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The MICHELIN Guide Great Britain and Ireland, 2021: a new year, a new chapter
News & Views 1 minute 08 September 2020
Great Britain Ireland Michelin Guide
The MICHELIN Star Revelation Great Britain and Ireland 2021 will be held on January 25, 2021.
All the new MICHELIN Stars will be unveiled at the event, which will take place at The Dorchester Hotel in London, and which will highlight the resilience, quality and potential of the industry across the UK and Ireland.
The event also marks the launch of the MICHELIN Guide Great Britain and Ireland 2021, which will be a digital-only publication to take into account industry recovery from Covid-19 and to offer users more modern and engaging resources via the app and on guide.michelin.com.
"Moving the publication and launch from our usual October date will not only allow time for the hospitality industry to get back on its feet, but will also let us prepare a full and comprehensive guide for our readers."
Gwendal Poullennec, International Director of the MICHELIN Guides, said: "Our inspectors are eating out and supporting the industry across Great Britain and Ireland. Moving the event to January and launching a digital-only guide gives us the time we need to be fair to restaurateurs and to ensure the consistent, expert advice our customers demand.
"Restaurants have been through challenges we previously couldn't even have imagined – and I want to applaud chefs and owners for the passion, ingenuity and entrepreneurship they have shown this year."
"Our inspectors have been out and about for some weeks now, eating twice a day in restaurants and pubs. They all love being back out there and have been busy sharing their joy and posting about their experiences on our social media platforms. We hope this has enticed many customers back into eating out and supporting their local restaurants and the restaurant industry at large."
The talent, adaptability and commitment shown by chefs and restaurateurs are the signs of an industry with huge potential, the MICHELIN Guide teams believe – and the MICHELIN Guide wants to support that potential by showcasing excellent restaurants to hundreds of thousands of people across its digital channels.
"The future may look different but our goal remains constant: to connect customers to the best restaurants around the country, whatever the occasion, and to celebrate this most magnificent of industries - one that brings so much joy to so many."
Access to the MICHELIN Star Revelation – both physical and digital – will constantly be reviewed to ensure as many people as possible can enjoy it in complete safety. No further details will be available until closer to the event.
About Michelin
Michelin, the leading mobility company, is dedicated to enhancing its clients' mobility, sustainably; designing and distributing the most suitable tyres, services and solutions for its clients' needs; providing digital services, maps and guides to help enrich trips and travels and make them unique experiences; and developing high-technology materials that serve a variety of industries. Headquartered in Clermont-Ferrand, France, Michelin is present in 170 countries, has more than 127,000 employees and operates 69 tyre production facilities which together produced around 200 million tyres in 2019. (www.michelin.com)
David Johnson: d.johnson@michelin.com; 0044 (0)1782 402341
News & Views 10 minutes
30% of MICHELIN Starred Restaurants open in Week 1_2021 (01/04 - 01/10)
The MICHELIN Guide has developed an international barometer to facilitate analysis of the impact of the Covid-19 crisis on Michelin Starred restaurants.
Michelin Guide restaurant Michelin Stars
News & Views 2 minutes
The MICHELIN Guide Spain & Portugal 2021 presents its latest selection of starred restaurants
In an exclusive virtual gala broadcast from La Puerta Del Sol in Madrid, the MICHELIN Guide Spain & Portugal unveiled its new 2021 restaurant selection. Featuring 252 starred establishments, including 3 new two Star restaurants and 21 that won their first Star, the 2021 edition also welcomed a new distinction, the MICHELIN Green Star which aims to highlight exemplary establishments operating at the forefront of sustainable gastronomy with virtuous initiatives.
Michelin Stars Portugal Spain
News & Views 1 minute
MICHELIN launches the MICHELIN Guide Main cities of Europe 2020
Cracow and Ljubljana have their first ever MICHELIN Starred restaurants ; Austria has two new 1 Star restaurants.
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Inspectors Michelin Guide United Kingdom
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A UK Michelin Inspector wants his old life back.
Inspectors Michelin Guide Chefs and the Community
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"Good morning, everyone, First of all, on behalf of the whole MICHELIN Guide team, I want to express my deepest sympathy for the families and loved ...
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MICHELIN Guide Croatia 2020: LD Terrace and Boškinac earn one Star
Michelin is pleased to unveil the new selection for the fourth edition of the MICHELIN Guide Croatia 2020. With this selection, the MICHELIN Guide celebrates the Croatian culinary scene and demonstrates its richness and diversity with four outstanding new addresses.
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Samsung relies on MicroLED and 8K for its premium TVs 2023
Four years after unveiling The Wall at CES 2018, Samsung is ready to take its MicroLED technology mainstream. At CES 2023, the company announced that it will be offering 50-, 63-, 76-, 89-, 101-, 114- and 140-inch MicroLED models, significantly expanding consumer choices with the new display technology would expand . Samsung hasn't provided pricing and availability information for the expanded line, but the company claims the new models are the most affordable MicroLED TVs yet. Since some of the devices are smaller than any MicroLED TVs Samsung has offered in the past, you also don't have to pay for a professional to install them in your home.
Samsung claims its MicroLED line will set the standard for picture quality in 2023. And judging by the 76-inch model's top-line features, that's not just a boast from the company. The set has a variable refresh rate of 240 Hz and a response time of 2 nanoseconds. It also offers 20-bit black detail for "intense" contrast.
Samsung is also promising upgrades to its Neo QLED sets, starting with the line's new flagship. The QN900C features an 8K Quantum MiniLED backlit panel that can reach a maximum brightness of 4,000 nits. As with Samsung's 2022 Neo QLED sets, the QN900C features a 14-bit backlight. However, thanks to a tweak the company has made to its 8K Real Depth Enhancer Pro software, the TV offers even better contrast. Samsung also promises improved picture quality when watching older movies and TV shows thanks to the inclusion of its new Auto HDR remastering algorithm, which can automatically apply HDR effects to standard dynamic range content.
For those who prefer a 4K set, there's also the QN935C. Samsung's new 4K flagship features a redesigned power supply board that eliminates the need for an external junction box and allows for bezels less than 20mm thick. The QN935C also features top-firing speakers, allowing the set to produce Dolby Atmos sound without a dedicated soundbar. You can use both the QN900C and QN935C as a smart home hub, as Samsung's entire 2023 Neo QLED lineup will have built-in Zigbee and Matter Thread all-in-one modules.
At last year's CES, Samsung introduced its new QD-OLED technology. This year, the company is adding additional models to the lineup, including an all-new 77-inch model. As previously announced, the 2023 QD OLED TV lineup will offer up to 2,000 nits of peak brightness thanks to Samsung's new HyperEfficient EL OLED material and IntelliSense AI. Samsung says its latest panels are also more energy efficient, supporting 144Hz refresh rates. In addition, the company has made every effort to have the panels AMD FreeSync Premium Pro certified. Samsung hasn't announced pricing and availability for its QD OLED line. Expect these details to emerge in the coming weeks.
All products recommended by Engadget are selected by our editorial team independently from our parent company. Some of our stories contain affiliate links. If you buy something through one of these links, we may receive an affiliate commission. All prices are correct at time of publication.
The best products we found at CES 2023, from giant TVs to tiny pee sensors News After a two-year hiatus, CES is back in great shape. The world's biggest tech companies came to Las Vegas once again, bringing with them some notable merchandise. Among other things, we saw an $800,000 flying car, a crazy giant wireless TV (more on that below), and yes, a urine sensor…
Eamonn Holmes relies on crutches as he leaves GB News studios in London following return to TV Entertainment Eamonn Holmes was finally back on GB News on Monday morning (Picture: Getty) Eamonn Holmes has been photographed relying on a pair of crutches as he left the GB News studios in London after returning to his breakfast show following a back operation. The 63-year-old hasn't been on air for…
BREAKING: Samsung confirms Galaxy S23 launch date on its website News After a series of leaks and rumors surrounding the Galaxy S23 series, Samsung has confirmed the launch date of the Galaxy S23. The company released the information through its own website, and it's pretty certain that the South Korean company will unveil the Galaxy S23 at its Galaxy Unpacked 2023…
Fitbit Sense 2, Versa 4 get new premium watch faces News At launch, Fitbit's new smartwatches lacked any third-party apps, but that appears to be changing. Fitbit also recently released new premium watch faces for the Sense 2 and Versa 4. At launch, the Apps tab of the Gallery on the Sense 2 and Versa 4 contained only Fitbit-developed apps: EDA…
Post Premium: Top stories for the week of Jan. 2-8 News Conditions on the drying Colorado River are worsening faster than expected. States can't agree on how to divide water cuts. Native American officials say they're still largely shut out from the bargaining table and murmurs of a dystopian "water war" scenario now punctuate the conversations. The crisis is over a century…

redpajama_set_name: RedPajamaCommonCrawl


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text:
\section{INTRODUCTION}

Since the ${\rm C}_{60}$ solid and $A_3{\rm C}_{60}$$^{1}$ with the novel high
temperature superconductivity were discovered, fullerenes have
been intensively investigated.  As the $\pi$ electrons are
delocalized on their surfaces, the fullerenes show optical
responses that are similar to those in $\pi$ conjugated polymers.$^{2}$
For example, the absorption spectra of ${\rm C}_{60}$ (Refs. 3 and 4)
and ${\rm C}_{70}$ (Ref. 3) reflect the existence of excitons (mainly
Frenkel excitons) which are important when the excitation
energy is larger than the order of 1eV.  The nonlinearity of
${\rm C}_{60}$ in the third harmonic generation (THG) is of the order
$10^{-11}$esu,$^{5,6}$ and the similar magnitudes have been
observed in polydiacetylenes.

