relative to U6 snRNA, β- actin, were calculated using the 2-ΔΔCT method. The real-time PCR primers are shown in Table 1.
The cell viability was evaluated by CCK8 assay according to the manufacturer's instructions. In brief, cells were seeded into 96-well plates with 4,000 cells/well at 12 h post-transfection. Afterward 10 μl of CCK8 was added to each well, and the culture medium was incubated at 37°C for additional 3 h. Absorbance at 450 nm wavelength was detected using Eliasa. The absorbance at 450 nm shows mitochondrial activity, and it indirectly reflects living cell number, which indicates cell viability. Each experiment was performed at least in triplicate.
Cell cycle analysis was performed using the standard propidium iodide (PI) method. In brief, cells were trypsinized, washed with cold PBS, fixed in 70% ethanol for 24 h, and stained with PI for 30 min. Finally, the cells were analyzed by flow cytometry (FCM). Each experiment was performed at least in triplicate.
The apoptosis was analyzed by FCM. Apoptotic cells were differentiated from viable or necrotic ones by combined application of annexin V-FITC and PI. The samples were washed twice and adjusted to a concentration of 1×106 cells/ml with cold PBS. 10 μl of annexin V-FITC and 10 μl PI were added into 100 μl of cell suspension, incubated for 15 min at room temperature in the dark. Finally, 400 μl of binding buffer was added to each sample without washing and analyzed using FCM. Each experiment was performed at least in triplicate.
Protein samples were separated by 12% SDS-PAGE and transferred to PVDF membranes (Millipore). After blocked 2 h with 5% milk, the PVDF membranes were then incubated with TP53INP1 (rabbit anti-human polyclonal antibody; 1:100, Santa Cruz), pro-caspase-3 (rabbit anti-human monoclonal antibody; 1:1,000, epitomics), cleaved-caspase-3 (rabbit anti-human monoclonal antibody; 1:1,000, Beyotime), caspase-8 (rabbit anti-human monoclonal antibody; pro-caspase-8: 1:1,000, cleaved-caspase-8: 1:500, Beyotime), caspase-9 (mouse anti- human monoclonal antibody; 1:1,000, Beyotime), and p21 (mouse anti- human monoclonal antibody; 1:200, Beyotime) overnight at 4°C. Following extensive washing for a total of 45 min, membranes were incubated with secondary antibody goat anti-rabbit IgG (1:5,000, Beijing 4A Biotech Co.Ltd) and goat anti-mouse IgG (1:5,000, Beijing 4A Biotech Co.Ltd) for 2 h. After washing three times for a total of 45 min with TBST at room temperature, chemiluminescent detection was performed with the BeyoECL Plus kit (Beyotime), and membranes were exposed to Kodak XBT-1 films.
The pYr-MirTarget-Report plasmids for miR-155 target TP53INP1 3'UTR were constructed as pMIR-TP53INP1-3U containing the wild-type 3'UTR of TP53INP1 and as pMIR-TP53INP1-3U-M containing the mutant 3'UTR of TP53INP1. The pMIR-TP53INP1-3U vector and pMIR-TP53INP1-3U-M vector contain Firefly luciferase and Renilla luciferase. 293 cells were seeded into 96-well plates, and each was co-transfected with pMIR-TP53INP1-3U vector or pMIR-TP53INP1-3U-M vector and 50 nM miR-155 m or miR-155 m NC according to the manufacturer's protocol. After 48 h transfection, luciferase activity was measured with the Dual Luciferase Assay system (Promega). Renilla luciferase activity was normalized to Firefly luciferase activity (Renilla LUC/ Firefly LUC). Each experiment was performed at least three times.
Each experiment was performed in triplicate, and repeated at least three times. The data were shown as the mean ± SD, and differences were compared between 3-group using one-way ANOVA and 2-group using t tests. The P ≤ 0.05 was considered to be statistically significant.
ERalpha expression was monitored by western blot analysis, using as control extracts from ERalpha (−) MDA-MB-231 cells (Figure 1A). In our study, we found that miR-155 expression was significantly higher in MCF-7 cells with ERalpha (+) compared with MDA-MB-231 cells with ERalpha (−) using real-time PCR analysis. As shown in Figure 1B, the expression of miR-155 in MCF-7 cells was 3.05-fold that of MDA-MB-231 cells and the difference had statistical significance.
The expression levels of ERalpha and miR-155 in MCF-7 and MDA-MB-231 cells. (A) Expression level of ERalpha was detected by western blot of protein extracts from ERalpha (+) MCF-7 and ERalpha (−) MDA-MB-231 cells. (B) Differential expression of miR-155 between MCF-7 and MDA-MB-231 cells. The expression of miR-155 in MCF-7 cells was 3.05-fold that of MDA-MB-231cells. **P < 0.01. All experiments were repeated at least three times.