Recently, we have studied the linear absorption and the THG of
${\rm C}_{60}$ by using a tight binding model$^{7}$ and a model with
a long ranged Coulomb interaction.$^{8,9}$  A free electron
model yields the THG magnitudes which are in agreement with the
experiments of ${\rm C}_{60}$.$^{5,6}$  However, when the Coulomb interactions
are taken into account, the THG magnitudes decrease.$^{8}$  We
have discussed that the local field correction would be necessary
in order to recover the agreement.  The model with Coulomb interactions
has turned out to describe well the linear absorption spectra of
${\rm C}_{60}$ and ${\rm C}_{70}$ in solutions.$^{9}$

In the first half part of this paper, we perform calculations for
${\rm C}_{60}^{6-}$ and consider the optical spectra of $A_6{\rm C}_{60}$
($A =$ K, Rb, Cs, etc).  This compound is a fulleride maximumly
doped with alkali metals, and is an insulator like the neutral ${\rm C}_{60}$.
The optical spectra have been measured in several papers.$^{10,11}$
The peak structures in the energy dependence are quite different from
those of the neutral ${\rm C}_{60}$, even though each peak could be explained
as an optical transition between molecular orbitals.  The absorption
spectra of the neutral ${\rm C}_{60}$ and $A_3{\rm C}_{60}$ are rather similar, but
the data of $A_6{\rm C}_{60}$ are largely different.  It seems that main peaks
of the neutral ${\rm C}_{60}$ spectra move to lower energies.  This fact could
be explained by reduction of the effective Coulomb interaction strengths.
The principal purpose of the present calculations is to confirm this
viewpoint.  The calculation method is the same as that used in the
previous paper.$^9$  We start from the Hartree-Fock approximation and
perform configuration interaction calculations which are limited to
single electron-hole excitations (single CI).  In the experiments
of $A_6{\rm C}_{60}$,$^{10,11}$ intermolecular interactions seem to
be relatively weak, because there is not a peak structure owing
to the intermolecular interactions (like the 2.8eV structure in
the ${\rm C}_{60}$ films$^{4}$).  Therefore, we shall limit our
considerations to the system ${\rm C}_{60}^{6-}$.  This is a reasonable
approximation for systems where single molecular contributions
(Frenkel exciton effects) are dominant.

The reduction of the Coulomb interactions means that dielectric
constants of the doped systems increase.  Actually, the
electron-energy-loss spectroscopy (EELS) studies of Rb$_x {\rm C}_{60}$
and Rb$_x {\rm C}_{70}$ ($x = 0, 3, 6$)$^{12}$ have revealed that the real part
of the dielectric function at zero frequency increases from
$x=0$ to 6, and the magnitude becomes about twice.  In our calculations
by single molecule models, this enhancement of the dielectric
functions is represented by the decrease of the phenomenological
parameters in Coulomb interactions.

In the last part of the paper, we consider ${\rm C}_{70}^{6-}$ in order
to discuss optical properties of the insulating phase $A_6{\rm C}_{70}$.
Frenkel excitons would be dominant in this system again.
We demonstrate that the picture of the reduction of Coulomb
strengths is relevant to ${\rm C}_{70}$ also.  Therefore, the reduction
of Coulomb interaction strengths is a common property seen in
two kinds of fullerenes.  In fact, the EELS studies$^{12}$
have concluded that $\pi$ electronic systems in Rb$_x{\rm C}_{60}$
and Rb$_x{\rm C}_{70}$ show a non-rigid-band behavior and excitation
energies become smaller upon doping.

In the next section, we explain our model briefly.  In Sec. III,
we show results of ${\rm C}_{60}^{6-}$ and give discussion relating
with molecular orbital structures. In Sec. IV, we consider ${\rm C}_{70}^{6-}$.
The final section is devoted to the summary.

\noindent
{\bf omitted afterwards ...}

\end{document}

redpajama_set_name: RedPajamaArXiv


A less obvious snafu, closer to home, is the seven-person competition for the three-vacancy Warkworth Subdivision of the Rodney Local Board. Supporters of the need for much younger representatives to be elected will enthusiastically give their 'first' tick to Tessa Berger, but if they go on to get their money's worth by ticking another couple of candidates, they could well spoil the chances for their favourite. Regrettably, first-past-the-post forces thinking voters to vote strategically, and discipline themselves to support only one candidate.


text:
Ltd great time and a great life coaching basketball parties
by | Mar 11, 2020 | Uncategorized | 0 comments
This is not any cause for glee and comfort, Madame Speaker said, as her giddy caucus used the floor of the House of Representatives – the very same place where President Franklin Roosevelt asked for a declaration of war following the Day of Infamy on Dec.
I think it's really about what they do at the quarterback position.
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Aided by a scathing halftime analysis from Custom Derek Carr Jersey TNT crew, Whiteside's Custom Dalvin Cook Jersey was heavily scrutinized.
They'll spread you out.
From there, her worries only grew.
Powell pulled down seven rebounds and added an assist, accumulating a plus-minus of +15 in his 30 on the floor.
If someone lost their tickets or the dog ate them, they called Leo.
Ronda Rousey 9.
James White – New England opened the game in a one-back set with White in the backfield and a no-huddle approach.
A Democrat, he served in the United States House of Representatives from 1993 to 1995 and then again from 1999 to 2012.
By the Numbers: Both the Devils and Senators haven't had the greatest success in games where they've scored the first goal this season.
The 21-year-old followed up his 28-goal rookie season with a 41-goal breakout year in 2018 and picked up right where he left off in Friday's season opener.
Woods and Pierre-Louis specifically bring a lot of value to special teams.
Boqvist was already expected to play in the OHL this season instead of his native Sweden, but his inclusion at training camp should aid his development.
• MLB and its 30 clubs have launched a charity auction, with net proceeds benefiting various charities that support ALS awareness, research and care efforts including ALS CURE Project, Healey Center for ALS at Mass General, ALS Association, Project Main Street and ALS Therapy Development Institute.
She went with only players I put marks next to, creating this roster of sleepers.
We would have been lucky to scrimmage and play once a week.
• TransPerfect, a New York-based global provider of language and technology solutions for business, has signed a multiyear agreement to become title sponsor for the Music City Bowl college football game in Nashville's Nissan Stadium beginning in 2020.
He notched three points in seven contests at the 2019 IIHF Under-18 Men's World Championship in Sweden to help the United States capture the bronze medal, and he earned a gold medal with Team USA at the 2018 Under-17 World Hockey Challenge, tallying one point in six outings.
I was not into working out before.
The left used to understand this.
You think about we've had success all year, it's being able to shut down other teams.
It was a good day and a good step forward for New England's two veteran complementary receivers.
The character himself has got a really aggressive drive, Boreanaz says.
That's just the type of guy he is, and I think that's helping him in these playoffs.
Thompson's doctor believes he could begin doing drills as soon as OTAs, enough for Thompson to catch some passes during the individual portions of practice and work on the side fields, slowly initiating him back into football shape.
In 2009, the team acquired Zach Randolph from the Clippers, and one year later they signed Tony Allen.
If you know someone who's trypophobic, it's key to not judge how they react or how triggering images make them feel.
Ran for 1 rushing yards and eight touchdowns on 219 attempts; 13th time a Bulldog rushed for 1 yards in a season.
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Watson will try and bounce back after throwing two interceptions for the second straight week in a 30 loss to Indianapolis last week which knocked Houston out of first place in the AFC South.
I think he came over from the Swedish Elite League and was really amazing over there last season so what a performance by him and a great start to his career.
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Thunder Waive Devon Hall From Two-Way Contract Dec 12 4 PM The Oklahoma City Thunder has waived guard Devon Hall.
Oil revenues pay for almost everything.


text:
Gás de síntese - é uma mistura combustível de gases, produzida a partir de processos de gaseificação, ou seja de combustão incompleta de combustíveis sólidos. Utiliza-se, por exemplo, madeira, carvão ou outros combustíveis, geralmente ricos em carbono, usando oxigênio insuficiente para a queima completa e (em alguns casos) vapor de água. Dependendo das matérias-primas usadas, os gases gerados também são conhecidos como: gás de madeira, gás de cidade ("gás de carvão"), gás de água e, por seu baixo nível calorífico, "gás pobre".

História

O aparelho para a produção deste gás, o gasogênio, foi inventado nos anos 1920 pelo francês Georges Imbert.

A produção de gás de síntese tem maior importância histórica em períodos de escassez energética. Foi empregada em diferentes regiões do mundo, com intuito de produzir estrategicamente combustíveis aptos ao uso em motores de combustão interna e turbinas a gás.

Por exemplo, durante a Segunda Guerra Mundial, houve dificuldade de importação de petróleo principalmente pela Alemanha, devido a restrições de comércio marítimo. Foram usados muitos equipamentos embarcados nos próprios veículos, permitindo produzir gás de síntese e alimentar motores de automóveis e tratores, ficando num reboque e consumindo principalmente madeira.

No Brasil, nesta mesma época, a dificuldade na importação do petróleo causou  racionamento de gasolina. Esta era destinada principalmente a táxis e veículos oficiais.  O gás de síntese tornou-se então a única opção para muitos veículos de passeio particulares. Seu uso era incentivado pelo governo.

Com o mesmo objetivo de suprir demandas energéticas de veículos, construiu-se plantas capazes de produzir óleo combustível. Em alguns casos se empregam processos de pirólise, que produz prioritariamente combustíveis líquidos como o bio-óleo. Já no processo de Fischer-Tropsch, o gás de síntese pode ser transformado em combustíveis líquidos, como gasolina. O gás obtido a partir da gaseificação de materiais orgânicos, pode ser empregado na produção de plásticos, usado diretamente como combustível ou transformado em combustíveis líquidos como o etanol.

A partir da década de 1990, a produção de gás de síntese passou a receber novo destaque devido a novos tipos de plantas de geração de energia elétrica, que envolvem gaseificação integrada a ciclo combinado. Esta configuração produz gás de síntese e alimenta turbinas a gás que, por sua vez, rejeitam calor. Este calor faz funcionar caldeiras a vapor que movimentam turbinas a vapor, gerando outra parcela de energia elétrica.

O gás de síntese tem também importância em processos industriais envolvendo combustão. Da mesma forma que em veículos, o gás pode ser mais adequado para o consumo em fornos industriais, proporcionando queima mais rápida e limpa em relação a combustíveis sólidos.

Folha artificial
Uma folha artificial replica a fotossíntese, o processo pelo qual uma folha converte dióxido de carbono, água e luz solar em energia. Mas, a folha artificial melhora o modelo da natureza. Por um lado, converte o excesso de dióxido de carbono da nossa atmosfera em energia utilizável. Uma planta, em sua máxima eficiência, converte 1% da luz solar em alimentos. Ao usar dióxido de carbono puro, algumas folhas artificiais da década de 2010 conseguiram atingir uma eficiência de 10%. Além disso, a energia gerada por suas folhas pode ser armazenada para uso posterior. E, finalmente, quando substituíram a bactéria usada na folha artificial por outra, produziu fertilizante à base de nitrogênio, um subproduto valioso para usuários em climas deficientes em nutrientes.

Em 2019, uma folha artificial é uma alternativa para produzir syngas e combustível líquido sustentável. Alimentada pela luz solar, a folha funciona com eficiência mesmo nos dias nublados. E, em comparação com os processos industriais atuais para a produção de syngas, o dispositivo não libera dióxido de carbono adicional na atmosfera. A folha artificial é equipada com dois absorvedores de luz com um catalisador feito a partir do elemento naturalmente abundante cobalto. Após a imersão em água, um absorvedor de luz usa o catalisador para produzir oxigênio, enquanto outros realizam a reação química que reduz o dióxido de carbono e a água em monóxido de carbono e hidrogênio, formando a mistura de syngas.

Produção

O gás de síntese é produzido, por exemplo, a partir de resíduos agrícolas (biomassa) e carvão, em usinas para geração de energia elétrica. Industrialmente, usa-se o oxigênio e/ou ar para queima, adicionando-se também vapor de água em alguns casos, que reage com o material aquecido a ~700°C reduzindo a parcela de monóxido de carbono e ampliando a parcela de hidrogênio.

 2C + O2 ⇌ 2CO + 58 000 cal por mol (44g).
 C + O2 ⇌ CO2 + 97 000 cal por mol.
 2CO + O2 ⇌ 2CO2 + 136 000 cal por mol.
 CO2 + C ⇌ 2CO - 38 000 cal por mol.

Quando vapor de água é injetado no ar de combustão, tem suas moléculas "quebradas" térmicamente (ver: reação de mudança do vapor de água). Tal reação produz grandes quantidades de hidrogênio obtendo-se misturas gasosas com as seguintes composições:

 C + H2O ⇌ CO + H2 ⇌ - 28 000 cal por mol.
 C + 2H2O ⇌ CO2 + 2H2 - 18 000 cal por mol.
 CO + H2O ⇌ CO2 + H2 + 9 400 cal por mol.

Ver também
 Bioenergia (combustíveis)
 Combustíveis alternativos
 Combustível sintético
 Energia renovável
 Fontes alternativas de combustíveis
 Fotossíntese artificial
 gás de cidade
 Gaseificação
 Pirólise
 Processo de Fischer-Tropsch

Ligações externas
 Carroantigo - GASOGÊNIO, um quebra-galho do tempo da guerra para a falta de gasolina
 Best Cars Web Site - Gasogênio, o motor movido a carvão
  APVGN - Veículos a gás do passado

 Samba-choro.com TUDO AGORA É SONHO - Letra de Samba sobre o carro a gasogênio
 Redetec - Unidade Gaseificadora. Bernardo Cruz Gondim o especialista em Gasogênio
 Novomilenio - Um caminhão movido a gasogênio, em 1941

Misturas químicas
Gás combustível
Gases industriais

redpajama_set_name: RedPajamaWikipedia


Dundalk Youth team manager Alan Browne has expressed his delight at Ciaran O'Connor's progress to the first-team squad at Oriel Park.
'Gazza' signed a professional contract at the club on July 1 after hitting 16 goals for the club's U18 team last season.
The youngster was honoured with the Youth team Top Goalscorer award at a presentation function prior to last Friday night's game with Bohemians.
And Browne said that if the 17-year-old can repeat his performances from last season, then it will not be long before he is pushing for a place in Stephen Kenny's first-team.
While the first-team approach the final third of their season, Browne is currently starting the preparations for the Youths' upcoming DDSL campaign which begins in September.
Browne's new batch will be predominantly made up of young players who can stay with the Youths for two seasons before progressing to the club's U19 squad.
"I think thats the way to look at it", said Browne. "I don't think there is any use in having an U18 squad full of 'U18s' who only have one year with us before they progress.
"Going from what I've seen in the trials, it will be a very young team but I don't mind that because some of the fellas I have seen have impressed me already and they are very young.
The former junior football star is approaching his third season as Youths manager and he said he is enjoying the experience, despite the demands of the job.
"Don't get me wrong, I enjoy it all year round but I always love the break at the end of the season", said Browne with a smile.
"It is a long season. Depending on how well you do in cup competitions you could either finish in May or the end of June.
"For us as coaches, to see the lads win a trophy and then when you see the likes of Gazza getting through to the first-team and the likes of Paul Finnegan on the fringes of the first-team, it makes it all worthwhile.