MCF-7 cells were transfected with TP53INP1 overexpression plasmid or control plasmid, and CCK8 assay showed there was a significant reduction in the growth of TP53INP1-expressing cells compared to those transfected with the control plasmid (Figure 2A).
TP53INP1 inhibits MCF-7 cell proliferation and promotes cell apoptosis. (A) CCK8 analysis of cell viability after transfection with TP53INP1 overexpression plasmid or control plasmid. *P < 0.05 and **P < 0.01 versus control. (B) FCM analysis of cell cycle after transfection with TP53INP1 overexpression plasmid or control plasmid. **P < 0.01 versus control. (C) FCM analysis of cell apoptosis rate after transfection with TP53INP1 overexpression plasmid or control plasmid. **P < 0.01 versus control. Data are presented as mean ± SD. All experiments were repeated at least three times.
To elucidate the mechanism of growth inhibition of TP53INP1-expressing MCF-7 cells, we analyzed their cell cycle and cell apoptosis rate. Overexpression of TP53INP1 resulted in a significant increase in the percentage of cells in the G1 phase indicating a G1 cell cycle arrest (Figure 2B). FCM assay showed that overexpression of TP53INP1 increased cell apoptosis rate compared with the control plasmid (Figure 2C). These data revealed that TP53INP1 inhibited the growth of MCF-7 cells by inducing cell apoptosis and inhibiting cell cycle progression.
To study the mechanisms and carcinogenic function of miR-155 on the development of breast cancer, TargetScan, Pictar-Vert, and microRNA.Org were using for this purpose. In combinational predicted with the three softwares, we found that TP53INP1 was a potential target of miR-155. The TP53INP1 3'UTR carries a binding site for miR-155 (Figure 3A), suggesting that TP53INP1 might be a direct target of miR-155. Therefore, we determined that whether overexpression of miR-155 led to down-regulation of TP53INP1 expression in human breast cancer cells.
TP53INP1 is a target of miR-155 in MCF-7 cells. (A) Putative binding sites of miR-155 in the TP53INP1 3'UTR (white sequences) predicted by TargetScan. (B) Sketch of the construction of pMIR-TP53INP1-3U or pMIR-TP53INP1-3U-M vectors. The mutant binding site is underlined. (C) TP53INP1 protein level was measured by western blot at 72 h post-transfection. Data are presented as mean ± SD. **P < 0.01 and *P < 0.05 versus Con, miR-155 m NC or miR-155i NC. (D) miR-155 m down-regulated luciferase activity controlled by wild-type TP53INP1 3'UTR, but did not affect luciferase activity controlled by mutant TP53INP1 3'UTR. *P < 0.05 versus pMIR-TP53INP1-3U+155 m NC and **P < 0.01 versus pYr-MirTarget+155 m, pMIR-TP53INP1-3U-M+155 m. All experiments were repeated at least three times.
Real-time quantitative PCR analyses showed that the TP53INP1 mRNA level was down-regulated by ~45% when miR-155 was overexpression ~69-fold (Figure 4A, 4C). In contrast, the TP53INP1 mRNA level was up-regulated by ~1.5-fold when miR-155 was down-regulated by ~87% (Figures 4B, 4D). Moreover, western blot assay indicated that transfection with miR-155 m led to nearly 78% down-regulation of TP53INP1 in MCF-7 cells compared with Con or miR-155 m NC group, whereas treatment with miR-155i induced 359% increase of TP53INP1 compared with Con or miR-155i NC group (Figure 3C). To understand these negative regulate relationship between miR-155 and TP53INP1, we constructed plasmids containing wild-type or mutant 3'UTR of TP53INP1 fused to the luciferase gene (Figure 3B). The wild-type or mutant plasmid was co-transfected into 293 cells with miR-155 m or miR-155m NC. As shown in Figure 3D, miR-155 significantly decreased the luciferase activity of wild-type 3'UTR of TP53INP1, whereas the luciferase activity with mutant 3'UTR of TP53INP1 was not repressed by miR-155, suggesting that miR-155 could directly bind to the 3'UTR of TP53INP1. Taken together, these findings showed that TP53INP1 is a direct target of miR-155 in breast cancer cells and mRNA degradation is involved in miR-155-suppressing TP53INP1.
Analysis of the expression levels of miR-155 and TP53INP1 mRNA after differential treatment. (A) Expression level of miR-155 after transfection with miR-155 m. Data are presented as mean ± SD. **P < 0.01 versus Con, miR-155 m NC. (B) Expression level of miR-155 after transfection with miR-155i. **P < 0.01 versus Con, miR-155i NC. (C) Expression level of TP53INP1 mRNA after transfection with miR-155 m. *P < 0.05 versus Con, miR-155 m NC. (D) Expression level of TP53INP1 mRNA after transfection with miR-155i. *P < 0.05 versus Con, miR-155i NC. All experiments were repeated at least three times.