text:
Re-expression of miR-200s in claudin‐low mammary tumor cells alters cell shape and reduces proliferation and invasion potentially through modulating other miRNAs and SUZ12 regulated genes
K. Simpson1,
G. Conquer-van Heumen1,
K. L. Watson1,
M. Roth1,
C. J. Martin1 &
R. A. Moorehead ORCID: orcid.org/0000-0001-9539-62951
MicroRNAs are a class of non-coding RNAs that regulate gene expression through binding to mRNAs and preventing their translation. One family of microRNAs known as the miR-200 family is an important regulator of epithelial identity. The miR-200 family consists of five members expressed in two distinct clusters; the miR-200c/141 cluster and the miR-200b/200a/429 cluster. We have found that murine and human mammary tumor cells with claudin-low characteristics are associated with very low levels of all five miR-200s.
To determine the impact of miR-200s on claudin-low mammary tumor cells, the miR-200c/141 cluster and the miR-200b/200a/429 cluster were stably re-expressed in murine (RJ423) and human (MDA-MB-231) claudin-low mammary tumor cells. Cell proliferation and migration were assessed using BrdU incorporation and transwell migration across Matrigel coated inserts, respectively. miRNA sequencing and RNA sequencing were performed to explore miRNAs and mRNAs regulated by miR-200 re-expression while Enrichr-based pathway analysis was utilized to identify cellular functions modified by miR-200s.
Re-expression of the miR-200s in murine and human claudin-low mammary tumor cells partially restored an epithelial cell morphology and significantly inhibited proliferation and cell invasion in vitro. miRNA sequencing and mRNA sequencing revealed that re-expression of miR-200s altered the expression of other microRNAs and genes regulated by SUZ12 providing insight into the complexity of miR-200 function. SUZ12 is a member of the polycomb repressor complex 2 that suppresses gene expression through methylating histone H3 at lysine 27. Flow cytometry confirmed that re-expression of miR-200s increased histone H3 methylation at lysine 27.
Re-expression of miR-200s in claudin-low mammary tumor cells alters cell morphology and reduces proliferation and invasion, an effect potentially mediated by SUZ12-regulated genes and other microRNAs.
MicroRNAs (miRNAs) are small, non-coding RNA molecules that can be found in both introns and exons [1]. MiRNAs regulate mRNA translation by binding to mRNAs and in turn, target the mRNA for degradation or impair ribosome mediated translation of the mRNA [2, 3]. The interaction between miRNA and mRNA is primarily regulated by nucleotides 1–8 of the miRNA. This region, known as the seed sequence, binds to complementary sequences in the 3' untranslated regions of mRNAs [4,5,6,7,8,9,10,11,12]. However, more recent studies have shown that non-canonical sites can also influence miRNA-mRNA interaction [13, 14] and miRNAs have been found to directly regulate gene transcription, activating Toll-like receptors and upregulating protein expression [15, 16], further increasing their complexity.
A number of miRNAs have been implicated in regulating breast cancer including the miR-200 family. The miR-200 family consists of 5 members organized into two clusters, the miR-200c/141 cluster and the miR-200b/200a/429 cluster [17,18,19]. miR-200b, miR-200c, and miR-429 share a common seed sequence (AAUACUG) while miR-200a and miR-141 share the same seed sequence (AACACUG) that differs from the seed sequence of miR-200b, miR-200c and miR-429 by one nucleotide [20]. Therefore, both miR-200 clusters express miRNAs with the AAUACUG and AACACUG seed sequences and thus are predicted to share mRNA targets. The most extensively characterized function of the miR-200 family is the regulation of epithelial and mesenchymal phenotypes. miR-200 family members negatively regulate mesenchymal transcription factors such as Zeb1/2, Twist1/2, Snai1/2, [19, 21,22,23] while inducing the expression of epithelial genes [24, 25]. Therefore, loss of miR-200s facilitates epithelial to mesenchymal transition (EMT).
Consistent with their roles in epithelial identity, most studies found that miR-200s are expressed in human luminal breast cancer but lost in triple negative breast cancer (TNBC) [26,27,28,29,30]. TNBCs lack expression of the estrogen receptor (ER), progesterone receptor (PR), and HER2, and have a poorer prognosis than luminal breast cancer [31,32,33,34]. Claudin-low breast cancers share features with TNBC including frequent loss of ER, PR and HER2 expression, high expression of mesenchymal genes and features of an epithelial–mesenchymal transition (EMT) phenotype [35]. In addition, claudin-low tumors express low levels of cell-cell adhesion and tight junction genes including claudins 3, 4, 7.
Our previous work with miR-200s in a murine claudin-low mammary tumor cell line, RJ423, revealed that re-expression of the miR-200b/200a/429 cluster reduced mesenchymal gene expression, restored a more epithelial-like morphology, reduced migration and inhibited tumor growth and metastasis in vivo [36]. In this current study, both the miR-200b/200a/429 and miR-200c/141 clusters were re-expressed in RJ423 cells as well as the human claudin-low breast cancer cell line, MDA-MB-231. Our data showed that when miR-200s are expressed at sufficiently high levels, they can restore a more epithelial like morphology and impair cell migration. miRNA sequencing revealed that re-expression of miR-200s significantly influenced the expression of > 50 miRNAs and RNA sequencing revealed significant alterations in > 1000 mRNAs. One of the molecular features associated with the differentially expressed mRNAs was an alteration in SUZ12 regulated genes. SUZ12 is a core protein of the polycomb repressor complex 2 PRC2 that regulates gene transcription through methylating histone H3 at lysine 27 (H3K27). Therefore, miR-200s appear to influence cell morphology and migration through (i) directly targeting mRNAs, (ii) altering the expression of other miRNAs, and (iii) regulating methylation of histone H3.
Cell lines and transfections
MCF-7 (cat #HTB-22) and MDA-MB-231 (cat #HTB-26) cells were purchased from ATCC (Manassas, VA). MDA-200c141 and MDA-200ba429 cells were created by transfecting MDA-MB-231 cells with pLenti 4.1 Ex miR200c-141 (cat #35,534, Addgene, Watertown, MA) or pLenti 4.1 Ex miR200b-200a-429 (cat #35,533, Addgene, Watertown, MA), respectively. MDA-231EV cells were created by infecting MDA-MB-231 cells with copGFP control lentiviral particles (cat #sc-108,084, Santa Cruz Biotechnology Inc, Dallas, TX). MCF-7, MDA-MB-231 and variants of MDA-MB-231 cells were maintained in DMEM media supplemented with 10% FBS, 1 mM sodium pyruvate, 4 mM glutamine, 1% antibiotic/antimycotic and 60 µg/ml puromycin (InvivoGen, San Diego, CA). RJ345 and RJ423 cells were created from mammary tumors that developed in distinct MTB-IGFIR transgenic mice and have previously been described [36,37,38]. RJ423-200c/141 cells were created by transfecting RJ423 cells with pLenti 4.1 Ex miR200c-141 (cat #35,534, Addgene, Watertown, MA). RJ423-200c/141 cells were cultured in DMEM media containing 10% FBS, 1 mM sodium pyruvate, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 4 mM glutamine, 2 mM hydrocortisone, 5 µg/ml estrogen, 5 µg/ml prolactin, 10 µg/ml EGF, 10 µg/ml insulin, and 1% antibiotic-antimycotic (all supplements purchased from ThermoFisher Scientific, Waltham, MA) as well as 300 µg/ml of puromycin (InvivoGen, San Diego, CA). RJ345, RJ423EV and RJ423-200ba429 cells have been described previously as have the culture conditions for these cells [36].
Crystal violet staining and transwell invasion assays
Crystal violet staining and transwell invasion assays were performed as described in [36] using 25,000 human tumor cells and 30,000 murine tumor cells for the transwell assays.
RNA extraction, taqman qRT-PCR for microRNA expression and qRT-PCR for gene expression
RNA extraction, Taqman qRT-PCR and qRT-PCR for gene expression were performed as described in [37]. All miR-200 Taqman probes were obtained from ThermoFisher Scientific (Waltham, MA). miR-200 expression for the murine mammary tumor cell lines was normalized to sno202 and sno234 while the miR-200 expression of the human breast cancer cell lines was normalized to RNU44 and RNU48. All gene primers were obtained from Bio-Rad Laboratories (Mississauga, ON); Cdh1 (qMmuCID0005843), Hprt (qMmuCED0045738), Snai1 (qMmuCID0024342), Snai2 (qMmuCED0046072), Twist1 (qMmuCED0004065), Twist2 (qMmuCID0009652) Vim (qMmuCID0005527), Zeb1 (qMmuCID0009095), and Zeb2 (qMmuCID0014662). Hprt was used as the housekeeping gene.
miRNA sequencing
miRNA sequencing libraries were generated using NEB Multiplex small RNA library Prep Set for Illumina and sequencing quality was determined using an Agilent 2100 Bioanalyzer. Libraries were sequenced using an Illumina NextSeq 500 instrument. The Q30 scores for all samples were above 93%. Reads were then 3′-adaptor trimmed and filtered ≤ 15 bp reads with cutadapt software (v1.14). Trimmed reads were aligned to the reference genome with bowtie software. miRNA expression levels were calculated using mirdeep2 (v0.0.8) and differentially expressed miRNAs were performed with edgeR (v3.18.1). Library preparation, sequencing and data analysis were performed by Arraystar Inc. (Rockville, MD). Four independent samples were sequenced.
RNA sequencing for one set of RJ423EV samples and the RJ423ba429 samples was performed at the Genome Quebec Innovation Centre at McGill University using the Illumina Hiseq 2500 v4 PE125 as previously described [37]. RNA sequencing for a second set of RJ423EV samples as well as RJ423-200c/141, MDAEV, MDA-200c/141 and MDA-200ba429 were performed by Arraystar Inc (Arraystar Inc., Rockville MD). All Fastq files were processed using Genialis software (Genialis Inc, Houston, TX) following the standard RNA-seq pipeline which uses BBDuk to remove adapters and trim reads, STAR to align the reads, and feature counts to generate gene level counts. RNA sequencing has been uploaded to GEO under accession number GSE150107. Note that our original data for RJ423EV and RJ423-200ba429 samples found at GSE113162 [36] were analyzed by the Genome Quebec Innovation Centre at McGill University and thus might differ from the data in this manuscript that was analyzed using Genialis software. Three independent samples were sequenced for the RJ423 variants and four independent samples were sequenced for the MDA-231 variants.
BrdU and H3K27me3 flow cytometry
For the murine cell lines, a FITC BrdU flow kit (BD Biosciences, San Jose, CA, cat #559,619) and for the human cells an APC BrdU flow kit (BD Biosciences, San Jose, CA, cat #552,598) were used following the manufacturer's protocol. The APC kit was required for the human cell lines as MDA-231EV cell lines express GFP. Briefly, cells were incubated with 1 mM BrdU in fully supplemented media for 45 min. Cells were then fixed, washed and analyzed on an Accuri C6 cytometer (BD Biosciences, San Jose, CA) using a flow rate of 35 μl/min and 20,000 events were collected. For the H3K27me3 analysis, cells were fixed in 4% paraformaldehyde for 15min and then permeabilized in 90% methanol for 10 min. Cells were then incubated with a 1:200 dilution of anti-H3K27me3 (Cell Signaling cat #9733S) or IgG KP isotype control (Cell Signaling cat #3900s) for 1 h followed by a 30 min incubation with the appropriate secondary antibody (Alexa Fluor 488 for the mouse cells and APC for the human cells). Cells were then resuspended in 5% 7-AAD solution for 5 min. Cells were analyzed on an Accuri C6 cytometer (BD Biosciences, San Jose, CA) using a flow rate of 35 μl/min and 20,000 events were collected.
Venn diagrams were generated using Venny 2.1.0 [38].
KEGG pathway, gene ontology and ChEA
For RNA sequencing data Enhrichr [39, 40] was used to identify KEGG and Gene Ontology Pathways affected as well as transcription factors implicated in regulating the differentially expressed genes (ENCODE and ChEA Consensus TFs from ChIP-X).
miRNA binding site analysis
Mouse genes with a target score ≥ 50 for either the miR-200c/141 cluster (miR-200c-3p, miR-200c-5p, miR-141-3p, miR-141-5p) or the miR-200b/200a/429 cluster (miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-429-3p, miR-429-5p) were identified in miRDB [41, 42] and compared to the mRNAs differentially expressed in the RNA-seq data of RJ423c141 or RJ423ba429 cells. The same approach was used for the human genes.
Statistical significance was determined using an ANOVA followed by a Dunnett's test using GraphPad Prism 9 (San Diego, CA). For the MCF-7 and MDA-231 variants, all comparisons were made using MDA-231EV cells as the control and for the RJ345 and RJ423 variants, all comparisons were made using RJ423EV cells as the control.
miR-200 expression and the impact on mesenchymal gene expression and cell shape
As shown in Fig. 1, the murine (RJ345, Fig. 1a) and human (MCF-7, Fig. 1c) mammary tumor cell lines with epithelial characteristics expressed high levels of all 5 miR-200 family members compared to the murine (RJ423/RJ423EV) and human (MDA-MB-231/MDA-231EV) claudin-low mammary tumor cell lines, respectively. Transfection of either the miR-200c/141 cluster or miR-200b/200a/429 cluster in RJ423 or MDA-MB-231 cells significantly increased the expression of one or more of the appropriate miR-200s. For example, RJ423ba429 cells expressed significantly higher levels of miR-200b, miR-200a and miR-429 compared to RJ423EV and MDA-231c141 cells expressed significantly higher levels of miR-200c and miR-141 than MDA-231EV cells.
MicroRNA and gene expression. Expression of the miR-200 family (a, c) and epithelial and mesenchymal genes (b, d) in RJ345, RJ423, RJ423EV, RJ423c141 and RJ423ba429 cells (a, b) and MCF-7, MDA-MB-231, MDA-231EV, MDA-231c141 and MDA-231ba429 cells (c, d). miR-200 expression was determined using Taqman qRT-PCR while gene expression was determined using qRT-PCR. The graphs represent the mean ± SEM (n = 3) and the * indicates as significant difference (p < 0.05) from RJ423EV cells (a, b) or MDA-231EV cells (c, d)
Since the miR-200 family has been implicated in regulating epithelial to mesenchymal transition (EMT) [19, 21,22,23,24,25], the expression of the epithelial gene Cdh1, and several mesenchymal genes were evaluated. The altered miR-200 expression in RJ423 and MDA-MB-231 cells did not significantly increase the expression of the epithelial gene, Cdh1 and had inconsistent effects on mesenchymal genes (Fig. 1b, d). Only Twist2 and Vim were significantly reduced by miR-200 expression in RJ423 cells (Fig. 1c) and only Snai1 was significantly reduced by miR-200 expression in MDA-MB-231 cells (Fig. 1d) however, the expression of Snai1 was extremely low in all the cell lines. Somewhat surprisingly, re-expression of miR-200c/141 in both RJ423 and MDA-MB-231 cells induced a significant increase in Snai2 expression (Fig. 1b, d). Zeb1, Zeb2 are predicted targets of all five miR-200s while Snai2 is a predicted target of miR-200b, miR-200c and miR-429 according to miRDB (mirdb.org). Zeb1 was reduced approximately twofold in both RJ423ba429 cells compared to RJ423EV cells and MDA-231c141 cells compared to MDA-231EV cells but these differences did not reach statistical significance. The mesenchymal genes evaluated in this study are regulated by other miRNAs in addition to the miR-200 family and several transcription factors and thus it is the balance of transcriptional induction and repression as well as post-transcriptional regulation that would determine gene expression levels.
During routine culture of these cells it was observed that RJ345 (Fig. 2a) and MCF-7 (Fig. 2f) cells displayed a more rounded cell shape typically associated with epithelial cells while RJ423 (Fig. 2b) and MDA-MB-231 (Fig. 2g) cells have a more spindle shaped morphology typically associated with mesenchymal cells. Re-expression of miR-200b/200a/429 in RJ423 cells (Fig. 2e and [36]) and re-expression of miR-200c/141 in MDA-MB-231 cells (Fig. 2i) reverted these cells to a more epithelial morphology. This morphological change was observed in all RJ423ba429 (Fig. 2e) and MDA-231c141 (Fig. 2i) cells and was maintained when the cells were passaged.
Cell morphology. Crystal violet staining of murine (a–e) and human (f–j) mammary tumor cells. Mammary tumor cells with a luminal phenotype (a, f) display a more rounded/cuboidal morphology while mammary tumor cells with a claudin-low phenotype (b, g) display a more spindle-like morphology. Re-expression of the miR-200b/200a/429 cluster in RJ423 cells (e) and the miR-200c/141 cluster in MDA-MB-231 cells (i) reverted these cells to a more luminal morphology. Scale bars are 25 µm
To evaluate whether miR-200 re-expression altered cell proliferation, BrdU incorporation was assessed using flow cytometry. Re-expression of miR-200c/141 or miR-200b/200a/429 in murine tumor cells significantly reduced proliferation (Fig. 3a). In the MDA-MB-231 cells re-expression of the miR-200c/141 cluster but not the miR-200b/200a/429 cluster significantly reduced proliferation in MDA-MB-231 cells (Fig. 3b).
Cell proliferation and invasion. Proliferation (a, b) and transwell migration across Matrigel coated inserts (c–l) in murine (a, c–g) and human (b, h–l) mammary tumor cells. Proliferation was determined using BrdU incorporation while transwell migration was determined by calculating the percentage of the lower transwell membrane covered with crystal violet stained cells. Representative images of the crystal violet stained cells on the bottom of the transwell insert have been provided for murine (c–f) and human (h–k) mammary tumor cells. Graphs (a, b, g, l) represent mean ± SEM with n = 3 for the BrdU assay and n = 4 for the transwell assay. * Indicate values significantly different (p < 0.05) from either RJ423EV or MDA-231EV cells
To determine the impact of miR-200s on migration, matrigel-coated, transwell assays were performed. In RJ423 cells, re-expression of either the miR-200c/141 cluster or the miR-200b/200a/429 cluster significantly reduced cell migration and invasion however, these cells still migrated and invaded more efficiently than the RJ345 cells (Fig. 3c–g). Re-expression of the miR-200c/141 cluster but not the miR-200b/200a/429 cluster in MDA-MB-231 cells significantly reduced migration and invasion and re-expression of the miR-200c/141 cluster returned migration and invasion capacity to a level similar to MCF-7 cells (Fig. 3h–l).
Re-expression of miR-200s influence the expression of other miRNAs
miRNA sequencing was performed to confirm that the appropriate miR-200 family members had been upregulated in RJ423c141, RJ423ba429, MDA-231c141 and MDA-231ba429 cells and to determine whether re-expression of miR-200s influenced the expression of other miRNAs. The complete miRNA sequencing files for each cell line compared to their respective control line are presented as Additional file 1 and the top 10 upregulated and downregulated miRNAs based on p-values are presented in Table 1. In each cell line, the miR-200s expressed by the transfected plasmid were found within the top 10 upregulated miRNAs confirming appropriate miR-200 expression. In contrast to the qPCR data (Fig. 1a, c), miRNA-seq found a significant increase in miR-200a and miR-429 in the MDA-231ba429 cells compared to the MDA-231EV cells. The miRNA sequencing data also found a significant increase in miR-200c in RJ423ba429 cells and significant increase in miR-200b and miR-429 in MDA-231c141 cells (Additional file 1).
Table 1 Top 10 upregulated and downregulated miRNAs as determined by miRNA-seq
To further explore miRNAs that were unique and shared in RJ423ba429 cells compared to RJ423c141 cells and MDA-231c141 cells compared to MDA-231ba429 cells, Venn diagrams were created (Additional file 2). These diagrams show miRNAs that were significantly, differentially expressed (log2FC ≥ 1, p < 0.01) in RJ423c141 and RJ423ba429 cells compared to RJ423EV cells (Additional file 2A) and MDA-231c131 and MDA-231ba429 cells compared to MDA-231EV cells (Additional file 2B). Re-expression of the miR-200b/200a/429 cluster altered the expression of more miRNAs than re-expression of the miR-200c/141 cluster in RJ423 cells and only a small number of miRNAs were shared (Additional file 2A). In the human cancer cells, re-expression of the miR-200c/141 cluster altered more miRNAs than re-expression of the miR-200b/200a/429 cluster and like the murine cells, a relatively small proportion of miRNAs were shared (Additional file 2B). Additional file 2C illustrates the number shared and unique miRNAs in the mouse and human miR-200 modified cells.
Re-expression of miR-200s altered the expression of genes regulated by SUZ12
RNA sequencing was also performed to identify genes altered by miR-200s. Hierarchical clustering revealed that the cell types segregated into discrete clusters and RJ423c141 cells clustered more closely with RJ423EV cells than RJ423ba429 cells (Fig. 4a). The analysis of this data was complicated by the fact that the RNA sequencing for the RJ423c141 and RJ423ba429 cells was performed on different days. In each RNA sequencing run, the same RNA from RJ423EV cells was used. However, the hierarchical clustering reveals some batch effects from the different runs as the two sets of RJ423EV samples (RJ423EVB samples that were utilized with the RJ423ba49 cells and RJ423EVC samples that were utilized with the RJ423c141 cells) showed distinct RNA expression profiles (Fig. 4b). Using a of log2FC ≥ 1 and FDR ≤ 0.01, 494 genes were upregulated and 1002 genes were downregulated in RJ423c141 cells compared to RJ423EV cells and 186 (~ 12%) of these genes had predicted miR-200c or miR-141 binding sites (using miRDB, Fig. 4b). When RJ423ba429 cells were compared to RJ423EV cells, 1691 genes were upregulated and 1899 were downregulated and 437 (~ 12%) of these genes had predicted miR-200b, 200a or miR-429 binding sites (Fig. 4b). Of the genes differentially regulated in RJ423c141 vs. RJ423EV and RJ423ba429 vs. RJ423EV, 913 were shared and 583 and 2677 were unique to RJ423c141 and RJ423ba429 cells, respectively (Fig. 4b).
Hierarchical clustering of RNA sequencing data (n = 4). Hierarchical clustering (a, c) and Venn diagram (b, d) analysis of mRNAs differentially expressed in RJ423c141 and RJ423ba429 cells relative to RJ423EV cells (a, b) as well as in, MDA-231c141 and MDA-231ba429 cells relative to MDA-231EV cells (c, d). The Venn diagrams also indicate the percentage of differentially expressed genes with predicted miR-200 binding sites as determined by the miRDB database
RNA sequencing was also performed on MDA-231EV, MDA-231c141 and MDA-231ba429 cells. Again, hierarchical clustering revealed that the 3 cell types segregated into discrete clusters and MDA-231b429 cells were more similar to MDA-231EV cells than MDA-231c141 cells (Fig. 4c). Using a of log2FC ≥ 1 and FDR ≤ 0.01, 1733 genes were upregulated, and 1663 genes were downregulated in MDA-231c141 cells compared to MDA-231EV cells and 409 (~ 12%) have predicted miR-200c or miR-141 binding sites (Fig. 3d). When MDA-231ba429 cells were compared to MDA-231EV cells, 506 genes were upregulated and 948 were downregulated and 159 (~ 11%) of these genes had predicted miR-200b, miR-200a or miR-429 binding sites (Fig. 4d). Of the genes differentially regulated in MDA-231c141 vs. MDA-231EV and MDA-231ba429 vs. MDA-231EV, 766 were shared and 2630 and 688 were unique to MDA-231c141 and MDA-231ba429 cells, respectively (Fig. 4d).
Since RJ423ba429 cells and MDA-231c141 cells display a change in cell morphology (Fig. 2), the genes unique to these two cell lines were further examined using Enrichr [40, 41] to identify pathways associated with the unique genes in RJ423ba429 and MDA-231c141 cells (Fig. 5a, b). Although there were only a small number of pathways shared between RJ423ba429 and MDA-231c141 cells, the top ChEA category was the same in both cell lines, SUZ12 (Fig. 5c). The Encode and ChEA Consensus TFs from ChIP-X database from Enrichr lists 4439 predicted Suz12 targets and it was found that 863 of the 2677 (~ 32%) unique genes in RJ423ba429 cells and 706 of the 2630 (~ 27%) unique genes in MDA-231c141 cells were predicted targets of SUZ12.
Pathway analysis of genes regulated by miR-200s. KEGG Pathways (a), Gene Ontology (b), and ENCODE/ChEA analysis (c) of genes differentially expressed in RJ423ba429 cells (white bars) or MDA-231c141 cells (grey bars)
When comparing the RJ423ba429 unique gene profile to the MDA-231c141 unique gene profile, it was determined that 370 genes were shared between the unique gene profiles of the two cell lines (Fig. 6a). The KEGG and Gene Ontology Pathways as well as the ChEA pathways of these shared genes are presented in Fig. 5b, c and suggest that altering miR-200 expression in these cells influences pathways such as hippo signaling, endothelial migration and metallopeptidase activity (Fig. 6d). The top ChEA pathways remained SUZ12 and 194 of the 370 shared genes (~ 52%) were predicted SUZ12 targets.
Venn diagrams and pathway analysis of shared genes. Venn diagram (a), KEGG Pathways (b), Gene Ontology (c) and ENCODE/ChEA analysis (d) of genes differentially expressed in both RJ423ba429 and MDA-231c141 cells
H3K27me3 levels are elevated in RJ423ba429 and MDA-231c141 cells
SUZ12 is a member of the polycomb repressor complex 2 (PRC2) which mono-, di- and tri-methylates lysine 27 on histone H3 [43,44,45,46,47,48,49,50]. To further investigate differences in SUZ12 activity, flow cytometry was used to quantify trimethylated histone H3 (H3K27me3). As shown in Fig. 7, RJ423ba429 cells had significantly higher levels of H3K27me3 than RJ423EV cells (Fig. 7a) and MDA-231c141 cells had significantly higher levels of H3K27me3 than MDA-231EV cells (Fig. 7b). The luminal breast cancer cell line, MCF-7, also had significantly elevated levels of H3K27me3 compared to the claudin-low breast cancer cell line MDA-231EV while RJ345 cells that have both luminal and basal-like characteristics, did not have higher levels of H3K27me3 compared to the claudin-low RJ423EV cells.
Quantification of H3K27me3 levels using flow cytometry. The levels of H3K27me3 in RJ345, RJ423c141, and RJ423ba429 cells were expressed relative to the levels of H3K27me3 in RJ423EV cells (a) while the levels of H3K27me3 in MCF-7, MDA-231c141 and MDA-231ba429 cells were expressed relative to the levels of H3K27me3 in MDA-231EV cells (b). The graphs represent the mean ± SEM (n = 5) and the * indicates p < 0.05 relative to RJ423EV cells (a) and MDA-231EV cells (b)
Re-expression of miR-200s in human and murine claudin-low mammary tumor cells is sufficient to induce a partial reversion to an epithelial morphology and to reduce cell migration. The miR-200b/200a/429 cluster but not the miR-200c/141 cluster was able to induce these alterations in cell shape and migration in the murine mammary tumor cell line, RJ423, while it was the miR-200c/141 cluster, and not the miR-200b/200a/429 cluster, that was capable of inducing these alterations in MDA-MB-231 cells. While cluster-specific effects in murine and human cell lines are possible, it is also possible that miR-200 expression levels are an important factor. In RJ423 cells, the miR-200b/200a/429 cluster was expressed at higher levels than the miR-200c/141 cluster while in the MDA-MB-231 cells the miR-200c/141 cluster was expressed at higher levels than the miR-200b/200a/429 cluster. Therefore, miR-200s may require expression levels beyond a certain threshold in order to have significant cellular effects. It also appears that if one miR-200 cluster is expressed at sufficiently high levels, it can positively influence the expression from the other miR-200 cluster. This was demonstrated in both RJ423ba429 where re-expression of the miR-200b/200a/429 cluster also induced a significant increase in miR-200c while in MDA-231c141 cells re-expression of the miR-200c/141 cluster induced a significant increase in miR-200b and miR-429 expression as determined by miRNA sequencing.
While the impact of miRNAs regulating the expression of mRNAs has been extensively investigated, the regulation of miRNAs by other miRNAs has largely been ignored. A small number of papers have shown that miRNAs can regulate other miRNAs [51,52,53], however, no paper describing a comprehensive examination of miRNAs regulated by the miR-200 family could be found. The miRNA sequencing results from our study demonstrate that simply re-expressing miR-200s induced alterations in the expression of a number of other miRNAs. Not only was the number of miRNAs regulated by miR-200 re-expression unexpected (> 100 miRNAs regulated in RJ423ba429 and MDA-231c141 cells), so was the small overlap (16–18%) of miRNAs regulated by the miR-200c/141 and miR-200b/200a/429 clusters in both the murine and human cancer cell lines. The small overlap in expressed miRNAs suggest that the miR-200c/141 and miR-200b/200a/429 clusters regulate the expression of different miRNAs.
Our observations that re-rexpression of miR-200s in claudin-low mammary tumor cells can inhibit proliferation and migration/invasion as well as altering cell shape is consistent with several published studies. These studies showed that re-expression of miR-200c in MDA-MB-231 cells significantly suppressed cell migration and promoted a more rounded cell morphology [54, 55], while treatment of MDA-231 cells with a miR-200c mimic was shown to significantly inhibit transwell invasion [56,57,58,59] or proliferation [57, 60]. Although our current study did not find that re-expression of the miR-200b/200a/429 cluster in MDA-MB-231 cells significantly impacted proliferation or invasion, other groups have shown that treating MDA-MB-231 cells with a miR-200a mimic [61], a miR-200b mimic [62,63,64,65], a miR-429 mimic [63], or overexpressing miR-200b [66], significantly reduced migration/invasion and treating MDA-MB-231 cells with a miR-200b or miR-429 mimic significantly reduced proliferation [63, 64]. It should be noted that two studies found miR-200 effects that contracted our findings. The first study stably expressed either the miR-200b/200a/429 or miR-200c/141 clusters in MDA-MB-231 cells and found that both clusters reduced cell growth (the miR-200c/141 cluster was more effective than the miR-200b/200a/429 cluster) and both clusters promoted migration and invasion [67]. The second study showed that re-expression of miR-200c significantly promoted invasion in a transwell assay [68]. Studies on murine claudin-low mammary tumor cells are far rarer and the only study outside our lab on murine claudin-low mammary tumor cells was performed in vivo and found that re-expression of the miR-200c/141 cluster in p53null mammary tumors reduced tumor growth and metastasis to the lungs [69].
RNA sequencing revealed a similar pattern with RJ423ba429 and MDA-231c141 cells expressing more differentially expressed mRNAs than RJ423c141 or MDA-231ba429 cells, respectively. It also revealed that the miR-200c/141 and miR-200b/200a/429 clusters targeted distinct mRNAs despite both clusters sharing identical seed sequences. Cluster specific mRNA (and miRNA) expression profiles were observed in both murine and human mammary tumor cell lines reducing the possibility of this being an artifact. Another interesting observation from the RNA sequencing study was that no more that 12% of the differentially expressed mRNAs in any of the cell lines were predicted targets of the miR-200 family. This is not completely unexpected given the large number of miRNAs also regulated by the miR-200 family as described above. However, this data suggests that studies focusing only on predicted mRNA targets may miss a majority of mRNAs regulated by their miRNA of interest .
To reduce the complexity, differentially expressed mRNAs unique to RJ423ba429 and MDA-231c141 cells (the two lines showing changes in cell shape) were further examined. In both cell lines, the top ChEA pathway identified was SUZ12. SUZ12 is a component of the Polycomb repressive complex 2 (PRC2) that mono-, di- and trimethylates lysine 27 of histone H3 (H3K27) [43,44,45,46,47,48,49,50]. Trimethylation of H3K27 promotes chromosome compaction preventing transcription factors from accessing transcription start sites in promoters [70,71,72]. miR-200b and miR-200c have been reported to target SUZ12 [73,74,75], downregulating its expression and leading to hypomethylation of H3K27 and loss of H3K27 trimethylation. However, re-expression of the miR-200b/200a/429 cluster in RJ423 cells and re-expression of the miR-200c/141 cluster in MDA-MB-231 cells resulted in a significant increase in H3K27me3 levels. In normal mammary epithelial cells, higher levels of H3K27me3 are observed in mature luminal cells compared to mammary stem cells and H3K27me3 levels have been observed to increase during mammary epithelial differentiation during pregnancy [76]. Therefore, re-expression of the miR-200b/200a/429 cluster in RJ423 cells and re-expression of miR-200c/141 cluster in MDA-MB-231 cells may reduce the stem cell features of these cells and drive them to a more differentiated phenotype. Evaluation of gene specific H3K27 methylation patterns are required to confirm this and identify the genes affected.
Understanding how miRNAs influence cellular function is already complex due to the large number of predicted targets and false positives in existing databases, however, our study demonstrates that miR-200s can regulate other microRNAs as well as epigenetic modulators thus adding additional complexity to this system. We have also shown that re-expression of miR-200s in claudin-low mammary tumor cells can revert claudin-low cells to a more epithelial phenotype provided the level of miR-200 re-expression is sufficiently high and this reversion is associated with alterations in SUZ12 regulated genes and reduced cell migration.
RNA sequencing has been uploaded to GEO under accession number GSE150107 and the miRNA sequencing files are presented as Additional file 1.
BrdU:
5-Bromo-2′-deoxyuridine
FITC:
Fluorescein isothiocyanate
H3K27:
Lysine 27 of histone H3
Trimethylated lysine 27 of histone H3
HER2:
Human epidermal growth factor receptor 2
MDA-231ba429:
MDA-MB-231 cells stably expressing the miR-200b/200a/429 cluster
MDA-231c141:
MDA-MB-231 cells stably expressing the miR-200c/141 cluster
MDA-231EV:
MDA-MB-231 cells stably expressing an empty vector
miRNA:
PCR2:
Polycomb repressor complex 2
Progesterone receptor
RJ423ba429:
RJ423 cells stably expressing the miR-200b/200a/429 cluster
RJ423c141:
RJ423 cells stably expressing the miR-200c/141 cluster
RJ423EV:
RJ423 cells stably expressing an empty vector
TNBC:
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This work was funded by a CIHR operating grant (MOP-136970) and a CHIR project grant (PJT-162218) awarded to RAM.
Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada
K. Simpson, G. Conquer-van Heumen, K. L. Watson, M. Roth, C. J. Martin & R. A. Moorehead
K. Simpson
G. Conquer-van Heumen
K. L. Watson
M. Roth
C. J. Martin
R. A. Moorehead
KS performed the crystal violet staining, transwell migration assay, flow cytometry for BrdU and H3K27me3 and qRT-PCR for the human breast cancer cells as well as editing the manuscript. GC-VH performed the crystal violet staining, transwell migration assay and flow cytometry for BrdU and H3K27me3 for the murine mammary cells as well as editing the manuscript. CJM performed the qRT-PCR for the mRNAs in the control and miR-200 transfected murine cells. MR performed the qRT-PCR for the microRNAs in the control and miR-200 transfected murine cells. KLW assisted with the qRT-PCR for both the miRNAs and the mRNAs. RAM performed the analysis of the miRNA sequencing and mRNA sequencing data, wrote the manuscript and ran the project. All authors read and approved the final manuscript.
Correspondence to R. A. Moorehead.
miRNA sequencing files for RJ423c141 vs. RJ423EV, RJ423ba429 vs. RJ423EV, MDA-231c141 vs. MDA-231ba429 and MDA-231ba429 vs. MDA-231EV cell lines.
Venn diagrams showing unique and shared miRNAs in the mouse cell lines (A), the human cell lines (B), and a combination of the mouse and human cell lines (C).
Simpson, K., Conquer-van Heumen, G., Watson, K.L. et al. Re-expression of miR-200s in claudin‐low mammary tumor cells alters cell shape and reduces proliferation and invasion potentially through modulating other miRNAs and SUZ12 regulated genes. Cancer Cell Int 21, 89 (2021). https://doi.org/10.1186/s12935-021-01784-4
miR-200
Claudin‐low breast cancer
H3K27me3