To investigate the biologic effects of miR-155, cell proliferation, cell cycle, and cell apoptosis were measured after differential transfection. As shown in Figure 5A, treatment with miR-155 m significantly promoted the growth of MCF-7 cells compared with Con or miR-155 m NC. MiR-155i, but not the miR-155i NC significantly inhibited the growth of MCF-7 cells compared with Con (Figure 5B). Next, we detected the effect of miR-155 on cell cycle, found that miR-155 significantly increased S and G2 phase population, but had more profound effect on G1 phase population compared with Con or miR-155 m NC (Figure 6A). G1 phase population of MCF-7 cells transfected with miR-155i was increased, and S and G2 phase population was reduced compared with Con or miR-155i NC (Figure 6B). Moreover, FCM assay was performed to detect the effect of miR-155 on cell apoptosis of MCF-7 cells. As shown in Figures 6C, 6D, transfected with miR-155 m could significantly reduce cell apoptosis rate of MCF-7 cells compared with Con or miR-155 m NC, and transfected with miR-155i increased cell apoptosis rate compared with Con or miR-155i NC. These data suggested that miR-155 might function as an oncogene in MCF-7 cells.
Cell proliferation of MCF-7 cells after differential treatment. (A) CCK8 analysis of cell viability after transfection with miR-155 m at differential times. *P < 0.05 and **P < 0.01 versus Con, miR-155 m NC. (B) CCK8 analysis of cell viability after transfection with miR-155i at differential times. *P < 0.05 and **P < 0.01 versus Con, miR-155i NC. The OD value reflects cell viability indirectly. All experiments were repeated at least three times.
Cell cycle and cell apoptosis rate of MCF-7 cells after differential treatment. (A) FCM analysis of cell cycle after transfection with miR-155 m. (B) FCM analysis of cell cycle after transfection with miR-155i. (C) FCM analysis of cell apoptosis rate after transfection with miR-155 m. (D) FCM analysis of cell apoptosis rate after transfection with miR-155i. Data are presented as mean ± SD. **P<0.01 and *P < 0.05 versus Con, miR-155 m NC or miR-155i NC. All experiments were repeated at least three times.
To verify the role of miR-155 in protein expression of caspase-3, -8, -9, and p21, MCF-7 cells were transfected with miR-155 m or miR-155i. Treatment with miR-155 m induced 87%, 65%, 68%, and 33% reduced in cleaved-caspase-3, -8, -9, and p21 compared with Con or miR-155 m NC, whereas transfection with miR-155i led to nearly 97%, 90%, 159%, and 60% up-regulation of cleaved-caspase-3, -8, -9, and p21 compared with Con or miR-155i NC (Figure 7). These data suggested that cell cycle and apoptosis-related protein caspase-3, -8, -9, and p21 were possible the potential downstream genes of TP53INP1.
MiR-155 negatively regulated protein expression of cleaved-caspase-3, -8, -9, and p21 in MCF-7 cells. (A) Protein expression levels of p21 and cleaved-caspase-3 after transfection with miR-155 m. (B) Protein expression levels of p21 and cleaved-caspase-3 after transfection with miR-155i. (C) Protein expression levels of cleaved-caspase-8, -9 after transfection with miR-155 m. (D) Protein expression levels of cleaved-caspase-8, -9 after transfection with miR-155i. The protein expression levels of cleaved-caspase-3, -8, -9, and p21 were detected by western blot and normalized to that of β- actin. Data are presented as mean ± SD. **P < 0.01 and *P < 0.05 versus Con, miR-155 m NC or miR-155i NC. All experiments were repeated at least three times.
The mechanisms of miR-155 functions as an oncomiR are largely unknown. Our study here was to investigate the effects of miR-155 on cell proliferation, cell cycle and cell apoptosis of ERalpha (+) breast cancer cells and to verify whether TP53INP1 is a target of miR-155, and tried to explore the mechanisms of miR-155 in this process.
In our study, we found that miR-155 expression was significantly higher in ERalpha (+) MCF-7 cells than ERalpha (−) MDA-MB-231 cells. Moreover, we observed that up-regulation of miR-155 significantly stimulated cell growth and inhibited cell apoptosis in MCF-7 cells, which was consistent with results that blocking miR-155 using miR-155 inhibitors inhibited cell growth and promoted cell apoptosis. But, how miR-155 might functions inside cells accounting for the effects of miR-155 on biologic behavior of MCF-7 cells. To study the mechanisms and carcinogenic function of miR-155 on the development of breast cancer, TargetScan, Pictar-Vert, and microRNA.Org were using for this purpose. In combinational predicted with the three softwares, we found that TP53INP1 was a potential target of miR-155. Therefore, we focused our attention on TP53INP1 signal pathways.