Some stocks rallied between 10% and 20% in the past month against a 1% gain in Sensex.
Bajaj Holdings holds 31.54 per cent in Bajaj Auto and 39.29 per cent stake in Bajaj Finserv. The company also has an investment portfolio with a market value of nearly Rs 7,000 crore in other equity and fixed income securities. The company's dividend payout ratio is nearly 14 per cent for the FY18. If the discounted value shrinks from current 50 per cent to 40 per cent in near term, the stock would gain 20 per cent, said Baliga.


It's no secret that positive cash flow is crucial to your business because without it, you can't pay salaries or other expenses. Don't think that calculating cash flow is a luxury; it is a necessity. It is one of those things you must analyze on a regular basis in order to maintain a thriving business.
Here are five steps to calculate cash flow.
1. Confirm your bank statements.
Verify that the funds you expect to be in the bank are, indeed, there.
2. Make a list of monthly expected cash sources.
List all the recurring revenue that you can count on.
3. Determine your additional monthly income.
Study your sales forecast and determine which deals will close and how quickly you will receive payment.
4. Make a list of monthly expected expenditures.
List all the recurring expenses that you pay each month.
5. Determine additional items you need or would like to spend on this month.
This is the one area that you can juggle up or down, depending on what the numbers from the three items above indicate.


text:
CCK Sees Social Security Clearly (And Helps Bush)
"John Thune Wants to Steal Your Social Security Money," by Chad Shuldt, Clean Cut Kid, 29 April2005, http://www.cleancutkid.com/2005/04/29/john-thune-want-to-steal-your-social-security-money/.
"Social Security's Progressive Paradox," by Julian Sanchez, Reason, 2 May 2005, http://www.reason.com/links/links050205.shtml (from South Dakota Politics).
Key: Blue, rejectionists; Red, reformers; Orange, rejectionists ready to deal; Dark Blue, extreme rejectionists; solid lines, mutual support; arrow lines, mutual opposition
It shows a successul 4GP network attack on a 2GP defending network. It shows the current state of the social security debate, with the (mostly Republican) Reformists splitting the (mostly Democrat) Rejectionist camp by offering progressive indexing.
The success comes from Bush splintering the Democrat network. While mainstream liberals such as the New York Times, USA Today, and Washington Post are urging Democrats to compromise, netroots such as CCK have different ideas
There is one proposal out there that is based around "progressive" indexing, and it's likely to be presented to Congress since "everything is on the table" (other than keeping Social Security as close to possible in its current state). But make no mistake about it, progressive indexing is just another scheme to rob working people of the money they have already paid into the system through taxation on every dollar they worked to earn.
Now, CCK is right that progressive indexing is an attempt to re-welfare-ize social security..
Neither is the notion of Social Security as "insurance" terribly coherent, if it ever was. Even when Social Security was first instituted, over half of Americans who reached the age of 21 would survive past age 65. As of 1990 the percentages were over 72 percent for men and 83 percent for women. Aging is not a "risk" to "insure" against; it's a normal part of life to plan for.
What worries liberals about progressive indexing, and about the shift to a more overtly welfare-like Social Security system, is that welfare benefits tend to be politically unpopular—and much easier to cut than benefits perceived as universal. Social Security, in other words, is a massive Rube Goldberg device, an ornate and utterly superfluous system of transfers from the middle and upper classes to themselves, the sole purpose of which is to construct—and conceal—a much smaller welfare machine for elderly retirees nestled deep in the guts of the meta-contraption. Some defenders of the status quo are now attempting—though they scarcely seem to believe it themselves—to argue that Social Security is no less vital for the middle class. But corner a progressive over a quiet drink and he'll probably admit that, in fact, the only defensible purpose of Social Security is to ensure that nobody retires in poverty. There may be political reasons for cutting a monthly check to Bill Gates when he turns 65, but there are no sane policy reasons.
Yet here the irony. CCK sees the issue clearly — proressive indexing will "weaken" the ageist everyone-to-elderly income transfer aspect of Social Security. But his actions — begging the liberal leadership to get back in line and scaring the "masses" with pseudo-facts — disrupt his own network and make Bush's job easier.
President Bush laid a political trap that SS Rejectionists jumped in to. That's the power that comes from understanding modern politics clearly.
clean cut kidPublic Financessocial security