The main pathway in human breast cancer involve p53, which acts as a multifunctional transcription factor, exerts its tumor suppressor function mainly through transcriptional induction of target genes involved in several processes, including cell cycle checkpoints and apoptosis . TP53INP1, one of the p53 target genes, is a p53-inducible cell stress response gene [24, 25] located on the chromosome 8q22 . Its expression is dependent on the activation of wide-type p53 . Originally, TP53INP1 expression is strongly induced in mice with acute pancreatitis in vivo , and also in several cell lines under exposure to various stress agents in vitro [25, 27, 28]. Overexpression of TP53INP1, which is a tumor suppressor gene, induces cell cycle arrest as well as enhances the p53-mediated apoptosis . In our study, we have also showed that ectopic expression of TP53INP1 inhibited growth of MCF-7 cells by inducing cell apoptosis and inhibiting cell cycle progression. Moreover, recent findings have shown a significant reduction of the expression of TP53INP1 during the development of breast cancer , stomach cancer , and pancreas cancer . TP53INP1 expression was investigated immunohistochemically in 81 cases of breast carcinoma, compared with normal breast tissue, decreased TP53INP1 expression was found in 45 cases (55.6%) . The expression level of TP53INP1 was inversely linked to high histological grade, tumor size, positive lymph node metastasis, and aberrant p53 expression . Besides, it had been reported that TP53INP1-positive rate decreased with the progression of gastric cancer; and TP53INP1 protein negativity was significantly associated with aggressive pathological phenotypes of gastric cancer [30, 31]. These observations suggest that TP53INP1 plays a crucial role in suppression of tumor progression including breast cancer through its anti-proliferative and pro-apoptotic activities. Our data showed that overexpression of miR-155 not only led to the down-regulation of TP53INP1 but also inversed the effect of TP53INP1 by promoting cell proliferation and suppressing cell apoptosis. Restoration of TP53INP1 by knockdown of miR-155 using miR-155 inhibitors was accompanied by decreased cell proliferation and enhanced cell apoptosis. The conclusion that miR-155 negatively regulated the protein expression of TP53INP1 was further supported by data showing that miR-155 significantly decreased the relative luciferase activity of TP53INP1 3'UTR. MiR-155 can directly repress TP53INP1 expression through binding to the binding sites in the 3'UTR of TP53INP1, thereby negatively regulating TP53INP1 function. Thus, we have reasons to believe that TP53INP1 is possible the target gene of miR-155 in MCF-7 cells.
Apoptosis is a process of programmed cell death that plays a crucial role in both normal growth and maintaining cellular homeostasis, and regulated by numerous signaling pathways . Dysregulated apoptosis is caused by the changes in the expression and activation of key apoptotic regulators . The caspase family of cysteine proteases plays a key role in coordinating the stereotypical events that occur during apoptosis. Caspases are classified into two groups according to their structure and function: the upstream initiator, such as caspase-8, -9, and −10, and the downstream executioner, such as caspase-3, -6, and-7 . Cell cycle arrest is closely linked to apoptosis. P21 is cyclin-dependent kinase inhibitor located in p53 gene downstream, and plays a key role in cell cycle G0/G1 arrest. Our data showed that down-regulation of TP53INP1 by miR-155 correlated with down-regulation of cleaved-caspase-3, -8, -9, and p21. It indicated that TP53INP1 regulates cell cycle and induces cell apoptosis may be by down-regulating p21 and activating caspase way.
In summary, our researches suggest that miR-155 functions as an oncomiR by targeting TP53INP1 and contributes to the control of cell survival and growth in breast cancer cells. Tumor suppressor gene TP53INP1 is negatively regulated by miR-155 and mediates miR-155 functions in inducing cell proliferation and inhibiting cell apoptosis. Therefore, TP53INP1 is identified to be the direct target of miR-155. The findings may be providing a new therapeutic strategy for breast cancer.
Tumor protein 53-induced nuclear protein 1.
We are grateful to the Stem Cell and Tissue Engineering Lab at Chongqing Medical University for providing experimental equipments, and thank all staff in this lab for their help and advice. We also thank the Institute of Life Sciences for providing the technical assistance of cell cycle analysis, cell apoptosis analysis, and real-time quantitative PCR.
CMZ and HYD designed experiments. CMZ performed major experiments and data analysis. CMZ and HYD drafted manuscript. JZ provided financial support. All authors read and approved the final manuscript.