Notes: The Black Reel Awards, inaugurated in 2000, annually recognize and celebrate the achievements of African-Americans in feature, independent and television films, ranging from their character portrayals in front of camera, to their technical artistry behind the camera.
The FAAAF/Black Reel Awards benefit The Foundation for the Advancement of African Americans in Film (FAAAF), a non-profit arts organization whose mission is to provide educational opportunities to the next generation of minority film executives. Through the FAAAF "Producer's Institute," scholarships are awarded to graduate students pursuing a business career in the movie and television industries.


Hmm, I think I've seen this picture at a site somewhere....not sure tho. I don't think he -made- it all by himself.
decent man not bad at all use photoshop?
is that a snake on his staff?
hey affe, is that supposed to be our Giran-house? kinda small eh?
Is it just me, or does the frase 'Snake on his staff' sound really...ummm...strange...?


The insurance affiliate of Lighthouse Guild International has signed a lease with an ESRT property in the Billionaires' Row neighborhood.
New York—Empire State Realty Trust (ESRT), a real estate investment trust that owns, manages, operates, acquires and repositions office and retail assets in the greater New York area, recently signed a new tenant at 250 W. 57th St. in Manhattan.
ESRT has leased the entire tenth floor and 37,680 square feet on the ninth floor of the building to GuildNet, the insurance affiliate of Lighthouse Guild International Inc. CBRE's Brian Gell and Laurence Briody, Jr. represented GuildNet in the lease negotiations, while Keith Cody of ESRT, alongside Harry Blair and Sean Kearns of Cushman & Wakefield provided landlord representation.
The 26-story office tower at 250 W. 57th St. is conveniently located just steps from the Time Warner Center, Carnegie Hall, the Lincoln Center, and Nordstrom's future flagship location. The building is currently undergoing extensive renovations, and will feature a new Gensler-designed lobby, building entrance, modernized elevator system and elevator cabs, retail storefronts and a cooling tower. Part of the city's upscale Billionaires' Row neighborhood, the property also offers access to five subway lines and the soon-to-open TurnStyle.


text:
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Mark Zuckerberg asks governments to help control internet content
Mark Zuckerberg says regulators and governments should play a more active role in controlling internet content.
In an op-ed published in the Washington Post, Facebook's chief says the responsibility for monitoring harmful content is too great for firms alone.
He calls for new laws in four areas: "Harmful content, election integrity, privacy and data portability."
It comes two weeks after a gunman used the site to livestream his attack on a mosque in Christchurch, New Zealand.
"Lawmakers often tell me we have too much power over speech, and frankly I agree," Mr Zuckerberg writes, adding that Facebook was "creating an independent body so people can appeal our decisions" about what is posted and what is taken down.
He also describes a new set of rules he would like to see enforced on tech companies.
These new regulations should be the same for all websites, he says, so that it's easier to stop "harmful content" from spreading quickly across platforms.
What does Mark Zuckerberg want?
In brief, Mr Zuckerberg calls for the following things:
Common rules that all social media sites need to adhere to, enforced by third-party bodies, to control the spread of harmful content
All major tech companies to release a transparency report every three months, to put it on a par with financial reporting.
Stronger laws around the world to protect the integrity of elections, with common standards for all websites to identify political actors
Laws that not only apply to candidates and elections, but also other "divisive political issues", and for laws to apply outside of official campaign periods
New industry-wide standards to control how political campaigns use data to target voters online
More countries to adopt privacy laws like the European Union's General Data Protection Regulation (GDPR), which came into force last year.
A "common global framework" that means these laws are all standardised globally, rather than being substantially different from country to country Clear rules about who's responsible for protecting people's data when they move it from one service to another.
The open letter, which will also be published in some European newspapers, comes as the social network faces questions over its role in the Cambridge Analytica scandal around data misuse during election campaigns.
The site has also been criticised for failing to stop the spread of footage of the Christchurch killings, in which 50 Muslims died as they prayed.
The video was livestreamed to the attacker's Facebook page on 15 March, before being copied 1.5 million times.
Mr Zuckerberg's letter did not specifically name these incidents.
However, the site earlier announced that it was considering introducing restrictions on live-streaming in the wake of the Christchurch attacks. On Thursday, it also said that it would ban white nationalism and separatism from the site.
On Friday it also started labelling political ads appearing on Facebook in EU countries, showing who the advertiser is, how much they paid and who they've targeted.
"I believe Facebook has a responsibility to help address these issues, and I'm looking forward to discussing them with lawmakers around the world," Mr Zuckerberg says.
China rushes to stamp out virus outbreak in city of 11 million
United States surpasses 20 million confirmed coronavirus cases
Chinese economy to overtake US 'by 2028' due to Covid
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Ref watch: Bradford City (h)
Match officials announced for second leg playoff
Nottinghamshire based referee David Coote will be the man in the middle for the Sky Bet League One Play-Off Semi-Final second leg against Bradford City on Sunday.
Mr Coote has primarily officiated Sky Bet Championship matches this season and has covered the EFL Trophy.
Coote has taken charge of one Fleetwood Town game this season, and that was the EFL Trophy defeat against Leeds United back in August, which ended with a penalty shoot-out.
This season, Mr Coote has refereed 38 games and has given out 127 yellow cards, as well as seven reds.
He will be assisted by Adam Matthews and Philip Dermott while Geoff Eltringham will be on fourth official duty.


Veronika requested minimalistic circle logo design with purple colour including her initials and if possible butterfly.
Anything is possible so I started working on the design. After I got nearly done the first version, I sent it to Veronika and she told me that's the one.
Without any hard feelings, I finished the logo really fast and easily.
Veronika was using her new logo couple months in her completely new blog. But currently, I am not sure If she is recreating her project or If she completely finished and started to work on something else. Anyway, this is was nice and easy going cooperation satisfying both of us.


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